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Costa-Hurtado, Mar, Alexandre Olvera, Verónica Martinez-Moliner, Nuria Galofré-Milà, Paloma Martínez, Javier Dominguez, and Virginia Aragon. "Changes in Macrophage Phenotype after Infection of Pigs with Haemophilus parasuis Strains with Different Levels of Virulence." Infection and Immunity 81, no. 7 (April 15, 2013): 2327–33. http://dx.doi.org/10.1128/iai.00056-13.
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ABSTRACTHaemophilus parasuisis a colonizer of healthy piglets and the etiological agent of Glässer's disease. Differences in virulence among strains ofH. parasuishave been widely observed. In order to explore the host-pathogen interaction, snatch-farrowed colostrum-deprived piglets were intranasally infected with 4 strains ofH. parasuis: reference virulent strain Nagasaki, reference nonvirulent strain SW114, field strain IT29205 (from a systemic lesion and virulent in a previous challenge), and field strain F9 (from the nasal cavity of a healthy piglet). At different times after infection, two animals of each group were euthanized and alveolar macrophages were analyzed for the expression of CD163, CD172a, SLA I (swine histocompatibility leukocyte antigen I), SLA II, sialoadhesin (or CD169), and CD14. At 1 day postinfection (dpi), virulent strains induced reduced expression of CD163, SLA II, and CD172a on the surfaces of the macrophages, while nonvirulent strains induced increased expression of CD163, both compared to noninfected controls. At 2 dpi, the pattern switched into a strong expression of CD172a, CD163, and sialoadhesin by the virulent strains, which was followed by a steep increase in interleukin 8 (IL-8) and soluble CD163 in serum at 3 to 4 dpi. The early increase in surface expression of CD163 induced by nonvirulent strains went along with higher levels of IL-8 in serum than those induced by virulent strains in the first 2 days of infection. Alpha interferon (IFN-α) induction was observed only in animals infected with nonvirulent strains. Overall, these results are compatible with a delay in macrophage activation by virulent strains, which may be critical for disease production.
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Fabriek, Babs O., Machteld M. J. Polfliet, Rianka P. M. Vloet, Roel C. van der Schors, Antoon J. M. Ligtenberg, Lehn K. Weaver, Christiaan Geest, et al. "The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor." Blood 109, no. 12 (June 15, 2007): 5223–29. http://dx.doi.org/10.1182/blood-2006-08-036467.
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Abstract Erythropoiesis occurs in erythroblastic islands, where developing erythroblasts closely interact with macrophages. The adhesion molecules that govern macrophage-erythroblast contact have only been partially defined. Our previous work has implicated the rat ED2 antigen, which is highly expressed on the surface of macrophages in erythroblastic islands, in erythroblast binding. In particular, the monoclonal antibody ED2 was found to inhibit erythroblast binding to bone marrow macrophages. Here, we identify the ED2 antigen as the rat CD163 surface glycoprotein, a member of the group B scavenger receptor cysteine-rich (SRCR) family that has previously been shown to function as a receptor for hemoglobin-haptoglobin (Hb-Hp) complexes and is believed to contribute to the clearance of free hemoglobin. CD163 transfectants and recombinant protein containing the extracellular domain of CD163 supported the adhesion of erythroblastic cells. Furthermore, we identified a 13–amino acid motif (CD163p2) corresponding to a putative interaction site within the second scavenger receptor domain of CD163 that could mediate erythroblast binding. Finally, CD163p2 promoted erythroid expansion in vitro, suggesting that it enhanced erythroid proliferation and/or survival, but did not affect differentiation. These findings identify CD163 on macrophages as an adhesion receptor for erythroblasts in erythroblastic islands, and suggest a regulatory role for CD163 during erythropoiesis.
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Schaer, D. J., G. Schoedon, and A. Schaffner. "Assignment of the CD163 antigen (Cd163) to mouse chromosome 6 band F2 by radiation hybrid mapping." Cytogenetic and Genome Research 98, no. 2-3 (2002): 231B. http://dx.doi.org/10.1159/000069812.
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Nishiwaki, Satoshi, Seitaro Terakura, Tatsunori Goto, Aika Seto, Keisuke Watanabe, Nobuhiko Imahashi, Shokichi Tsukamoto, et al. "Macrophage Infiltration of Skin Lesions Correlates to Prognosis of Gvhd; A Clue to Refractory Gvhd." Blood 112, no. 11 (November 16, 2008): 1178. http://dx.doi.org/10.1182/blood.v112.11.1178.1178.
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Abstract The pathophysiology of acute graft-versus-host disease (aGVHD) is a complex process. Although it is considered an alloimmune attack on host tissues mounted mainly by donor CD8-positive T cells, skin infiltration of T cells is not always the case in biopsy specimens of skin aGVHD, suggesting the involvement of other cellular components. In detailed observation, CD163-positive macrophages which have abilities of antigen presentation and releasing cytokines were frequently observed. We retrospectively reviewed 66 biopsy specimens of skin lesions clinically thought to be aGVHD performed within 100 days after allogeneic stem cell transplantation, and analyzed the relationship between types of infiltrating cells and clinical outcomes. Paraffin-section immunohistochemical analysis was performed using a monoclonal antibody against CD8 and CD163. Counting the total number of CD8-positive T cells and CD163-positive macrophages in 4 fields with 200-fold magnification, median number of CD-8 positive T cells was 78.5 (range; 2–305), and that of CD163-positive macrophages was 149 (range; 38–372). Infiltration of over 100 cells of CD8-positive T cells (CD8ov100) correlated with HLA class I mismatch and grade III–IV a GVHD, while infiltration of over 200 cells of CD163-positive macrophages (CD163ov200) correlated with grade III–IV aGVHD and refractory aGVHD. In logistic analyses, CD163ov200 (Odds 6.29 (95%CI 1.57–25.0); p=0.009) was identified as the only negative predictors of aGHVD outcome. Overall survival of patients with CD163ov200 was significantly lower than that of those with infiltration of under 200 cells of CD163-positive macrophages (63.0% vs. 40.6% at 1 year, respectively; p=0.02). In 23 patients treated with steroid for aGVHD, CD163ov200 was the only significant predictor of refractory aGVHD outcome (Odds 10.9 (95%CI 1.37–83.3); p=0.02), and overall survival of patients with CD163ov200 was significantly lower than that of those with infiltration of under 200 cells of CD163-positive macrophages (63.5% vs. 12.5% at 1 year, respectively; p=0.0004). These results indicate that CD163-positive macrophages may provide a clue for the pathophysiology of refractory GVHD and new treatment strategies.
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Santegoets, Saskia J., Chantal L. Duurland, Ekaterina J. Jordanova, Vanessa J. van Ham, Ilina Ehsan, Nikki M. Loof, Vipin Narang, et al. "CD163+ cytokine-producing cDC2 stimulate intratumoral type 1 T cell responses in HPV16-induced oropharyngeal cancer." Journal for ImmunoTherapy of Cancer 8, no. 2 (August 2020): e001053. http://dx.doi.org/10.1136/jitc-2020-001053.
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BackgroundHuman papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is a distinct clinical entity with a much better prognosis after (chemo)radiotherapy than HPV-negative OPSCC, especially in patients with a concomitant intratumoral HPV-specific and type-1 cytokine-oriented T cell response. However, knowledge on the type of myeloid cells and their coordination with intratumoral T cells and influence on patient outcome in OPSCC is lacking.MethodsWe analyzed the presence of intratumoral myeloid cells and their relationship to tumor-infiltrating T cells and patient outcome in a well-described cohort of HPV16+ patients with OPSCC using multispectral immunofluorescence, flow cytometry and functional analyses.ResultsWe show that the tumor microenvironment of HPV16+ OPSCC tumors with such an ongoing HPV16-specific T cell response is highly infiltrated with a newly defined CD163+ cytokine-producing subset of conventional dendritic cell type 2 (cDC2), called DC3. These CD163+ cDC2 predominantly stimulated type 1 T cell polarization and produced high levels of interleukin-12 (IL-12) and IL-18, required for IFNγ and IL-22 production by T cells after cognate antigen stimulation. Tumor-infiltration with these CD163+ cDC2 positively correlated with the infiltration by Tbet+ and tumor-specific T cells, and with prolonged survival.ConclusionsThese data suggest an important role for intratumoral CD163+ cDC2 in stimulating tumor-infiltrating T cells to exert their antitumor effects.
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Gandhi, Maher K., David Arpon, Colm Keane, Erica Han, Josh Tobin, Robert Bird, Mark S. Hertzberg, et al. "A Novel Anti-Lymphoma Immune Evasion Mediated By the Interaction Between PD-1 Enriched NK-Cells and CD163+PD-L1+PD-L2+ Tumor Associated Macrophages, That Is More Prominent in Hodgkin Lymphoma Than Diffuse Large B-Cell Lymphoma." Blood 128, no. 22 (December 2, 2016): 918. http://dx.doi.org/10.1182/blood.v128.22.918.918.
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Abstract PD-L1/PD-L2 are immunomodulatory molecules that engage with the PD-1 receptor on immune effector T and NK-cells to inhibit anti-lymphoma immunity. PD-1/PD-L1/PD-L2 axis molecules are prognostic in Hodgkin Lymphoma (HL, Roemer et al J Clin Oncol 2016) and Diffuse Large B-cell Lymphoma (DLBCL, Keane et al Lancet Haem 2015). Importantly, blockade of the axis is associated with particularly potent clinical responses in relapsed/refractory HL (Ansell et al NEJM 2015), as well as response in DLBCL (Armand et al J Clin Oncol 2013). Focus has been on the interaction of PD-L1 on malignant B-cells with PD-1 on HLA-class I restricted CD8+ effector T-cells. This is despite considerable evidence that: A) malignant B-cells in HL and DLBCL frequently lack the ability to present HLA-class I due to mutations in b2M and associated antigen presenting molecules (Challa-Malladi et al Cancer Cell 2013). This makes them insensitive to direct lysis by CD8+ T-cells (Zaretsky et al NEJM 2016) but potentially enhances their sensitivity to NK-cells; B) PD-L1/PD-L2 are expressed by inhibitory CD163+ monocytes/macrophages as well as by malignant B-cells (Chen et al CCR 2013). Here, we seek to establish the contribution of NK-cells and inhibitory CD163+ expressing monocytes/macrophages in the setting of HL and DLBCL. CD163/PD-1/PD-L1/PD-L2 gene expression was quantified by nanoString in 194 patients and was elevated in HL relative to DLBCL tissues (P<0.01, <0.01, <0.0001, <0.0001 respectively). By FACS, intratumoral tumor associated macrophages (TAMs) demonstrate pronounced protein expression of PD-L1/PD-L2 within HL and DLBCL diseased lymph nodes (Fig A). Pre-therapy blood was tested in 114 patients. Interestingly levels in each of total monocytes, CD14+HLA-DRlo monocytoid derived suppressor cells (moMDSC) and CD163+CD14+ monocytes were equivalent between lymphoma sub-types. However, consistent with tissue findings, there was marked increase in PD-L1 expression on CD14+ monocytes, moMDSC and CD163+CD14+ monocytes in HL compared to DLBCL patients (P<0.001, <0.0001 and 0.0086 respectively). The NK-cell marker CD56 were higher in HL compared to DLBCL tissues (P<0.0001). Levels of PD-1 on circulating NK-cells were 7-fold elevated in HL relative to DLBCL (P<0.0001), whereas CD4+ and CD8+ T-cell PD-1 levels were equivalent between lymphoma sub-types. NK-cells can be subdivided into CD3-CD56dimCD16+ and CD3-CD56hiCD16- subsets. The CD16- subset produces abundant cytokines but are only weakly cytotoxic before activation. Although CD16- NK-cells are typically <10% of all NK-cells in the healthy circulation, we show their relative proportion is markedly expanded by 3.5-fold in HL patients. This is of particular importance since CD16- NK-cells are enriched in secondary lymphoid tissues, i.e. the context in which lymphoma resides. Notably, CD3-CD56hiCD16- NK-cells had substantially higher PD-1 expression relative to CD3-CD56dimCD16+ cells (P<0.0001, Fig B). A similarly aberrant NK-cell phenotype was observed in DLBCL. An in-vitro functional model of TAM-like monocytes was developed to demonstrate the potential impact of inhibitory CD163+ expressing monocytes/macrophages on NK-cells in HL and DLBCL. Monocytes were cultured with the M6 TAM inducing cytokine cocktail of M-CSF and IL-6. Consistent with an inhibitory phenotype, M6 cultured monocytes were highly enriched for CD163 (P<0.001) and PD-L1 (P=0.0024). Critically, M6 cultured monocytes suppressed activation of primary NK-cells in direct cytotoxicity and ADCC assays against lymphoma targets. In line with these findings, depletion of circulating monocytes from the blood of pre-therapy HL and DLBCL patients enhanced NK-cell activation relative to monocyte intact PBMC, whereas this was not observed in age/gender-matched healthy control participants. Interestingly, the increase in NK-cell activation following monocyte depletion was most pronounced in the CD16-CD56hiCD3- NK-cell subset. We describe a hitherto unrecognised immune evasion strategy mediated via skewing towards an exhausted PD-1 enriched CD16-CD56hiCD3- NK-cell phenotype. In addition to inhibition of NK-cells by the malignant B-cell, suppression of NK-cells occurs by PD-L1/PD-L2 expressing tumor associated macrophages. This mechanism is more prominent in HL than DLBCL. Figure. Figure. Disclosures No relevant conflicts of interest to declare.
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Sánchez-Martín, Lorena, Ana Estecha, Rafael Samaniego, Silvia Sánchez-Ramón, Miguel Ángel Vega, and Paloma Sánchez-Mateos. "The chemokine CXCL12 regulates monocyte-macrophage differentiation and RUNX3 expression." Blood 117, no. 1 (January 6, 2011): 88–97. http://dx.doi.org/10.1182/blood-2009-12-258186.
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Abstract Monocytes are versatile cells that can express different functional programs in response to microenvironmental signals. We show that primary blood monocytes secrete the CXCL12 chemokine, and express the CXCR4 and CXCR7 receptors, leading to an autocrine/paracrine loop that contribute to shape monocyte differentiation to a distinct type of macrophages, with an enhanced expression of CD4, CD14, and CD163, or dendritic cells, with a reduced functional ability to stimulate antigen-specific T-lymphocyte responses. The in vivo relevance of CXCL12 production by mononuclear phagocytes was studied in metastatic melanoma tissues by a thoroughly immunofluorescence phenotyping of CXCL12high expressing cells, which were CD45+, coexpressed the macrophage antigens CD68, CD163, and CD209 and constituted the 60%-90% of tumor-associated macrophages. Microarray analysis of primary monocytes revealed that the vascular endothelial growth factor and the angiogenic chemokine CCL1 mRNA levels were up-regulated in response to CXCL12, leading to enhanced expression of both proteins. In addition, we found that CXCL12 autocrine/paracrine signaling down-regulates the expression of the transcription factor RUNX3 and contributes to maintain the long-term CD4 and CD14 expression in monocytes/macrophages. Together, these results suggest that autocrine CXCL12 production modulates differentiation of monocytes toward a distinct program with proangiogenic and immunosuppressive functions.
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Sun, Zhaoyu, Richard Nyberg, Yaping Wu, Brady Bernard, and William L. Redmond. "Developing an enhanced 7-color multiplex IHC protocol to dissect immune infiltration in human cancers." PLOS ONE 16, no. 2 (February 17, 2021): e0247238. http://dx.doi.org/10.1371/journal.pone.0247238.
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The TSA Opal multiplex immunohistochemistry (mIHC) protocol (PerkinElmer) has been used to characterize immune infiltration in human cancers. This technique allows multiple biomarkers to be simultaneously stained in a single tissue section, which helps to elucidate the spatial relationship among individual cell types. We developed and optimized two improved mIHC protocols for a 7-color panel containing 6 biomarkers (CD3, CD8, CD163, PD-L1, FoxP3, and cytokeratin (CK)) and DAPI. The only difference between these two protocols was the staining sequence of those 6 biomarkers as the first sequence is PD-L1/CD163/CD8/CK/CD3/FoxP3/DAPI and the second sequence is FoxP3/CD163/CD8/CK/CD3/PD-L1/DAPI. By comparing PD-L1/FoxP3 staining in mIHC and singleplex PD-L1/FoxP3 staining on the adjacent slide, we demonstrated that the staining sequence does not affect the staining intensity of individual biomarkers as long as a proper antigen retrieval method was used. Our study suggests that use of an antigen retrieval buffer with higher pH value (such as Tris-EDTA pH9.0) than that of the stripping buffers (such as citrate buffer pH6.0) is helpful when using this advanced mIHC method to develop panels with multiple biomarkers. Otherwise, individual biomarkers may exhibit different intensities when the staining sequence is changed. By using this protocol, we characterized immune infiltration and PD-L1 expression in head and neck squamous cell carcinoma (HNSCC), breast cancer (BCa), and non-small cell lung cancer (NSCLC) specimens. We observed a statistically significant increase in CD3+ cell populations within the stroma of NSCLC as compared to BCa and increased PD-L1+ tumor cells in HNSCC as opposed to BCa.
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Gemei, Marica, Rosa Di Noto, Peppino Mirabelli, and Luigi Del Vecchio. "Cytometric Profiling of CD133+ Cells in Human Colon Carcinoma Cell Lines Identifies a Common core Phenotype and Cell Type-specific Mosaics." International Journal of Biological Markers 28, no. 3 (July 2013): 267–73. http://dx.doi.org/10.5301/jbm.5000020.
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In colorectal cancer, CD133+ cells from fresh biopsies proved to be more tumorigenic than their CD133– counterparts. Nevertheless, the function of CD133 protein in tumorigenic cells seems only marginal. Moreover, CD133 expression alone is insufficient to isolate true cancer stem cells, since only 1 out of 262 CD133+ cells actually displays stem-cell capacity. Thus, new markers for colorectal cancer stem cells are needed. Here, we show the extensive characterization of CD133+ cells in 5 different colon carcinoma continuous cell lines (HT29, HCT116, Caco2, GEO and LS174T), each representing a different maturation level of colorectal cancer cells. Markers associated with stemness, tumorigenesis and metastatic potential were selected. We identified 6 molecules consistently present on CD133+ cells: CD9, CD29, CD49b, CD59, CD151, and CD326. By contrast, CD24, CD26, CD54, CD66c, CD81, CD90, CD99, CD112, CD164, CD166, and CD200 showed a discontinuous behavior, which led us to identify cell type-specific surface antigen mosaics. Finally, some antigens, e.g. CD227, indicated the possibility of classifying the CD133+ cells into 2 subsets likely exhibiting specific features. This study reports, for the first time, an extended characterization of the CD133+ cells in colon carcinoma cell lines and provides a “dictionary” of antigens to be used in colorectal cancer research.
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Poderoso, Teresa, Paloma Martínez, Belén Álvarez, Ana Handler, Sara Moreno, Fernando Alonso, Ángel Ezquerra, Javier Domínguez, and Concepción Revilla. "Delivery of antigen to sialoadhesin or CD163 improves the specific immune response in pigs." Vaccine 29, no. 29-30 (June 2011): 4813–20. http://dx.doi.org/10.1016/j.vaccine.2011.04.076.
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Wahyuningtyas, Rika, Yin-Siew Lai, Mei-Li Wu, Hsin-Wei Chen, Wen-Bin Chung, Hso-Chi Chaung, and Ko-Tung Chang. "Recombinant Antigen of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) Promotes M1 Repolarization of Porcine Alveolar Macrophages and Th1 Type Response." Vaccines 9, no. 9 (September 10, 2021): 1009. http://dx.doi.org/10.3390/vaccines9091009.
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The polarization status of porcine alveolar macrophages (PAMs) determines the infectivity of porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV infection skews macrophage polarization toward an M2 phenotype, followed by T-cells inactivation. CD163, one of the scavenger receptors of M2 macrophages, has been described as a putative receptor for PRRSV. In this study, we examined two types of PRRSV-2-derived recombinant antigens, A1 (g6Ld10T) and A2 (lipo-M5Nt), for their ability to mediate PAM polarization and T helper (Th1) response. A1 and A2 were composed of different combination of ORF5, ORF6, and ORF7 in full or partial length. To enhance the adaptive immunity, they were conjugated with T cells epitopes or lipidated elements, respectively. Our results showed that CD163+ expression on PAMs significantly decreased after being challenged with A1 but not A2, followed by a significant increase in pro-inflammatory genes (TNF-α, IL-6, and IL-12). In addition, next generation sequencing (NGS) data show an increase in T-cell receptor signaling in PAMs challenged with A1. Using a co-culture system, PAMs challenged with A1 can induce Th1 activation by boosting IFN-γ and IL-12 secretion and TNF-α expression. In terms of innate and T-cell-mediated immunity, we conclude that A1 is regarded as a potential vaccine for immunization against PRRSV infection due to its ability to reverse the polarization status of PAMs toward pro-inflammatory phenotypes, which in turn reduces CD163 expression for viral entry and increases immunomodulation for Th1-type response.
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Li, Wei, Yaomei Wang, Huizhi Zhao, Huan Zhang, Yuanlin Xu, Shihui Wang, Xinhua Guo, et al. "Identification, Isolation and Transcriptome Analyses of Mouse, Rat and Man Erythroblastic Island Central Macrophages." Blood 132, Supplement 1 (November 29, 2018): 841. http://dx.doi.org/10.1182/blood-2018-99-114188.
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Abstract Erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, is the first hematopoietic niche discovered for erythropoiesis. Yet, the identity of the central macrophage has so far remained elusive. Based on the previous findings that F4/80, VCAM1 and CD169 are potential mouse central macrophage markers, we first calculated the number of F4/80+VCAM1+CD169+ mouse macrophages in the mouse bone marrow and compared it to the number of Ter119+ erythroblasts. We found that the ratio of F4/80+VCAM1+CD169+ macrophage and erythroblasts is about 1:2. Given the fact that one central macrophage is surrounded by multiple erythroblasts, the above finding suggests that it is unlikely that all the F4/80+VCAM1+CD169+ macrophages are central macrophages. Erythropoietin (Epo) is essential for erythropoiesis. It has been reported that the Epo receptor (Epor) is expressed in peritoneal macrophages. These findings promoted us to speculate that EBI central macrophages may express Epor so that Epo acts on both erythroid cells and the central macrophages simultaneously in the niche to ensure efficient and optimal red cell production. To test this notion, we first examined whether mouse bone marrow and fetal liver macrophages express Epor using the Epor-GFPcre knockin mouse model. We found that ~5% of bone marrow F4/80+ macrophages and ~35% of fetal liver F4/80+ macrophages express Epor-GFP. As negative control, no Epor-GFP macrophages are noted in wild type F4/80+ macrophages. Importantly, ImageStream analyses revealed the native EBIs in bone marrow and fetal liver are formed by Epor+ but not Epor- macrophages. Bioinformatics analyses of RNA-seq data on the sorted Epor+ and Epor- macrophage populations revealed that molecules involved in central macrophage-erythroblast association such as VCAM1, CD169, and molecules known to be important for central macrophage function such as Dnase2a, ferroportin, are highly expressed in Epor+ macrophages. In marked contrast, highly expressed pathways in Epor- macrophages are associated with immune responses including antigen process and presentation. Intriguingly, the immune related pathways are dramatically downregulated in the Epor+ macrophages, suggesting that the Epor+ macrophages in bone marrow and fetal liver have evolved a specialized function in supporting erythropoiesis. To examine whether expression of Epor in EBI central macrophages is a conserved feature across species, we generated Epor-GFPcre knockin rat using the CRISP/Cas9 technology. Using CD163 as rat macrophage marker, we found that a subpopulation of rat bone marrow CD163+ macrophages expresses Epor-GFP. As a negative control, no Epor-GFP macrophages are noted in wild type CD163+ macrophages. To examine whether EPOR is expressed in human EBI central macrophages, antibody specificity for human EPOR is critical. To this end, we employed CRISP/Cas9 approach to knock out EPOR in K562 and Hela cell lines and validated the specificity of a commercially available anti-human EPOR antibody. Using CD163, CD169 as human macrophage markers, we found that EPOR is also expressed in a subpopulation of human macrophages. Moreover, in vitro EBI formation assay revealed that human EPOR+ but not EPOR- macrophages form EBIs with erythroid cells and that the EBI formation is enhanced by EPO. In summary, we for the first time, after discovery of the EBIs 60 years ago, have identified Epor+ macrophages in mouse bone marrow and fetal liver as EBI central macrophages. Our findings provide solid foundation for studying the mechanisms by which erythropoieis is supported EBI central macrophages. A better understanding of such mechanisms will provide extensive new knowledge on basic biology of erythropoiesis. It is also important to understand the pathology of erythropoietic disorders as well as to improve ex vivo erythrocyte production. Disclosures No relevant conflicts of interest to declare.
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Najem, Hinda, Anantha Marisetty, Craig Horbinski, Jared Burks, and Amy B. Heimberger. "LMD-20. Immune Suppressive Macrophages and Signal Transducer and Activator of Transcription 3 (STAT3) Expression are common in Melanoma Leptomeningeal Disease." Neuro-Oncology Advances 3, Supplement_3 (August 1, 2021): iii11—iii12. http://dx.doi.org/10.1093/noajnl/vdab071.045.
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Abstract Leptomeningeal disease (LMD) in melanoma patients is associated with significant neurological impairments and has a dismal outcome with a median survival of 1.8 months. Despite the therapeutic benefit of targeted therapies and immunotherapies for most kinds of Stage IV melanoma, patients with LMD do not typically benefit. A deeper understanding of the tumor microenvironment (TME) of LMD may provide more appropriate therapeutic selection. A retrospective analysis of subjects who underwent surgical resection with LMD (n=8) were profiled with seven color multiplex to evaluate the expression of the global immune suppressive hub - the signal transducer and activator of transcription 3 (STAT3) and for the presence of CD3 T cells, CD68+ monocytes, CD163 immune suppressive macrophages, CD11c+ antigen presenting cells (APCs) in association with the melanoma tumor marker S100B and DAPI for cellular nuclear identification. High-resolution cellular imaging and quantification was conducted using the Akoya Vectra Polaris. CD163+ macrophage is the most frequent immune cell population in the LMD TME. Occasional CD3+ T cells and CD11c+ APC are also identified, although the latter has concurrent expression of CD163. STAT3 nuclear localization is heterogeneously expressed in the various immune cell populations. Occasional immune cluster interactions can be seen in the tumor stroma and the tumor edge. In conclusion, the TME of LMD is largely devoid of CD3+ T cells, but is enriched for immune suppression and innate immunity.
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Fabriek, Babs O., Elise S. Van Haastert, Ian Galea, Machteld M. J. Polfliet, Ed D. Döpp, Michel M. Van Den Heuvel, Timo K. Van Den Berg, Corline J. A. De Groot, Paul Van Der Valk, and Christine D. Dijkstra. "CD163-positive perivascular macrophages in the human CNS express molecules for antigen recognition and presentation." Glia 51, no. 4 (2005): 297–305. http://dx.doi.org/10.1002/glia.20208.
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Niino, Daisuke, Yoshihiro Komohara, Yoshizo Kimura, Masanori Takeuchi, Hiroaki Miyoshi, Maki Yoshida, Ayako Ichikawa, et al. "M2 Macrophage Infiltration Is Closely Associated with Poor Prognosis for Adult T-Cell Leukemia/Lymphoma (ATLL),." Blood 118, no. 21 (November 18, 2011): 3672. http://dx.doi.org/10.1182/blood.v118.21.3672.3672.
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Abstract Abstract 3672 Background: Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell lymphoma caused by a retrovirus, human T-cell lymphotropic virus type I, and characterized by an aggressive clinical course and poor prognosis. Although several factors associated with this poor prognosis have been identified, including high-level Ki67 antigen expression and high serum levels of calcium, parathyroid hormone-related protein, soluble interleukin-2 receptor, β2-microglobulin, and neuron-specific enolase, the accuracy of current prognostic models for prediction of the outcome of treatment is inadequate, and clinically relevant biomarkers have not been established. Tumor-associated macrophages, which are known to possess the immunosuppressive M2 macrophage phenotype, contribute to tumor growth, invasion, and metastasis by producing various mediators. Macrophage polarization is divided into types M1 and M2 based on the expression of membrane receptors, cytokines, and chemokines. M1 expresses CD80, interleukin (IL)-6, IL-12, and chemokine receptor 7, while M2 expresses CD163, IL10, and chemokine ligand 22. The classically activated M1 macrophages exhibit antitumor functions and the alternatively activated M2 macrophages protumor functions, which contribute to the development and progression of tumors. CD163 is a monocyte/macrophage-restricted membrane protein belonging to the scavenger receptor cysteine-rich domain family and it functions as an endocytic receptor for a hemoglobin-haptoglobin complex. CD163 expression has been associated with an anti-inflammatory M2 macrophage phenotype and is believed to be useful for distinguishing M2 macrophages from pro-inflammatory M1 macrophages. Macrophages, especially M2 polarized macrophages, preferentially express CD163, but no studies have investigated macrophage phenotypes in ATLL. The aim of our study was therefore to investigate CD163 expression, which has been used as a marker for M2 macrophages, and the relationship between macrophage activation and prognosis for ATLL in order to gain new information about the therapeutic implications of this relationship for ATLL. Methods: Between 1985 and 2003, 75 cases of ATLL were examined. The male-to-female ratio was 1.34:1 and the median age 63 years (range: 35–87 years). Advanced clinical stages were identified in 75% of the patients, and lactic dehydrogenase was elevated in 25%. Most patients were treated with combination chemotherapy and the median survival period was 271 days (range: 4–3, 807 days). We performed a retrospective study on the immunohistochemical expression of macrophage markers (CD68, CD163) and their correlation with overall survival for the 75 ATLL patients. Paraffin sections were examined immunohistochemically by using anti-CD68 (PGM1) and anti-CD163 antibodies, and the absolute number of intratumoral macrophages in the ATLL specimens was determined. Kaplan-Meier survival estimates were subjected to comparative univariate analyses using the log-rank test. Cox proportional-hazard regression test was used for the multivariate analysis. P-values of less than 0.05 were considered significant. Results: The number of CD68-positive macrophages in ATLL tissues did not correlate with overall survival (P =0.25), whereas patients with a large number of CD163-positive macrophages (>250 cells/mm2 tumor area; n=37) had worse outcomes than those with a small number (<250 cells/mm2 tumor area; n=36) (P =0.05). Meanwhile, a higher ratio of CD163-positive to CD68-positive macrophages in ATLL significantly correlated with worse overall survival (P =0.039). Multivariate analysis confirmed that high CD163/CD68 ratio was an independent prognostic factor. Conclusions: Considering that high CD163/CD68 ratio reflects the proportion of macrophages polarized to the M2 phenotype, our findings further indicate that activation of macrophages towards the M2 phenotype correlates with worse prognosis. They also suggest that immunohistochemical analysis of M2 macrophages at the time of diagnosis can provide additional relevant prognostic information. However, these findings need to be further explored in future studies. Disclosures: No relevant conflicts of interest to declare.
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Lundgren, Sebastian, Carl Fredrik Warfvinge, Jacob Elebro, Bjorn Nodin, Agnieszka Krzyzanowska, Anders Bjartell, Emelie Karnevi, Jakob Eberhard, Karin Leandersson, and Karin Jirstrom. "Prognostic significance of professional antigen presenting cells according to morphological subtype of periampullary adenocarcinoma." Journal of Clinical Oncology 35, no. 7_suppl (March 1, 2017): 121. http://dx.doi.org/10.1200/jco.2017.35.7_suppl.121.
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121 Background: Dendritic cells (DCs) and macrophages play vital roles in tumorigenesis and may, depending on the context, promote or inhibit tumor progression. In this study, we examined the prognostic significance of DCs and macrophages in periampullary adenocarcinoma, with particular reference to morphological type. Methods: Immune cell-specific expression of CD68, CD163 and CD1a was analysed by immunohistochemistry on tissue microarrays with tumours from 175 consecutive cases of periampullary adenocarcinoma, 110 of pancreatobiliary type (PB-type) and 65 of intestinal type (I-type) morphology. Kaplan-Meier and Cox regression analysis were applied to determine the impact of the investigated cells on 5-year overall survival (OS). Results: Low density of CD68+ cells or CD163 + cells was significantly associated with an improved 5-year OS in unadjusted Cox regression analysis in the entire cohort (HR = 1.66; 95% CI 1.05-2.63 and HR = 1.84; 95 % CI 1.09-3.09, respectively), but not in adjusted analysis or in strata according to subtype. CD1a+ cell density was not prognostic in entire cohort or in I-type tumours. However, in PB-type tumors, high CD1a+ cell density was significantly associated with a reduced OS in unadjusted as well as in adjusted analysis (HR = 2.09; 95% CI 1.07-4.09 and HR = 2.35; 95% CI 1.13-4.87). The prognostic value of the investigated markers did not differ in strata according to adjuvant chemotherapy, neither in the entire cohort nor according to morphological type. Conclusions: The results from study demonstrate that the prognostic significance of dendritic cells, but not macrophages, differs by morphological subtype in periampullary adenocarcinoma. Hence, morphological subtype is an important factor to consider in studies on the prognostic and predictive role of the immune microenvironment in these types of cancer.
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Matthews, Kathryn, Irina Eberle-Ayres, Katherine Lu, Nishi Singh, Murray J. Cutler, and David Bell. "Therapeutic Targeting of CD163 on Hematopoietic Progenitor Cells By Novel Monoclonal Antibody TBI 304H Demonstrates an Increase in Erythropoiesis in Humanized Mice." Blood 126, no. 23 (December 3, 2015): 1163. http://dx.doi.org/10.1182/blood.v126.23.1163.1163.
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Abstract Hemoglobin (Hb) is known to stimulate erythropoiesis, a process that may be mediated by CD163. CD163 is a receptor for the hemoglobin-haptoglobin (Hb-Hp) complex expressed on monocyte/macrophages as well as a subpopulation of human CD34+ hematopoietic progenitor cells (HPCs). We have demonstrated that administration of ligands to the CD163 receptor can measurably stimulate erythropoiesis in human CD34+ cell-engrafted severe-combined immunodeficiency (SCID) mice. To better elucidate the role of CD163 in hematopoiesis, we investigated the effects of the natural ligand to CD163 (Hb-Hp) as well as a stimulatory antibody, TBI 304H, on HPCs in vivo. SCID mice engrafted with human CD34+ cells were used as a model system were used to investigate the effect of Hb and anti-CD163 monoclonal antibodies (TBI 304 and TBI 304H) on human hematopoiesis in vivo. In an initial study, NOD-SCID IL2R gammanull (NSG) mice were engrafted with human CD34+ cells and animals with < 30% human CD45+ cells in the peripheral blood were administered 2 mg Hb/mouse, or 100 or 500 µg/mouse TBI 304 every 4 days for a total of four doses. At study termination on day 14, bone marrow cells (BMCs) were examined by flow cytometry and CD34+ cells were recovered from the BMCs for enumeration in colony-forming assays. Hemoglobin administration resulted in an increase of human CD34+ cells ranging from 4% to 7% of BMCs and a corresponding 57% increase in colony-forming cells (CFCs) over control animals. In contrast, the monoclonal antibody (mAb), TBI 304, produced a dose-dependent decrease in CD34+ and colonies, possibly reflecting a depletion of CD34+/CD163+ cells as a result of overstimulation due to the much longer circulating half-life of the mAb compared to Hb.. To confirm this hypothesis, human CD34+ cell engrafted animals were given only a single dose of 10 or 100 µg/mouse of TBI 304 and BMCs were examined earlier on day 7. TBI 304 provided a 3.5-fold increase in human CD34+ cells as well as a 1.8 to 6.7-fold increase in bone marrow erythroid lineage engraftment (huGlyA+, huCD36+ and huCD71+) and a 2-fold increase in colony-forming cells. The ability of TBI 304 to stimulate erythropoiesis in preclinical models led to the creation of an anti-CD163 mAb suitable for human clinical use. TBI 304H was generated by grafting the complementarity-determining regions derived from TBI 304 onto a humanized IgG4 framework without altering antigen specificity. An IgG4 framework, as an antibody without Fc effector function, was deemed the most suitable for an agonistic mAb. In the single dose, 7 day Hu-SCID model human CD34+ cells were mobilized from the mouse bone marrow by TBI 304H, as reflected by dose dependent decreases in huCD34+, huCD71+, and huGlyA+ cells in the mouse marrow. At the highest dose tested (500 µg/mouse) the decrease in human HPCs was similar to that found in animals administered Hb (2 mg/mouse). In this model, human hematopoiesis derived from the engrafted human CD34+ cells is not sustained and these date may reflect a mobilization of human HPCs through stimulation by an anti-CD163 antibody. Therapure has received U.S. FDA approval to conduct a Phase I trial of the novel therapeutic antibody TBI 304H. The Phase I clinical trial is a single-center, open-label, intra-subject escalating dose study, which will evaluate the safety, tolerability and pharmacokinetics of TBI 304H following administration to subjects experiencing chemotherapy-induced anemia. Disclosures Matthews: Therapure Biopharma: Employment. Eberle-Ayres:Therapure Biopharma: Employment. Lu:Therapure Biopharma: Employment. Singh:Therapure Biopharma: Employment. Cutler:Therapure Biopharma: Employment. Bell:Therapure Biopharma: Employment.
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Florian, Stefan, Karoline Sonneck, Alexander W. Hauswirth, Maria-Theresa Krauth, Wolfgang R. Sperr, and Peter Valent. "Phenotyping of Neoplastic (CD34+/CD38−/CD123+) Stem Cells in Myeloid Malignancies Reveals Expression of Multiple Molecular Targets." Blood 106, no. 11 (November 16, 2005): 1381. http://dx.doi.org/10.1182/blood.v106.11.1381.1381.
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Abstract Recent data suggest that myeloid neoplasms are organized hierarchically in terms of self renewal and maturation of early progenitor cells, similar to normal myelopoiesis. In acute myeloid leukemia (AML), the NOD/SCID mouse-repopulating leukemic stem cells usually co-express CD123 with CD34, but lack CD38. So far, however, little is known about expression of other markers and targets on these progenitors. In the present study, expression of target antigens on CD34+/CD38− cells was analyzed by multicolor flow cytometry in patients with AML (n=18), myelodysplastic syndromes (MDS, n=6), chronic myeloid leukemia (CML, n=8), systemic mastocytosis (SM, n=9), and normal bone marrow (n=5). The IL-3Ra chain (CD123) was found to be expressed on CD34+/CD38− cells in a majority of all patients in all disease-categories. Independent of the type of disease, the vast majority of these stem cells also co-expressed aminopeptidase-N (CD13) and the target receptor CD44 in all patients. CD34+/CD38− progenitor cells expressed variable amounts of the Mylotarg® receptor CD33, KIT (CD117), HLA-DR, and AC133 (CD133). With regard to AC133, two distinct subpopulations of progenitor cells were detected in many cases, namely a CD133+ and a clearly CD133- cell-fraction. In patients with AML, the levels of CD33 varied from patient to patient with a broad range of reactivity, whereas in most patients with MDS, CML, and SM, CD33 was found to be consistently expressed on most progenitors. In most patients, neoplastic stem cells did not express substantial amounts of the GM-CSF receptor alpha chain (CD116), Thy-1 (CD90), E-NPP3 (CD203c), MDR-1 (CD243), or PAR-2. In the normal bone marrow, CD34+/CD38− cells co-expressed CD13, CD44 and CD45, but did not express CD33, CD116, or CD123. In conclusion, neoplastic stem cells in various myeloid neoplasms appear to express a similar phenotype including target receptors such as CD13, CD33, and CD44. These antigens may thus be attractive targets of therapy in AML. However, since many of these targets are not expressed on all stem cells in all patients, the elimination of the entire clone may require combinations of targeted antibodies or use of additional drugs. In other cases (CD13, CD44, CD45), the target antigen is also expressed on normal stem cells, so that targeted therapy is likely to be an ablative maneuver and thus would require a combined stem cell transplantation approach.
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Torres, SH, JB De Sanctis, L. M. de Briceno, N. Hernandez, and HJ Finol. "Inflammation and nitric oxide production in skeletal muscle of type 2 diabetic patients." Journal of Endocrinology 181, no. 3 (June 1, 2004): 419–27. http://dx.doi.org/10.1677/joe.0.1810419.
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An inflammatory process may be involved in nitric oxide production in skeletal muscle of type 2 diabetic patients. Nitric oxide generation in skeletal muscle was assessed in 14 non-complicated type 2 diabetic patients and in 12 healthy subjects. In samples of quadriceps femoris muscle, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), nitrite, nitrate and nitrotyrosine were determined. The macrophage-specific antigen CD163, the T-cell membrane factor CD154 and tumour necrosis factor-alpha (TNF-alpha) were also assayed. In six patients, ultrastructural analysis of muscle was performed. Nitrites and nitrates were increased in patients as compared to controls (22.7+/-4.5 and 32.7+/-7.0 vs 16.0+/-2.9 and 22.8+/-4.0 micromol/mg protein; P<0.001, Mann-Whitney U test). Endothelial NOS was similar in diabetic and control subjects (36.4+/-13.8 vs 36.3+/-6.8 ng/mg protein), contrasting with the significant increase of iNOS recorded in patients (34.3+/-13.0 vs 8.5+/-2.8 ng/mg protein, P<0.00002). Nitrotyrosine levels were higher in the patient than in the control group (42.1+/-24.4 vs 10.3+/-2.5 ng/mg protein, P<0.00002), as were CD163 (10-fold) and TNF-alpha (fourfold) levels. Furthermore, CD154 levels were detectable only in the patient samples (10.2+/-5.3 ng/mg protein). By multiple-regression analysis, changes in glycated haemoglobin values could predict 96% variation in nitrotyrosine. Macrophages were present in all muscle samples analysed by electromicroscopy. The increased levels of CD163, CD154 and TNF-alpha indicate that an inflammatory process occurs in skeletal muscle of type 2 diabetic patients. This may contribute to iNOS induction, muscle damage and insulin resistance.
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Tachibana, Mitsuhiro, Tadahiro Isono, and Yutaka Tsutsumi. "Adenocarcinoma with Neuroendocrine Differentiation of the Colon Accompanying Osteoclast-Like Giant Cells." Case Reports in Pathology 2020 (April 28, 2020): 1–5. http://dx.doi.org/10.1155/2020/1976319.
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Introduction. Neuroendocrine differentiation in colorectal cancer is reportedly associated with poorer grade of tumor differentiation, nodal and distant metastasis, and other unfavorable features, contributing to a worse clinical outcome. Colorectal cancer with osteoclast-like giant cells (OGCs) is extremely rare. Case Presentation. An 86-year-old woman was diagnosed as double cancer of the transverse and sigmoid colon. Both tumors were simultaneously removed. The transverse colon cancer directly invaded the area of the right gastroepiploic vessels and spread to the nodes and histologically consisted of both the tubuloglandular and solid components. CD8/granzyme B-positive tumor-infiltrating lymphocytes and CD163/CD68-positive macrophages, frequently forming OGCs, were observed particularly at the invasion front. The carcinoma cells were labeled focally for synaptophysin and diffusely for the DR locus of the human leukocyte antigen and programmed death-ligand 1 (PD-L1). Deficient expression of DNA mismatch repair (dMMR) proteins was immunohistochemically confirmed. The patient died 16 months after surgery. Conclusion. This is the first report of colonic adenocarcinoma with neuroendocrine differentiation accompanying OGCs. Histopathologic factors of the poor prognosis in the present case included (a) the presence of more than 2% cells with neuroendocrine differentiation, (b) infiltration of CD163/CD68-positive OGCs at the invasion front, (c) deficiency of dMMR proteins, and (d) PD-L1 expression.
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Zhou, Wenxing, Yiyang Zhang, Fei He, Shaohua Lv, Xiaodong Zhang, and Chunming Fei. "Abundance of CD163-Positive Tumor-Associated Macrophages in the Early Gastric Cancer Predicts the Recurrence after Curative Resection." Digestive Diseases 38, no. 6 (2020): 458–65. http://dx.doi.org/10.1159/000506122.
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<b><i>Background:</i></b> We aimed to investigate the prognostic value of M2 macrophages to predict the recurrence of early gastric cancer (EGC). <b><i>Methods:</i></b> A retrospective analysis was carried out among EGC patients (95 non-recurrence and 78 recurrence) who underwent surgery at Luhe People’s Hospital of Nanjing. A 5-year recurrence status was utilized to classify the patients into the recurrence group and non-recurrence group. CD163 and proliferating cell nuclear antigen were utilized as markers to detect M2 macrophages and proliferation. Cumulative tumor recurrence curve was adopted to analyze the association between the number of tumor-associated macrophages (TAMs) and the recurrence of EGC. Colony formation and invasion abilities of MKN45 (JCRB0254, human gastric epithelial cell line) with or without M2 macrophage coculture were detected in vitro, and the xenograft model was utilized to detect in vivo effect of M2 macrophages on tumor growth. <b><i>Results:</i></b> The number of CD163<sup>+</sup> macrophages and expression of transforming growth factor-β1, matrix metallopeptidase 9, and vascular endothelial growth factor A were significantly different between the EGC recurrence and non-recurrence group. The cumulative tumor recurrence rate was found to be dependent on the infiltration number of TAMs. M2 macrophages promoted the proliferation and invasion of human MKN45 cells in vitro, as well as tumor growth in the xenograft model. <b><i>Conclusion:</i></b> The abundance of CD163<sup>+</sup>-positive TAMs in EGC predicts the recurrence after curative resection.
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Cumberbatch, Marie, Nathan Elliott, Sarah Warren, Woo Ho Kim, Christopher Womack, Milan Bhagat, Lorenzo Colarossi, and Lorenzo Memeo. "Association of immune microenvironment to response in treatment-naïve non-small cell lung cancer (NSCLC) samples with follow-up second-line immunotherapy data." Journal of Clinical Oncology 38, no. 5_suppl (February 10, 2020): 49. http://dx.doi.org/10.1200/jco.2020.38.5_suppl.49.
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49 Background: Archival specimens collected months or years prior to starting immunotherapy are often used to identify patients for second line immune checkpoint inhibitor (ICI) treatment. PD-L1 expression and the immune microenvironment in these patients may have altered over time following multiple lines of failed standard of care (SOC) treatments. Methods: Formalin fixed paraffin embedded (FFPE) tumor samples, taken during resection performed as first line surgical treatment from a cohort of NSCLC patients (n = 18), were evaluated by Nanostring using the IO360 gene expression panel, and by immunohistochemistry (IHC) for CD3, CD8, PD-L1, CD68 and CD163. The resultant immune profiles were correlated with the clinical follow-up data for radiotherapy, SOC chemotherapy, and second line immunotherapy with the aim of understanding whether immune signatures predictive of response to ICI therapy may be identified in such samples. Results: Of the 18 cases, clinical follow-up data indicated objective response to ICI therapy for 4 patients, with the mean time from initial diagnosis to ICI treatment being 2.8 years (range: 0.4 to 8.5 years). Although pathologist PD-L1 IHC scores were not predictive of response, IHC image analysis data revealed significant increases in CD3 (2.3-fold) and CD8 (2.7-fold) T cell numbers in the responder population. In addition, although CD68+ macrophage frequencies did not differ significantly between responder and non-responder populations, reduced M2-like CD163+ macrophage/monocyte numbers were evident for responders. While the Tumor Inflammation Signature was not predictive of response, several gene expression signatures were significantly associated with response including increased abundance of CD8 T cells, cytotoxic cells, cytotoxicity, MHC class II antigen presentation and Melanoma-Associated Antigens (MAGE). Conclusions: Despite these patients having received various lines of radiotherapy and SOC chemotherapy prior to receiving immunotherapy, immune profiles associated with response to second line immunotherapy were detected in surgical first line resection samples.
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Colovic, Radoje, Nada Suvajdzic, Nikica Grubor, Natasa Colovic, and Tatjana Terzic. "Atipical immunophenotype in a littoral cell angioma." Vojnosanitetski pregled 66, no. 1 (2009): 63–65. http://dx.doi.org/10.2298/vsp0901063c.
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Background. Littoral-cell angioma (LCA) is a recently described benign vascular tumor of the spleen, whose imaging and pathologic characteristics have been discussed only by a few authors. The tumor is characterized by a mixture of papillary and cystic areas lined by neoplastic cells deriving from normal splenic lining - littoral cells. The neoplastic LCA cells express both endothelial and histiocytic antigens associated with CD8 negativity, compared with the normal endothelium of the venous sinuses of the spleen red pulp that only expresses endothelial antigens and CD8 positivity. Therefore, the typical and characteristic immunohistochemical pattern of the LCA is as follows: CD31, CD68, CD163, CD21, FVIII antigen positive; CD34, CD8 negative. Case report. We reported a 60-year-old male with moderate nodular splenomegaly with one large hypoechogenic solid lesion and mild thrombocytopenia in whom the diagnosis of LCA was made after the elective splenectomy. Namely, histopathological and immunohistochemical data allowed a final diagnosis of classical LCA in spite of CD21 negativity. As far as we know this is the first reported CD21-negative LCA patient. Histological specimens were presented and differential diagnoses discussed. Conclusion. Littoral-cell angioma is a very rare benign splenic neoplasm that should be considered in the differential diagnosis of multinodular splenomegaly, particularly if the patient has the signs of hypersplenism.
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Morotti, Denise, Massimiliano Cadamuro, Elena Rigoli, Aurelio Sonzogni, Andrea Gianatti, Cristina Parolin, Luisa Patanè, and David A. Schwartz. "Molecular Pathology Analysis of SARS-CoV-2 in Syncytiotrophoblast and Hofbauer Cells in Placenta from a Pregnant Woman and Fetus with COVID-19." Pathogens 10, no. 4 (April 15, 2021): 479. http://dx.doi.org/10.3390/pathogens10040479.
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A small number of neonates delivered to women with SARS-CoV-2 infection have been found to become infected through intrauterine transplacental transmission. These cases are associated with a group of unusual placental pathology abnormalities that include chronic histiocytic intervillositis, syncytiotrophoblast necrosis, and positivity of the syncytiotrophoblast for SARS-CoV-2 antigen or RNA. Hofbauer cells constitute a heterogeneous group of immunologically active macrophages that have been involved in transplacental infections that include such viral agents as Zika virus and human immunodeficiency virus. The role of Hofbauer cells in placental infection with SARS-CoV-2 and maternal-fetal transmission is unknown. This study uses molecular pathology techniques to evaluate the placenta from a neonate infected with SARS-CoV-2 via the transplacental route to determine whether Hofbauer cells have evidence of infection. We found that the placenta had chronic histiocytic intervillositis and syncytiotrophoblast necrosis, with the syncytiotrophoblast demonstrating intense positive staining for SARS-CoV-2. Immunohistochemistry using the macrophage marker CD163, SARS-CoV-2 nucleocapsid protein, and double staining for SARS-CoV-2 with RNAscope and anti-CD163 antibody, revealed that no demonstrable virus could be identified within Hofbauer cells, despite these cells closely approaching the basement membrane zone of the infected trophoblast. Unlike some other viruses, there was no evidence from this transmitting placenta for infection of Hofbauer cells with SARS-CoV-2.
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Kuijpers, TW, AT Tool, CE van der Schoot, LA Ginsel, JJ Onderwater, D. Roos, and AJ Verhoeven. "Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation." Blood 78, no. 4 (August 15, 1991): 1105–11. http://dx.doi.org/10.1182/blood.v78.4.1105.1105.
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Abstract Neutrophil research relies largely on studies with highly purified cells. Yet the isolation procedures induce changes in surface expression of several proteins. We used a large panel of monoclonal antibodies (MoAbs) to characterize in detail the phenotypic changes during isolation and stimulation of human neutrophils. Centrifugation on density gradients appears to be the crucial step that causes an increase in expression of antigens not detectable on neutrophils in whole blood samples (cytochrome b558 recognized by MoAb 7D5; and CD10) or expressed at significantly lower levels (CD11a, CD11b, CD11c, CD13, CD16, CD45, and CD67). Other antigens were unaffected by the density gradient centrifugation step (CD32, CD54, CD58, Leu-8, HLA class I). Upregulation of antigens was also determined by stimulation of purified neutrophils. Upregulation of CD63 was an excellent marker for release from azurophil granules. We subsequently related the surface antigen expression to functional activities of purified neutrophils. From these experiments, we concluded that 7D5-as “early activation” marker--does not necessarily discriminate between primed or resting neutrophils with respect to NADPH oxidase activity.
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Kuijpers, TW, AT Tool, CE van der Schoot, LA Ginsel, JJ Onderwater, D. Roos, and AJ Verhoeven. "Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation." Blood 78, no. 4 (August 15, 1991): 1105–11. http://dx.doi.org/10.1182/blood.v78.4.1105.bloodjournal7841105.
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Neutrophil research relies largely on studies with highly purified cells. Yet the isolation procedures induce changes in surface expression of several proteins. We used a large panel of monoclonal antibodies (MoAbs) to characterize in detail the phenotypic changes during isolation and stimulation of human neutrophils. Centrifugation on density gradients appears to be the crucial step that causes an increase in expression of antigens not detectable on neutrophils in whole blood samples (cytochrome b558 recognized by MoAb 7D5; and CD10) or expressed at significantly lower levels (CD11a, CD11b, CD11c, CD13, CD16, CD45, and CD67). Other antigens were unaffected by the density gradient centrifugation step (CD32, CD54, CD58, Leu-8, HLA class I). Upregulation of antigens was also determined by stimulation of purified neutrophils. Upregulation of CD63 was an excellent marker for release from azurophil granules. We subsequently related the surface antigen expression to functional activities of purified neutrophils. From these experiments, we concluded that 7D5-as “early activation” marker--does not necessarily discriminate between primed or resting neutrophils with respect to NADPH oxidase activity.
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Romano, Emanuela, Monika Kusio-Kobialka, Periklis G. Foukas, Petra Baumgaertner, Christiane Meyer, Pierluigi Ballabeni, Olivier Michielin, Benjamin Weide, Pedro Romero, and Daniel E. Speiser. "Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients." Proceedings of the National Academy of Sciences 112, no. 19 (April 27, 2015): 6140–45. http://dx.doi.org/10.1073/pnas.1417320112.
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Enhancing immune responses with immune-modulatory monoclonal antibodies directed to inhibitory immune receptors is a promising modality in cancer therapy. Clinical efficacy has been demonstrated with antibodies blocking inhibitory immune checkpoints such as cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) or PD-1/PD-L1. Treatment with ipilimumab, a fully human CTLA-4–specific mAb, showed durable clinical efficacy in metastatic melanoma; its mechanism of action is, however, only partially understood. This is a study of 29 patients with advanced cutaneous melanoma treated with ipilimumab. We analyzed peripheral blood mononuclear cells (PBMCs) and matched melanoma metastases from 15 patients responding and 14 not responding to ipilimumab by multicolor flow cytometry, antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and immunohistochemistry. PBMCs and matched tumor biopsies were collected 24 h before (i.e., baseline) and up to 4 wk after ipilimumab. Our findings show, to our knowledge for the first time, that ipilimumab can engage ex vivo FcγRIIIA (CD16)-expressing, nonclassical monocytes resulting in ADCC-mediated lysis of regulatory T cells (Tregs). In contrast, classical CD14++CD16− monocytes are unable to do so. Moreover, we show that patients responding to ipilimumab display significantly higher baseline peripheral frequencies of nonclassical monocytes compared with nonresponder patients. In the tumor microenvironment, responders have higher CD68+/CD163+ macrophage ratios at baseline and show decreased Treg infiltration after treatment. Together, our results suggest that anti–CTLA-4 therapy may target Tregs in vivo. Larger translational studies are, however, warranted to substantiate this mechanism of action of ipilimumab in patients.
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Sadofsky, Laura R., Yvette A. Hayman, Jesse Vance, Jorge L. Cervantes, Simon D. Fraser, Holly N. Wilkinson, James D. Williamson, Simon P. Hart, and Alyn H. Morice. "Characterisation of a New Human Alveolar Macrophage-Like Cell Line (Daisy)." Lung 197, no. 6 (November 16, 2019): 687–98. http://dx.doi.org/10.1007/s00408-019-00288-3.
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Abstract Purpose There is currently no true macrophage cell line and in vitro experiments requiring these cells currently require mitogenic stimulation of a macrophage precursor cell line (THP-1) or ex vivo maturation of circulating primary monocytes. In this study, we characterise a human macrophage cell line, derived from THP-1 cells, and compare its phenotype to the THP-1 cells. Methods THP-1 cells with and without mitogenic stimulation were compared to the newly derived macrophage-like cell line (Daisy) using microscopy, flow cytometry, phagocytosis assays, antigen binding assays and gene microarrays. Results We show that the cell line grows predominantly in an adherent monolayer. A panel of antibodies were chosen to investigate the cell surface phenotype of these cells using flow cytometry. Daisy cells expressed more CD11c, CD80, CD163, CD169 and CD206, but less CD14 and CD11b compared with mitogen-stimulated THP-1 cells. Unlike stimulated THP-1 cells which were barely able to bind immune complexes, Daisy cells showed large amounts of immune complex binding. Finally, although not statistically significant, the phagocytic ability of Daisy cells was greater than mitogen-stimulated THP-1 cells, suggesting that the cell line is more similar to mature macrophages. Conclusions The observed phenotype suggests that Daisy cells are a good model of human macrophages with a phenotype similar to human alveolar macrophages.
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Herrmann-Hoesing, L. M., S. M. Noh, K. R. Snekvik, S. N. White, D. A. Schneider, T. Truscott, and D. P. Knowles. "Ovine Progressive Pneumonia Virus Capsid Antigen as Found in CD163- and CD172a-Positive Alveolar Macrophages of Persistently Infected Sheep." Veterinary Pathology 47, no. 3 (December 31, 2009): 518–28. http://dx.doi.org/10.1177/0300985809359605.
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Shao, Zonghong, Lijuan Li, Rong Fu, Huaquan Wang, Lanzhu Yue, Hui Liu, Jun Wang, et al. "Expression of Differentiation Antigens on Bone Marrow Myeloid Cells of the Patients with Myelodysplastic Syndromes and it's Clinical Significances." Blood 116, no. 21 (November 19, 2010): 4967. http://dx.doi.org/10.1182/blood.v116.21.4967.4967.
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Abstract Abstract 4967 Objective To study the abnormal differentiation of bone marrow myeloid cells in myelodysplastic syndromes (MDS) and its correlation with the prognosis of MDS patients. Methods Quantitative assessment of CD11b, CD13, CD16 and HLA-DR expression on the membrane of bone marrow granulocytes, and CD71 and glycophorin A on erythroblasts of 12 MDS patients in low-risk, 22 in high-risk and 31 normal controls was conducted with flow cytometry. The correlation between the abnormality of these antigen expression and the prognosis of MDS cases were analyzed. Results The granulocytic differentiation was analyzed with the combinations of CD13/CD11b, CD13/CD16 and CD11b/CD16. The “right hook”, “sickle” and “retroflex 7” shape expressions were found in normal controls while there were various changes in MDS groups. The ratios of CD11b-/CD11b+(0.39±0.34)and CD16-/CD16+(1.33 ±0.77)of high-risk MDS group were significantly higher than those of control group (0.07±0.05 and 0.39 ±0.31 respectively) (P<0.05). The MFI (mean fluorescence index) of SSC (side scatter) in the granulocyte gate of MDS groups was lower while their MFI of CD13 was higher. The mean percentages of CD11b-HLA-DR+ (3.88%±3.07%), CD11b- HLA-DR- (16.23%±15.59%), CD16-HLA-DR- (41.12%±24.53%), CD11b+CD16- (33.53%±17.26%) and CD13+CD16- (44.51%±21.99%) granulocytes of high-risk MDS group were significantly higher than those of low-risk and control groups (P<0.05). The erythroid cell lineage differentiation was analyzed with CD71/glycophorin A combination. Double antigen positive expression was found in all controls, but asynchronous expression of CD71/glycophorin A was found in some MDS cases. The mean percentage of double antigen positive cells in CD45- and glycophorin A+ cell population was significantly lower in low-risk and high-risk MDS groups. The abnormal numbers and patterns of the antigen expression of MDS cases correlated directly with their IPSS (international prognostic scoring system) (r=0.690, P=0.000) and WPSS (WHO adapted prognostic scoring system) (r=0.651, P=0.000) scores. Conclusion There were abnormal expressions of differentiation antigens on bone marrow myeloid cells of MDS patients. And the severity of these abnormal expressions was correlated with their prognosis. The abnormal differentiation of myeloid cells is probably involved in the pathogenesis of MDS. So the examination of these antigenic expressions with flow cytometry might be helpful for diagnosis and prognosis of MDS. Disclosures: No relevant conflicts of interest to declare.
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Oh, Chee Won, Carlos Torres-Cabala, Mikyoung Chang, and Madeleine Duvic. "Early Mycosis Fungoides Lesions with Increased CD3-CD4+ Histiocytes Are Associated with Dyslipidemia and Use of Statins." Blood 126, no. 23 (December 3, 2015): 4609. http://dx.doi.org/10.1182/blood.v126.23.4609.4609.
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Abstract Background The term "histiocyte" includes cells of the monocyte/macrophage series as antigen processing cells and the Langerhans cell/DC series as antigen-presenting cells. At least three DC subsets exist in skin: two expressing either CD1a or CD14 are dermal and Langerhans cells expressing CD1a are epidermal. Since the phenotype of histiocytic cells is typically CD3-CD4+, an estimation of the CD4+ histiocytic population can be made by comparing the numbers of CD3+ T cells with CD4+ cells. Programmed Cell Death 1 (PD-1) is an inhibitory receptor expressed on T cells, B cells, and some myeloid cells. During chronic antigen exposure, expression of PD-1 is sustained. Statins, inhibitors of cholesterol biosynthesis, are immunomodulatory agents acting on T cells and DCs, but their effects on skin immunology are unknown. Objectives To investigate whether infiltrates of CD3-CD4+histiocytes in early mycosis fungoides (MF) lesional skin biopsies are associated with any other factors, including history of medication and to reveal their histopathological pattern. Methods From Jan to Dec 2014, we identified cases of early MF from the clinic in which CD4+ cells exceeded CD3+ cells with biopsies to identify increased histiocytic population. Exclusion criteria included Sézary syndrome, granulomatous MF, T cell receptor beta monoclonality, abnormal T cell populations by flow cytometry, retinoid treatment, and progression of disease after treatment (n=12). Clinical and laboratory findings were retrospectively reviewed. Skin biopsies stained for H&E, CD3, CD4, CD7, and CD8 were reviewed. In 3 cases with paraffin blocks available, immunohistochemical stains for CD68, CD1a, CD163, PD-1, and PD-1 ligand PD-L1 were done. Results Clinical manifestations of early MF were pink scaly patches (9/12), capillaritis (2/12), and annular erythema - like patches (1/12). Eleven also had an increased monocytes in peripheral blood. All cases had a medication history of taking statins (atorvastatin 5/12; simvastatin 2/12; rosuvastatin 1/12) for dyslipidemia (hypercholesterolemia 7/12; both hypercholesterolemia and hypertriglyceridemia 3/12). In 9/12, symptoms persisted after MF treatment. A lichenoid or superficial perivascular lymphohistiocytic infiltration was observed in skin lesions. Focal basal vacuolization was found in all 12 patients. Upper dermal perivascular extravasation of RBCs suggesting vasculopathy was also found in 12/12 cases. All twelve cases showed predominant CD4+ T cells compared to CD8+ T cells in dermis and the CD4+ T cells were more prominent in dermis rather than in epidermis. CD7+ T cells were preserved (3/12) or partially lost (9/12). In all 3 cases, macrophage markers CD68 and CD163 were positive in dermal infiltrates. CD1a+ DCs were increased in both epidermis and dermis in all 3/3. Only one case of three showed PD1/PD-L1+ T cells in dermis. Discussion and Conclusion All our cases had a medication history of statins for dyslipidemia. Of interest, skin biopsies showed a vasculopathy previously reported during high-dose atorvastatin treatment (Tehrani et al, 2013) and infiltration of CD4/CD8+ T cells, CD1a+DCs and CD163/CD68+ macrophages. We hypothesize that statins or dyslipidemia in early MF were associated with cutaneous T cell immune reaction. In support of our hypothesis that dyslipidemia is associated with histiocytosis, we found a report of nine cases of granulomatous pigmented purpuric dermatosis with concurrent hyperlipidemia (Battle et al, 2015). Cholesterol induces monocytosis and M1 macrophages in mice. One study showed that predominant migration of mature CD1a+ DC is associated with release of IL-12p70 and efficient expansion of Th 1 cells and functional CD8+ T cells. On the contrary, IL-10 up-regulates migration of immature CD14+ DC, expression of the M2 macrophage marker CD163, poor expansion of CD4+ and CD8+ T cells, and skewing of Th responses conducive to expression of PD-L1. We cannot know whether skin lesions are secondary to hyperlipidemia or to treatment with statins. Although M1 and M2 macrophages can be distinguished by diverse markers, none of these antigens are suitable for single-marker identification by immunohistochemistry in paraffin embedded tissue blocks. Further study of the cutaneous effect and immunologic mechanisms leading to increased expression of DCs and T cell dysfunction after statin medication is necessary. Disclosures Duvic: Oncoceutics: Research Funding; Therakos: Research Funding, Speakers Bureau; Huya Bioscience Int'l: Consultancy; Tetralogics SHAPE: Research Funding; Innate Pharma: Research Funding; Cell Medica Ltd: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; MiRagen Therapeutics: Consultancy; Soligenics: Research Funding; Allos (spectrum): Research Funding; Array Biopharma: Consultancy; Spatz Foundation: Research Funding; Rhizen Pharma: Research Funding; Eisai: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin, Co: Membership on an entity's Board of Directors or advisory committees, Research Funding.
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Shabo, Ivan, Hans Olsson, Xiao-Feng Sun, and Joar Svanvik. "Expression of the macrophage antigen CD163 in rectal cancer cells is associated with early local recurrence and reduced survival time." International Journal of Cancer 125, no. 8 (October 15, 2009): 1826–31. http://dx.doi.org/10.1002/ijc.24506.
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Kawashima, Naomi, Satoshi Nishiwaki, Seitaro Terakura, Sonoko Kamoshita, Kyoko Watakabe, Emi Yokohata, Daisuke Koyama, et al. "Impact of Macrophage Activation on Delayed Engraftment Following Allogeneic Hematopoietic Stem Cell Transplantation: Mac Ratio, a New Predictive Index." Blood 122, no. 21 (November 15, 2013): 4527. http://dx.doi.org/10.1182/blood.v122.21.4527.4527.
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Introduction Delayed engraftment and subsequent engraftment failure cause fatal complications including severe infection and lead to poor prognosis after allogeneic hematopoietic stem cell transplantation (allo-SCT). We have previously reported that hemophagocytic syndrome (HPS) induced by uncontrolled macrophage activation in bone marrow had high mortality due to engraftment failure (BMT.2012;47:387–394). Macrophages are phagocytic cells with abilities of phagocytosis, antigen-presenting, and secretion of cytokines. Although phagocytosis reflects only a part of their activation, their morphological changes or increased cell counts can be used for evaluating their activation. Our hypothesis was that macrophage activation would have intrinsic value whether it would meet diagnostic criteria for HPS or not. In this study, we analyzed the clinical impact of activated macrophages in bone marrow during the peri-enrgaftment period in patients with delayed engraftment and engraftment failure. Patients and Methods We retrospectively reviewed 212 adult patients who received a first allo-SCT for hematological diseases from January 2006 to December 2011 in our institution. Delayed engraftment was defined that neutrophil engraftment was achieved later than day 15, 20 and 27 post peripheral blood stem cell, bone marrow and cord blood transplantation, respectively, whereas engraftment failure was defined that engraftment was never observed including those who received second allo-SCT. Bone marrow clot sections of day 14±7 and day 28±7 post allo-SCT were analyzed by staining macrophages with anti-CD163 monoclonal antibody. CD163 is a member of the scavenger receptor cystein-rich superfamily and is an exclusive marker for macrophages, playing a major role in the scavenging components of damaged cells. Recently macrophages with an unrestrained proinflammatory activation state along with highly expressed CD163 were reported (JCI.2011;121:985–97). Therefore, the total number of CD163 positive-macrophages was counted in three fields at a 200-fold magnification. We calculated the ratio of macrophages (mac ratio), dividing the number of macrophages by the total cell counts per field. Total area of CD163 positive-macrophages was measured with a digital microscope (BZ-9000, Keyence, Japan). The size of a macrophage was estimated total area of CD163+/ number of CD163 positive-macrophages. Results Delayed engraftment and engraftment failure were observed in 17 (8.0%) and 7 (3.3%) out of 212 patients, respectively. Median mac ratio of day 14 marrow was 0.09 (0.03-0.28), 0.54 (0.15-0.82) and 0.57 (0.46-0.65), whereas that of day 28 marrow was 0.05 (0.02-0.08), 0.25 (0.14-0.70) and 0.53 (0.33-0.82) in normal engraftment, delayed engraftment and engraftment failure groups, respectively. Both delayed engraftment and engraftment failure groups had significantly higher mac ratio in both day 14 and 28 than normal engraftment group (p=0.0002, 0.002 at day 14, p=0.0000, 0.0004 at day 28, respectively). Between delayed engraftment and engraftment failure groups, mac ratio was significantly higher in engraftment failure group in day 28 marrow (p=0.04), while no difference was observed in day 14 marrow (p=0.64). Higher mac ratio than 0.5 could predict delayed engraftment or engraftment failure in day14 marrow (p=0.002), and engraftment failure in day 28 marrow (p=0.000). Only 1 patient of 7 engraftment failure patients (14%) met the criteria of HPS, while mac ratio of 5 patients (71%) was >0.5 at day14. The size of macrophages at day 14 was significantly larger in delayed engraftment and engraftment failure groups than normal engraftment group (p=0.03; 1682.2 μm2 in engraftment failure, 1537.1 μm2 in delayed engraftment and 1054.3 μm2 in normal engraftment, respectively), which indicated the activation of macrophages. Conclusion Increased number of activated macrophages in bone marrow during the peri-engraftment period post allo-SCT was associated with subsequent delayed engraftment and engraftment failure. Our data also suggest that delayed engraftment and engraftment failure could be predicted by the mac ratio of day 14 and 28 marrow, which could be more sensitive marker than the criteria of HPS. This study suggests rationale for macrophage-targeted therapy among patients with the proliferation of activated macrophages in their bone marrow to prevent engraftment failure. Disclosures: No relevant conflicts of interest to declare.
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Koelzer, Viktor Hendrik, Kristi Baker, Daniela Kassahn, Daniel Baumhoer, and Inti Zlobec. "Prognostic impact of β-2-microglobulin expression in colorectal cancers stratified by mismatch repair status." Journal of Clinical Pathology 65, no. 11 (August 2, 2012): 996–1002. http://dx.doi.org/10.1136/jclinpath-2012-200742.
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Backgroundβ-2-microglobulin (B2M) is essential for antigen presentation, yet may also possess proto-oncogenic properties.AimTo determine the prognostic impact of B2M in patients with mismatch repair (MMR) proficient and deficient colorectal cancer (CRC) and to investigate whether this effect on outcome is dependent on the local immune response. MethodsB2M protein expression and tumour-infiltrating immune cells (CD3, CD16, CD163, CD20, CD4, CD45RO, CD56, CD68, CD8, FoxP3, GranzymeB, iNOS, mast cell tryptase, MUM1, PD1, TIA-1) were evaluated in a well characterised tissue microarray of 408 CRCs. The predictive value for clinicopathological features and the prognostic significance of B2M expression were analysed, stratified by MMR status and the immunohistological characteristics of immune cell infiltrates. ResultsInterobserver agreement for B2M staining was high (intra-class correlation coefficient=0.91). Complete B2M loss was more frequent in MMR-deficient (19.4%) compared to MMR-proficient (7.1%) tumours (p<0.001). In MMR-deficient cases, B2M loss predicted rare local recurrence (p=0.034), infrequent nodal-positivity (p=0.035), absence of distant metastasis (p=0.048; sensitivity=100%) and a trend towards favourable survival (p=0.124) independent of immune infiltrates. No associations between B2M and clinicopathological features were observed in MMR-proficient cases.ConclusionsOur data show for the first time that absence of B2M protein expression identifies MMR-deficient cancers with a favourable clinical course and absence of metastatic disease. Validation of B2M protein expression for sub-classification of MMR-deficient CRC is recommended for future clinical trials.
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Michalak, Zuzanna, Dima Obari, Matthew Ellis, Maria Thom, and Sanjay M. Sisodiya. "Neuropathology of SUDEP." Neurology 88, no. 6 (January 13, 2017): 551–61. http://dx.doi.org/10.1212/wnl.0000000000003584.
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Objective:To seek a neuropathologic signature of sudden unexpected death in epilepsy (SUDEP) in a postmortem cohort by use of immunohistochemistry for specific markers of inflammation, gliosis, acute neuronal injury due to hypoxia, and blood-brain barrier (BBB) disruption, enabling the generation of hypotheses about potential mechanisms of death in SUDEP.Methods:Using immunohistochemistry, we investigated the expression of 6 markers (CD163, human leukocyte antigen–antigen D related, glial fibrillary acid protein, hypoxia-inducible factor-1α [HIF-1α], immunoglobulin G, and albumin) in the hippocampus, amygdala, and medulla in 58 postmortem cases: 28 SUDEP (definite and probable), 12 epilepsy controls, and 18 nonepileptic sudden death controls. A semiquantitative measure of immunoreactivity was scored for all markers used, and quantitative image analysis was carried out for selected markers.Results:Immunoreactivity was observed for all markers used within all studied brain regions and groups. Immunoreactivity for inflammatory reaction, BBB leakage, and HIF-1α in SUDEP cases was not different from that seen in control groups.Conclusions:This study represents a starting point to explore by immunohistochemistry the mechanisms underlying SUDEP in human brain tissue. Our approach highlights the potential and importance of considering immunohistochemical analysis to help identify biomarkers of SUDEP. Our results suggest that with the markers used, there is no clear immunohistochemical signature of SUDEP in human brain.
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Cooper, Carina J., Stefan M. Keller, Luis G. Arroyo, Joanne Hewson, Daniel Kenney, and Dorothee Bienzle. "Acute Leukemia in Horses." Veterinary Pathology 55, no. 1 (August 16, 2017): 159–72. http://dx.doi.org/10.1177/0300985817720983.
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Leukemia is broadly divided into acute and chronic lymphocytic and myeloid types based on the proportion of blasts, morphology of cells, and expression of specific antigens on neoplastic cells. Classifying leukemia in horses can be challenging if blasts predominate and since few antibodies to identify cell types are available. The objective of this study was to describe in detail the clinical and pathologic features of acute leukemia in horses. Twelve horses ranging from 0.2 to 25.9 years of age were diagnosed with acute leukemia. Six cases were classified as acute lymphocytic leukemia (ALL) based on predominance of blasts, lack of granulocytic or monocytic differentiation, and detection of CD3, CD20, and/or CD79a antigens by immunohistochemistry. Six other cases were classified as acute myeloid leukemia (AML) with myelomonocytic ( n = 4), basophilic ( n = 1), and eosinophilic ( n = 1) differentiation based on > 20% bone marrow blasts and partial leukocytic differentiation. Reactivity with antibodies to Iba-1/AIF-1, CD172a, and CD163 was determined for all cases of AML. Eleven horses had thrombocytopenia, 10 had neutropenia, 8 had anemia, all had blasts on blood films, and none had leukocytosis. Ten horses had increased serum acute phase proteins. Bone marrow cellularity ranged from 30% to 100%, and the proportion of blasts ranged from 80% to 100% and 30% to 60% in ALL and AML, respectively. Horses were severely ill at diagnosis and euthanized within days or weeks. Unique features of acute leukemia in horses compared to other species were variable lymphocyte antigen expression (ALL) and frequent inflammation (ALL and AML).
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Nakamura, Mano, Elmira Amiri Souri, Gabriel Osborn, Roman Laddach, Jitesh Chauhan, Chara Stavraka, Sara Lombardi, et al. "IgE Activates Monocytes from Cancer Patients to Acquire a Pro-Inflammatory Phenotype." Cancers 12, no. 11 (November 15, 2020): 3376. http://dx.doi.org/10.3390/cancers12113376.
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IgE contributes to host-protective functions in parasitic and bacterial infections, often by monocyte and macrophage recruitment. We previously reported that monocytes contribute to tumour antigen-specific IgE-mediated tumour growth restriction in rodent models. Here, we investigate the impact of IgE stimulation on monocyte response, cellular signalling, secretory and tumour killing functions. IgE cross-linking on human monocytes with polyclonal antibodies to mimic formation of immune complexes induced upregulation of co-stimulatory (CD40, CD80, CD86), and reduced expression of regulatory (CD163, CD206, MerTK) monocyte markers. Cross-linking and tumour antigen-specific IgE antibody-dependent cellular cytotoxicity (ADCC) of cancer cells by cancer patient-derived monocytes triggered release of pro-inflammatory mediators (TNFα, MCP-1, IL-10, CXCL-10, IL-1β, IL-6, IL-23). High intratumoural gene expression of these mediators was associated with favourable five-year overall survival in ovarian cancer. IgE cross-linking of trimeric FcεRI on monocytes stimulated the phosphorylation of intracellular protein kinases widely reported to be downstream of mast cell and basophil tetrameric FcεRI signalling. These included recently-identified FcεRI pathway kinases Fgr, STAT5, Yes and Lck, which we now associate with monocytes. Overall, anti-tumour IgE can potentiate pro-inflammatory signals, and prime tumour cell killing by human monocytes. These findings will inform the development of IgE monoclonal antibody therapies for cancer.
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Filipovich, Alexandra H. "Hemophagocytic lymphohistiocytosis (HLH) and related disorders." Hematology 2009, no. 1 (January 1, 2009): 127–31. http://dx.doi.org/10.1182/asheducation-2009.1.127.
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Abstract Hemophagocytic lymphohistiocytosis (HLH), which has many genetic causes, is characterized by multi-system inflammation. HLH is a reactive process resulting from prolonged and excessive activation of antigen presenting cells (macrophages, histiocytes) and CD8+ T cells. Hemophagocytosis, which is mediated through the CD163 heme-scavenging receptor, is a hallmark of activated macrophages/histiocytes and is the characteristic finding for which the disorder was named. The majority of genetic causes identified to date affect the cytotoxic function of NK and T cells, crippling immunologic mechanisms that mediate natural immune contraction. The predominant clinical findings of HLH are fevers (often hectic and persistent), cytopenias, hepatitis and splenomegaly. Due to the life-threatening implications of the diagnosis of genetically determined HLH, antiinflammatory therapy, often consisting of steroids, etoposide or antithymocyte globulin (ATG), should be instituted promptly, followed by curative hematopoietic cell transplantation. Secondary HLH, associated with autoimmune disorders or viral infections in teens and adults, also carries a significant mortality rate and should be managed in consultation with specialists familiar with the diagnosis and treatment of such disorders.
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Cao, Ming, Camellia Eshoa, Christopher Schultz, Jennifer Black, Youli Zu, and Chung-Che Chang. "Primary Central Nervous System Histiocytic Sarcoma With Relapse to Mediastinum: A Case Report and Review of the Literature." Archives of Pathology & Laboratory Medicine 131, no. 2 (February 1, 2007): 301–5. http://dx.doi.org/10.5858/2007-131-301-pcnshs.
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Abstract Histiocytic sarcoma is a rare, malignant neoplasm of the lymphohematopoietic system that usually occurs in the skin, lymph node, and intestinal tract. Here we describe a unique case of primary central nervous system histiocytic sarcoma that initially showed an indolent clinical course following local resection and radiotherapy. However, relapse of disease within the mediastinum was noted 3½ years later. Biopsies of the initial brain lesion and subsequent mediastinal recurrence each revealed an identical, diffuse proliferation of histiocytes with expression of CD45, CD68, and CD163 but not pan-cytokeratin, epithelial membrane antigen, CD3, CD15, CD20, CD30, CD43, CD79a, CD138, myeloperoxidase, ALK-1, PAX-5, CAM 5.2, S100, CD1a, or glial fibrillary acidic protein. In the literature, central nervous system histiocytic sarcoma portends a poor prognosis with median survival of 4.5 months. To our knowledge, this case represents the first case of “low-grade” primary central nervous system histiocytic sarcoma with relatively indolent clinical course. A thorough discussion of the differential diagnosis of histiocytic sarcoma and a review of primary central nervous system histiocytic sarcoma are also presented.
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Kunk, Paul, and Sean Dougherty. "748 Myeloid cell infiltration correlates with prognosis and varies based on tumor location in cholangiocarcinoma." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A796—A797. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0748.
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BackgroundCholangiocarcinoma (CC) is a rare malignancy with an increasing incidence and poor prognosis. Immunotherapy represents one potential treatment for CC, however identification of immunotherapeutic targets requires a thorough characterization of the tumor immune microenvironment (TIME). Mesothelin, a tumor associated antigen, is abundantly expressed in other malignancies, though its expression in CC has not been well characterized. We hypothesized that (1) the TIME of CC would vary by primary tumor location and between primary and metastatic lesions, (2) high tumor infiltration by CD8+ T cells and low infiltration by M2 macrophages would be associated with improved survival, and (3) most CC would express mesothelin.Methods99 CC tumors from unique stage I-IV patients were included, of which 89 were primary tumors (24 intrahepatic (ICC), 65 extrahepatic (ECC - 30 hilar (H-ECC) and 35 distal (D-ECC))) and 10 were metastatic lesions. Tissue microarrays were constructed and immunohistochemistry (IHC) was performed for lymphoid and myeloid markers, as well as for PD-L1 and mesothelin. IHC+ cells were quantified by automated image analysis. Expression of mesothelin and PD-L1 by tumors cells were evaluated on a semiquantitative scale (0, +1, +2, or +3). Hypothesis testing was performed using Kruskal-Wallis test and survival analyses were performed with Univariate and Multivariate Cox Hazard Models.ResultsMost tumors were infiltrated by myeloid cells in addition to CD4+, CD8+, and FoxP3+ T-cells. Mesothelin was expressed (≥1+) in 68% of tumors (figure 1), while PD-L1 was expressed (≥1%) in only 16% of tumors. Higher densities of M1 macrophages (CD68+) were present in D-ECC relative to ICC and H-ECC (figure 2). M1 macropahges were also found in higher densities in metastatic tumors. Mesothelin and granzyme-B expression was significantly higher in D-ECC. Increasing density of myeloid cells (CD14+) and M2 macrophages (CD163+) was associated with worse survival (p= 0.02, 0.03, respectively) (figure 3). Intraepithelial and intratumoral T cell infiltration did not correlate with OS.Abstract 748 Figure 1Mesothelin expression by primary tumor locationA+C) Representative low Mesothelin expression at low (X10) (A) and higher power (X20) (C). B+D) Representative high Mesothelin expression at low (X10)(B) and higher power (X20)(D). E) Log(x+1) transformed Mesothelin Expression as determined by automated cell counting, median and IQR, all data points shown. Median: 5.5, 79.5, 146.0 for ICC, H-ECC, D-ECC, respective, p-value = 0.025. F-H) Mesothelin Expression determined by visual inspection and scoring for ICC (F), H-ICC (G), and D-ECC (D).Abstract 748 Figure 2Immune infiltration based on primary tumor locationIncrease in immune infiltrate in primary tumors as distance from liver increases. P-values determined by Jonckheere-Terpstra Test with FDR correctionsAbstract 748 Figure 3CD14 and CD163 Correlate with OSA+C) Kaplan Meier Curve of OS for (A) CD14 (Median OS: 20 vs. 90 months, log-rank p-value <0.01) and (C) CD163 (Median OS: 15 vs. 32 months, log-rank p-value<0.01). B+D) Multivariate Cox Hazard Models. Assumptions of Cox Hazard Model were checked with Schoenfeld residual values, significance level <0.01ConclusionsThe TIME of CC varies significantly by primary tumor location and between primary and metastatic lesions. D-ECC has a favorable immune profile compared to ICC and H-ECC, with a better milieu for antigen presentation including increased mesothelin and less suppressive macrophages, which may support better response to checkpoint blockade. The data supported the hypothesis that higher densities of intra-tumoral M2 macrophages and myeloid cells correlated with worse OS, even after controlling for clinical variables, suggesting that these cell populations may represent promising immunotherapeutic targets in CC.
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Reiss, David J., Trevor Do, David Kuo, Vanessa E. Gray, N. Eric Olson, Chung-Wein Lee, Mary H. Young, et al. "Multiplexed Immunofluorescence (IF) Analysis and Gene Expression Profiling of Biopsies from Patients with Relapsed/Refractory (R/R) Diffuse Large B Cell Lymphoma (DLBCL) Treated with Lisocabtagene Maraleucel (liso-cel) in Transcend NHL 001 Reveal Patterns of Immune Infiltration Associated with Durable Response." Blood 134, Supplement_1 (November 13, 2019): 202. http://dx.doi.org/10.1182/blood-2019-127683.
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Background: The availability of chimeric antigen receptor (CAR)-modified T cells (CAR T) has profoundly increased therapeutic options for patients (pts) with B cell malignancies, including DLBCL. Liso-cel is an investigational, anti-CD19, defined composition, 4-1BB, CAR T cell product administered at a target dose of CD4+ and CD8+ CAR T cells. To understand tumor microenvironmental (TME) factors affecting short-term and durable responses in pts with R/R DLBCL who received liso-cel in the TRANSCEND NHL 001 study, we conducted multiplexed IF analyses of 111 DLBCL biopsies for 83 pts obtained at baseline (n=58) and approximately 11 days (D11) (n=53; 28 paired) after liso-cel infusion (NCT02631044). Methods: We employed three 5-plex IF panels, consisting of antibodies detecting (1) B cell (CD19, CD20) and T cell lineage (CD4, CD8, EGFR) markers, (2) immunosuppressive markers (CD163, FoxP3, CD73, IDO1, PD-L1), and (3) functional markers (CD3, Ki67, GZMB, PD-1, EGFR). Liso-cel expresses a truncated EGFR (EGFRt), and fluorescent anti-EGFR was used to identify CAR T cells within the tumor biopsies. We also performed bulk tumor RNA profiling for an overlapping subset of 50 baseline biopsies and 37 D11 biopsies (11 paired). We investigated the association of differences in marker densities for pts with best overall response (BOR) of complete response (CR), and progressive disease (PD). Baseline and D11 biopsy findings were correlated with early responses at ~1 month (M1) posttreatment (PD n=16; CR n=42) and durable responses at ~9 months (M9) posttreatment (PD n=76; CR n=32; 55 pts evaluated at both M1 and M9). We investigated how baseline and D11 densities, with spatial distinction between tumoral and peritumoral regions, correlated with early and durable responses. All comparisons describe differences in median densities, and have statistical significance reported with uncorrected P values assessed via the (unpaired) Wilcoxon-Mann-Whitney nonparametric test. Results: Signals in baseline biopsies that correlated with early (M1) response differed from those that correlated with durable (M9) CR. A 21% higher baseline presence of PD-1+ T cells was associated with pts who achieved early CR at M1 vs pts who had PD at M1 (P=0.007). Pts with durable CR at M9 had 39% lower baseline levels of CD163+ macrophages (P=0.019) and 270% higher levels of CD73+ cells (P=0.028) than those with PD at M9. On-treatment (D11) tumors of pts with both early and durable CR had 28% higher levels of EGFRt+ (CAR T) CD8+ T cells (P=0.022), and 810% higher EGFRt- (non-CAR T) CD4+ (but notably, not CD8+; P=0.28) T cells (P=0.009). We also investigated changes in marker densities between baseline and on-treatment (D11) biopsies, and found that pts with durable CR at M9 had decreased on-treatment B cell densities (P=0.029), and increased densities of CD8+ GZMB+, Ki67+, and/or PD-1+ CAR (P=0.001) as well as non-CAR T (P=0.017) cells. Pts with durable CR also had a 29% increase in tumor-associated CD163+ macrophages at D11 relative to baseline (P=0.033). While the accessibility of spatial arrangements and multilabeled cells from IF enables a more nuanced picture of the TME, many of the general trends described above are concordant with those observed in bulk tumor RNA sequencing. Lower baseline expression of CD163 (P=0.021) and higher expression of CD73 (P=0.054) were seen in pts with durable CR. Additionally, elevated on-treatment (D11) expression of CD3E, CD4, and liso-cel (P<0.001) supports the IF finding of greater endogenous and CAR T cell infiltration in pts who responded to treatment. Moreover, pts with a CR at M9 had increased CD163 expression measured at D11 relative to baseline (P<0.001). Conclusions: Overall, these data suggest that increased infiltration of tumor-specific CAR T cells upon initial treatment with liso-cel helped establish an active immune response, and that recruitment of additional functional endogenous (particularly CD4+) T cells correlated with durable response. Higher numbers of activated/functional T cells and lower numbers of macrophages prior to treatment also correlated with durable response to liso-cel. Thus, tumors in responders may already have had a baseline TME in which T cells could infiltrate and respond to antigen. This may have promoted the success of CAR T cell entry into tumors and the subsequent recruitment and activation of endogenous lymphocytes that support their function. Disclosures Reiss: Celgene Corporation: Employment, Equity Ownership. Do:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Kuo:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Gray:Celgene Corporation: Employment, Equity Ownership. Olson:Celgene Corporation: Employment, Equity Ownership. Lee:Celgene Corporation: Employment, Equity Ownership. Young:Celgene Corporation: Employment, Equity Ownership. Srinivasan:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Gray:Celgene: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Couto:Celgene Corporation: Employment, Equity Ownership. Dubovsky:Celgene: Employment. Schmitz:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership.
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Wong, A., S. Mitra, and P. Gupta. "Targeting brain tumor stem cells using a bispecific antibody directed against CD133+ and EGFRvIII+." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 2022. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.2022.
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2022 Background: Using the marker CD133, cancer stem cells (CSCs) have been demonstrated for glioblastomas (GBMs) and medulloblastomas. However, CD133 is also present on normal neural stem cells. EGFRvIII is a tumor specific EGF receptor. We hypothesized that a recombinant bispecific antibody directed against CD133 and EGFRvIII would be a highly specific reagent. Methods: Single chain antibodies (scFv) were cloned for anti-EGFRvIII and anti-CD133 using existing hybridomas and published sequences. scFv were cloned into a bicistronic vector containing a human CH3 constant domain. The bispecific antibody (BsAb) contains the anti-CD133 (AC133) and anti-EGFRvIII single chain Fv; monospecific but bivalent reagents for anti-CD133 and anti-EGFRvIII were also constructed. Results: U87 cells were co-transfected with increasing amounts of CD133 and decreasing amounts of EGFRvIII cDNAs. The BsAb showed the highest binding for cells expressing both epitopes, whereas Di-EGFRvIII and Di-AC133 had the highest affinity for cells expressing high levels of individual antigens. When cells expressing both antigens were mixed with cells expressing high levels of either antigen alone, the BsAb only recognized CD133+/EGFRvIII+ cells. We then explored the efficiency of tumor cell killing. Using human CD16-expressing NK cells as the effectors at an effector:target ratio of 10:1 and an 83 nM antibody concentration, the BsAb induced 86% lysis of U87-EGFRvIII/CD133 cells, 42.5% in U87-vIII but only 27.3% in U87-CD133 cells. Di-EGFRvIII efficiently induced cytotoxicity in U87-EGFRvIII/CD133 and U87vIII cells but not in U87-CD133 cells. Di-CD133 was the least effective at inducing ADCC. Finally, we studied the ability of the BsAb to induce ADCC on tumor spheres and normal neurospheres. The BsAb showed at least 4X greater lysis on tumor spheres that were CD133+/EGFRvIII+ over normal neurospheres expressing CD133 alone at an E:T of 10:1. This was seen at concentrations as low as 0.83 nM. Conclusions: A recombinant bispecific antibody directed against CD133 and EGFRvIII is highly specific for cells that are positive for both antigens, but poorly targets cells expressing CD133 alone. It can potentially be used as a therapeutic agent for specifically targeting CD133+/EGFRvIII+ cancer stem cells. No significant financial relationships to disclose.
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Lee, S. H., P. R. Crocker, S. Westaby, N. Key, D. Y. Mason, S. Gordon, and D. J. Weatherall. "Isolation and immunocytochemical characterization of human bone marrow stromal macrophages in hemopoietic clusters." Journal of Experimental Medicine 168, no. 3 (September 1, 1988): 1193–98. http://dx.doi.org/10.1084/jem.168.3.1193.
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Stromal macrophages (M phi) have been localized in situ and isolated within erythroid clusters from human marrow. Stromal M phi arborize in an extensive network uniformly distributed throughout marrow interstitium, and express the phenotype CD4+, CD11a+, CD11c+, CD13+, CD14+, CD16+, CD18+, CD31+, CD32+, FcRI+, HLA-DR+, and CD35-, transferrin receptor-negative, and CD11b (weak). They express endocytic receptor antigens, but show significant differences in myeloid antigen expression compared with freshly harvested or cultured monocytes. Human stromal M phi are therefore specialized mature marrow M phi that are accessible for further investigations in infectious, storage, or hemopoietic disorders.
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MacDonald, Kelli P. A., David J. Munster, Georgina J. Clark, Andrzej Dzionek, Juergen Schmitz, and Derek N. J. Hart. "Characterization of human blood dendritic cell subsets." Blood 100, no. 13 (December 15, 2002): 4512–20. http://dx.doi.org/10.1182/blood-2001-11-0097.
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Dendritic cells (DCs) are key antigen-presenting cells for stimulating immune responses and they are now being investigated in clinical settings. Although defined as lineage-negative (Lin−) HLA-DR+ cells, significant heterogeneity in these preparations is apparent, particularly in regard to the inclusion or exclusion of CD14+, CD16+, and CD2+ cells. This study used flow cytometry and a panel of monoclonal antibodies (mAbs), including reagents from the 7th Leukocyte Differentiation Antigen Workshop, to define the cellular composition of 2 standardized peripheral blood mononuclear cell (PBMCs)–derived Lin− HLA-DR+preparations. Lin− cells were prepared from PBMCs by depletion with CD3, CD14, CD19, CD11b, and either CD16 or CD56 mAbs. Analysis of the CD16-replete preparations divided the Lin− HLA-DR+ population into 5 nonoverlapping subsets (mean ± 1 SD): CD123 (mean = 18.3% ± 9.7%), CD1b/c (18.6% ± 7.6%), CD16 (49.6% ± 8.5%), BDCA-3 (2.7% ± 1.4%), and CD34 (5.0% ± 2.4%). The 5 subsets had distinct phenotypes when compared with each other, monocytes, and monocyte-derived DCs (MoDCs). The CD85 family, C-type lectins, costimulatory molecules, and differentiation/activation molecules were also expressed differentially on the 5 Lin−HLA-DR+ subsets, monocytes, and MoDCs. The poor viability of CD123+ DCs in vitro was confirmed, but the CD16+ CD11c+ DC subset also survived poorly. Finally, the individual subsets used as stimulators in allogeneic mixed leukocyte reactions were ranked by their allostimulatory capacity as CD1b/c > CD16 > BDCA-3 > CD123 > CD34. These data provide an opportunity to standardize the DC populations used for future molecular, functional and possibly even therapeutic studies.
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Kern, Wolfgang, Marie-Christine Béné, and Anna Porwit. "Interlaboratory Validation of a 3-Tube 10-Color Antibody Flow Cytometry Panel for Diagnosis of Myelodysplastic Syndromes." Blood 120, no. 21 (November 16, 2012): 4941. http://dx.doi.org/10.1182/blood.v120.21.4941.4941.
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Abstract Abstract 4941 Background: Multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of patients with suspected myelodysplastic syndromes (MDS). Modern MFC allows the simultaneous detection of 10 fluorochrome-conjugated antibodies recognizing different antigens and thus provides the basis for a refined detection of MDS-related aberrant antigen expression. While efforts are made to harmonize MFC approaches to diagnose MDS, especially on the markers useful to investigate (Westers et al., Leukemia 2012), there has been no interlaboratory evaluation yet of uniform procedures including a common antibody panel and thus reproducibility remains to be proven. Aims: Prove the interlaboratory comparability of diagnostic read-out using a uniform MFC approach to diagnose MDS. Methods: A 10-color/3-tube panel was designed using the fluorochromes FITC, PE, ECD, PC5. 5, PC7, APC, APCA700, APCA750, Pacific Blue, and Krome Orange conjugated to the following antibodies: tube 1) CD14, CD13, CD38, CD123, CD117, CD11b, CD34, CD33, CD16, CD45; tube 2) CD71, CD4, CD64, CD56, CD117, CD36, CD34, CD33, HLA-DR, CD45; tube 3) CD7, CD10, CD8, CD5, CD2, CD3, CD34, CD19, CD15, CD45. Tube 1 was designed for the assessment of myeloid progenitor cells and granulocytes, tube 2 for monocytes and erythroid cells, tube 3 for lymphoid cells and granulocytes. Data acquisition was performed using Navios flow cytometers (Beckman Coulter, Miami, FL) in each of three laboratories from Germany, France, and Canada. Instrument settings were adjusted at all sites to match pre-defined target channels using beads as controls. During the pilot phase, data of a total of 10 MDS cases acquired at the different sites were analyzed by all of the 3 sites following systematic procedures agreed upon. The parameters evaluated included the percentages of respective cell compartments, expression patterns of CD13/CD16/CD11b in granulocytes, aberrant expression of myeloid markers, coexpression of lymphoid markers in granulocytes, monocytes, myeloid progenitor cells and erythroid cells. Coefficients of variation (CV) were calculated for each analyzed parameter for each patient. Results: The quantification of cell compartments was homogeneously performed by the different sites with median CVs amounting to 5. 7% for granulocytes, 10. 5% for monocytes, 7. 1% for lymphocytes, and 5. 0% for CD34 positive progenitor cells. The mean side scatter signal for granulocytes was consistently determined with a median CV of only 3. 4%. Mean fluorescent intensities (MFIs) for distinct markers in general were homogeneously determined between the three sites with the following median CVs: CD33 1. 8% in granulocytes; CD33 2. 6%, CD14 15. 6%, CD13 4. 8%, CD11b 3. 6%, CD56 23. 8%, HLA-DR 5. 1%, CD2 16. 7% in monocytes; CD71 6. 9%, CD36 2. 9% in erythroid cells; CD34 4. 8% in progenitor cells. This provides an important basis for the harmonization of rating aberrantly expressed antigens in MDS which often are identified by only relatively small changes in the respective MFIs. Importantly, also the percentages of positivity for cross-lineage expression of lymphatic antigens in myeloid progenitor cells, which have previously been shown to carry prognostic significance, were determined homogeneously between the three sites with median CVs of 22. 0%, 13. 4% and 33. 9% for the very small subsets of CD2, CD7 and CD5 positive cells. Another aim of the present pilot study has been the objective read-out of parameters which in general have been judged on subjectively. In this regard, the expression patterns of CD11b and CD16 as well as of CD13 and CD16 in granulocytes were subjectively judged aberrant or not aberrant with complete agreement between the three sites. Importantly, the separation of the giraffe-like pattern of CD11b/CD16 expression in granulocytes into 4 subpopulations was performed homogeneously (median CVs 4. 8%, 22. 1%, 18. 0%, 13. 6%) and the resulting MFIs for CD11b (median CVs 7. 2%, 12. 8%, 10. 8%, 11. 5%) in these 4 subpopulations also were consistently quantified between the three sites. Conclusions: This data indicates that harmonization of procedures across different sites in the flow cytometric evaluation of patients with MDS is feasible. While agreement for judgement on antigen expression patterns is possible by experienced scientists it may be complemented or even substituted by standardized data analysis procedures following testing rounds. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership; Beckman Coulter, Miami, Florida: Research Funding. Béné:Beckman Coulter, Miami, Florida: Research Funding. Porwit:Beckman Coulter, Miami, Florida: Research Funding.
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De Waele, Marc, Barbara Leus, Fabienne Trullemans, Inge Verschraegen, Montse Urbino, Kristin Jochmans, and Wim Renmans. "Diagnostic Potential of Immunophenotyping CD34+ Cells in Myelodysplastic Syndromes." Blood 108, no. 11 (November 16, 2006): 4836. http://dx.doi.org/10.1182/blood.v108.11.4836.4836.
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Abstract Myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal hematopoeitic stem cell disorders. They are characterized by abnormal bone marrow differentiation, peripheral blood cytopenia and a risk of transformation into acute myeloid leukemia (AML). The diagnosis of MDS depends on cytomorphology and cytogenetics and may be difficult especially in cases with normal numbers of blasts and without ringed sideroblasts in the bone marrow. Cytomorphology is subjective and dysplastic features may be present in other disorders than MDS. In this study we examined the potential of immunophenotyping CD34+ hematopoietic precursors for the diagnosis and classification of MDS. Bone marrow samples of 31 patients with low grade MDS (21 without and 10 with ringed sideroblasts), of 17 patients with refractory anemia with excess of blasts (RAEB), of 25 patients with AML and of 39 patients with cytopenia not due to MDS (controls) were examined. CD34+ cells were enumerated and the expression of B cell antigens (CD19), of myeloid antigens (CD13, CD33, CD117) and of immature antigens (CD133) was determined by flow cytometry. Statistical analysis was done with a Mann-Whitney test. A high number of CD34+ cells was found in MDS and AML. This was accompanied by an increase of the number of myeloid precursors and a decrease of the B cell precursors. CD117 appeared to be the best marker of myeloid precursors followed by CD13. A wide range of CD34+CD133+ and of CD34+CD33+ cells was found in all types of samples. Forty percent of the patients with low grade MDS showed an increased expression of CD117 on their CD34+ cells. In 25% of the cases without ringed sideroblasts a high expression of CD133 was present. Similar changes were more frequently found in RAEB and AML together with an increased expression of CD13 and CD33 and a low positivity for CD19. With a scoring system based on the expression of these antigens 57% of low grade MDS samples (score 1/6 or 2/6) could be distinguished from the controls (score 0/6). An elevated score was also found in respectively 84% and 100% of the RAEB and AML samples. 85% of them even had a score between 3/6 and 6/6. In conclusion, immunophenotyping of CD34+ cells is able to differentiate 60% of low grade MDS samples from other causes of cytopenia. Increased expression of CD117, CD133 and CD34 are the main differences. Similar changes are even more frequently found in RAEB and AML. A scoring system based on the antigen expression on the CD34+ cells is a powerful tool for the diagnosis and classification of MDS.
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van Dinther, Dieke, Miguel Lopez Venegas, Henrike Veninga, Katarzyna Olesek, Leoni Hoogterp, Mirjam Revet, Martino Ambrosini, et al. "Activation of CD8+ T Cell Responses after Melanoma Antigen Targeting to CD169+ Antigen Presenting Cells in Mice and Humans." Cancers 11, no. 2 (February 5, 2019): 183. http://dx.doi.org/10.3390/cancers11020183.
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The lack of tumor-reactive T cells is one reason why immune checkpoint inhibitor therapies still fail in a significant proportion of melanoma patients. A vaccination that induces melanoma-specific T cells could potentially enhance the efficacy of immune checkpoint inhibitors. Here, we describe a vaccination strategy in which melanoma antigens are targeted to mouse and human CD169 and thereby induce strong melanoma antigen-specific T cell responses. CD169 is a sialic acid receptor expressed on a subset of mouse splenic macrophages that captures antigen from the blood and transfers it to dendritic cells (DCs). In human and mouse spleen, we detected CD169+ cells at an equivalent location using immunofluorescence microscopy. Immunization with melanoma antigens conjugated to antibodies (Abs) specific for mouse CD169 efficiently induced gp100 and Trp2-specific T cell responses in mice. In HLA-A2.1 transgenic mice targeting of the human MART-1 peptide to CD169 induced strong MART-1-specific HLA-A2.1-restricted T cell responses. Human gp100 peptide conjugated to Abs specific for human CD169 bound to CD169-expressing monocyte-derived DCs (MoDCs) and resulted in activation of gp100-specific T cells. Together, these data indicate that Ab-mediated antigen targeting to CD169 is a potential strategy for the induction of melanoma-specific T cell responses in mice and in humans.
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Affandi, Alsya J., Joanna Grabowska, Katarzyna Olesek, Miguel Lopez Venegas, Arnaud Barbaria, Ernesto Rodríguez, Patrick P. G. Mulder, et al. "Selective tumor antigen vaccine delivery to human CD169+antigen-presenting cells using ganglioside-liposomes." Proceedings of the National Academy of Sciences 117, no. 44 (October 16, 2020): 27528–39. http://dx.doi.org/10.1073/pnas.2006186117.
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Priming of CD8+T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1+antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14+CD169+monocytes and Axl+CD169+DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169+moDCs and Axl+CD169+DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8+T cells. Finally, Axl+CD169+DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169+DCs to drive antitumor T cell responses.
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Lasho, Terra, Christy Finke, Teresa K. Kimlinger, Darci Zblewski, Dong Chen, Mrinal M. Patnaik, Curtis A. Hanson, Christopher Brooks, Ayalew Tefferi, and Animesh Pardanani. "Expression of CD123 (IL-3R-alpha), a Therapeutic Target of SL-401, on Myeloproliferative Neoplasms." Blood 124, no. 21 (December 6, 2014): 5577. http://dx.doi.org/10.1182/blood.v124.21.5577.5577.
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Abstract Background: CD123 (alpha chain of the interleukin-3 receptor [IL-3RA]) heterodimerizes with CD131 (βc, common beta chain) to constitute the high-affinity receptor for IL-3. CD123 is not expressed on hematopoietic stem/progenitor cells from normal bone marrow (BM). In contrast, CD123 is highly expressed in CD34+/CD38- cells from acute myeloid leukemia (AML) patients, which recapitulate the leukemic phenotype in NOD/SCID mice; CD123, therefore, is a marker for leukemic stem cells in AML (Jordan CT et al., Leukemia 2000). CD123 is also highly expressed on CD4+/56+ leukemic cells in patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive hematodermic neoplasm. The validity of CD123 as a rational therapeutic target was illustrated by clinical efficacy data for SL-401, a biologic target therapy directed to IL-3R, in patients with BPDCN and AML, respectively (Frankel AE et al., Blood 2014 andFrankel AE et al., J Clin Oncol 31, 2013 (suppl; abstr 7029)). Objectives: To study CD123 expression as a potential therapeutic target in myeloproliferative neoplasms (MPN). Methods: The study was approved by our institutional review board and patients provided written consent for sample collection. MPN diagnosis was based on WHO 2008 criteria. Surface antigen expression on hematopoietic cells of various lineages was interrogated using the 4-color multiparametric flow cytometer, FACSCaliber TM (BD Biosciences, San Jose, CA). Data analysis was performed using CellQuest Pro Software (BD Biosciences). Results: We studied a total of 21 MPN patients and 3 normal controls. Of the former, 14 patients had systemic mastocytosis (SM) (indolent SM=9, SM with associated myeloid neoplasm=4 and aggressive SM=1), 6 had primary myelofibrosis (PMF) and 1 had eosinophilic leukemia transformed to AML. Normal controls: Three normal BM samples were studied; the number of CD123 positive cells in the total population was <1%. Rare CD34+/CD38- cells were uniformly CD123-. Of the CD123+ cells, only a minor subset (<10%) coexpressed myeloid/granulocyte lineage (CD13+, CD15+ or CD16+) or monocyte/macrophage lineage (CD14+ or CD11b+) markers. Systemic mastocytosis: The data were informative for 6 patients (3 with peripheral blood [PB], 1 with BM, and 2 with paired PB and BM samples) for whom a sufficient number of mast cells (MC) were identified for immunophenotyping. Neoplastic MC were defined as CD117 hi/SSC hi/CD45 hi/CD34-/CD25+/FcεRI+. CD123 expression was seen in the majority of MC for 4 patients (#1-4, CD123 percentage positive, PB/BM): 91%/n.a. (#1), 75%/81% (#2), 84%/n.a. (#3), and 88%/n.a. (#4). In contrast, one patient (#5) had a minority of CD123+ MC (33%/28%). Clonal eosinophilia: PB was studied; the WBC differential count showed 41% leukemic blasts and 57% eosinophils (both confirmed to be clonally related based on FISH studies). Both cell populations were predominantly CD123+ (≥90%). Primary myelofibrosis: We studied PB samples from 6 PMF patients. Approximately 1-2% of circulating cells marked as CD123+; of these, expression was notable on CD34-/CD38+ cells (median 54%; range 41-74% positive). We found a larger proportion (30-50%) of CD123+ cells that coexpressed CD13+, CD16+ or CD11b+ representing monocytes, immature myeloid cells and granulocytes, as compared to normal BM controls. Studies on BM samples from PMF patients are ongoing. Conclusions: CD123 (IL-3RA) is expressed on relevant primary cells of interest in select MPNs, namely neoplastic mast cells in SM patients and eosinophils in clonal eosinophilia patients. In PMF, a minor population of circulating cells was CD123+ with a biased distribution on myeloid lineage cells as compared to normal BM samples. We are currently studying CD123 expression in additional MPN patients, and also analyzing CD123 expression on BM trephine biopsies by immunohistochemistry. In addition, the cytotoxicity assessment of SL-401 against MPN cell lines and primary cells is ongoing. Overall, the aforementioned data support the clinical development of SL-401 in patients with MPNs and clinical trials are currently being planned. Disclosures Brooks: Stemline Therapeutics: Employment, Equity Ownership. Pardanani:Stemline Therapeutics Inc.: Research Funding.
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Taylor, Joseph G., Andrew James Clear, Edward Truelove, Mariarita Calaminici, and John G. Gribben. "Beyond Exhaustion: The PDL1-PD1 Axis Shapes the Classical Hodgkin Lymphoma Microenvironment." Blood 134, Supplement_1 (November 13, 2019): 658. http://dx.doi.org/10.1182/blood-2019-125247.
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Background: PD1 inhibitor (PD1i) efficacy is taken as evidence of T cell exhaustion in Classical Hodgkin Lymphoma (CHL), however, direct evidence for exhaustion is limited. PD1 expression does not predict PD1i response in CHL. Additionally, PDL1 and PD1 expression are not correlated and are independently prognostic. This suggests a more complex picture than PD1 expression simply reflecting exhaustion induced by PDL1. Aim: To investigate PD1/PDL1 interaction in CHL Methods: 149 CHL cases were compared to 27 reactive lymph node controls (RLN) by immunohistochemistry (IHC). PD1 expression was validated with two clones. Multiplex IHC (mIHC) was performed in 47 CHL and 27 RLN by stripping, reprobing and image alignment. Imaging Mass Cytometry (IMC) was analysed by t-SNE clustering and spatial point pattern modelling (SPPM). SPPM is a powerful technique adopted from other disciplines and its use in multiplex image analysis is novel. Flow-based CFSE proliferation and cytokine assays were performed in 15 CHL and 5 RLN single cell suspensions (SCS) with CD3/28 and PMA/Ionomycin stimulation. Co-cultures were with KMH2 and L428 cell lines and healthy donor naïve T helper cells (TH). Results: We evaluated PDL1 expression by IHC and found that PDL1hi CHL cells were surrounded by PDL1+ macrophages (Mφ), consistent with the literature. Mφ PDL1 intensity correlated inversely with distance from CHL cells consistent with induction by a CHL-secreted factor (p <0.001), a finding we validated in vitro (p <0.001). Deep phenotyping using IMC identified CD14+CD16-CD163-PDL1+-, CD14-CD68+CD163-PDL1+ and CD14+CD16+CD163+PDL1- myeloid subsets by t-SNE. SPPM revealed that tumor and vessel location predicted their distribution with subsets one and two localising to tumor whilst subset three localised to vessel. Our data supports the hypothesis that CHL recruits and induces a PDL1+ myeloid environment. We next sought evidence of T cell exhaustion. PD1 expression by IHC was lower in CHL than RLN (p<0.001). Cells co-expressing PD1 with TIM3 or LAG3 were significantly reduced in CHL compared to RLN by mIHC. Cells co-expressing PD1 with TBET or EOMES were reduced in or showed no significant difference between CHL and RLN. No correlation was seen between PD1 and PDL1 expression. We performed functional validation, evaluating proliferative and IL2/IFNγ production capacity, and identified no significant difference between CHL and RLN SCS with or without the addition of Pembrolizumab or isotype control. This constitutes a more detailed assessment of exhaustion with higher case numbers than publications to date. Our data does not support the presence of an exhausted T population above that seen in reactive controls and finds no relationship between PD1 and PDL1 expression. The absence of exhaustion does not exclude a role for the PD1-PDL1 axis, which regulates multiple facets of T cell homeostasis including motility, differentiation (skewing towards T regulatory cells (TReg)), and TReg effector functions. Many of these are characterised by transient PD1 expression unlike the sustained expression seen in exhaustion. In contrast to PD1, CHL PDL1 and MHC class 2 expression do predict PD1i response. CHL MHC2 expression suggests antigen presentation and a role for TH cells. This led us to examine connections between PDL1 and the TH compartment. Co-culture of CHL cell lines with naïve TH led to enrichment of CD25hiCD127loFOXP3+ TReg which upregulated PD1 compared to TReg from paired CD3/28 controls (p<0.001). FOXP3+ cells were enriched by IHC compared to RLN (p<0.001). Unlike PD1, FOXP3 associated with CHL MHC2 expression (p=0.012), which in turn associated with PDL1 expression (p=0.02). SPPM of TC and TH subsets revealed that PDL1 expression predicted the location of FOXP3+ TReg and negatively predicted the location of TC and TH17. PDL1 was a stronger predictor than CHL cell density. These data support a role for PDL1 in shaping T differentiation and trafficking irrespective of observed PD1 expression. Conclusion: We find that CHL cells are PDL1hi and induce a PDL1+ myeloid environment, but detect little evidence of T exhaustion. Instead we find that PDL1 expression shapes T (particularly TReg) differentiation and trafficking. Our data challenges the narrative that PD1i in CHL act by reversing exhaustion and highlights novel mechanisms with the potential to provide insight into CHL biology and inform the hunt for biomarkers of response. Figure Disclosures Gribben: Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.