Academic literature on the topic 'TCD4+ Lymphocytes'

The host lymphocyte response is decisive in Pneumocystis pneumonia (PCP) pathophysiology but little is known of the specific roles of lymphocyte subpopulations in this fungal infection. Peripheral NK, NKT, B, TCD4+ and TCD8+ subpopulations were compared by immunophenotyping between 20 patients diagnosed with PCP (PCP(+)] and 20 uninfected immunosuppressed patients (PCP(−)). Among PCP(+) subjects, the lymphocyte populations were also compared between surviving and deceased patients. Low B cell count (<40 cells/µL) was more frequent in PCP(+) than in PCP(−) patients (p = 0.03), while there was no difference for the TCD4 count. Among the PCP(+) group, the 7 deceased patients had lower Th1 (p = 0.02) and Tc1 (p = 0.03) populations, higher Th2 response (p = 0.03), higher effector TCD8 (p < 0.01), lower central memory TCD8 (p = 0.04) and reduced NK cells (p = 0.02) compared with the 13 survivors. Th1/Th2 ratio < 17, CD8 Tc1 < 44%, effector TCD8 < 25%, central memory TCD8 < 4%, NK cells < 50 cells/µL and total lymphocytes < 0.75 G/L were associated with a higher risk of mortality (p = 0.003, p = 0.007, p = 0.0007, p = 0.004, p = 0.02 and p = 0.019, respectively). The traditional analysis of TCD4 and TCD8 populations may be insufficient in the context of PCP. It could be completed by using B cells to predict the risk of PCP, and by using lymphocyte subpopulations or total lymphocyte count, which are easy to obtain in all health care facilities, to evaluate PCP prognosis.

Abstract Adult T-cell leukemia/lymphoma (ATL) is a mature T-cell neoplasm of post-thymic lymphocytes linked to human T-cell lymphotropic virus type I (HTLV-I). ATL develops in 3–5% of HTLV-I carriers after a long period of clinical latency and presents chromosomes abnormalities resulting in aneuploidy usually seem in telomere dysfunction disease. We were interested whether the abnormalities of the telomere regulation in ATL and if its abnormalities are correlated with ATL pathogenesis. The telomere length of TCD4 and TCD8 lymphocytes in samples from HTLV-I carriers (n= 52) with median age of 49.5 years (18–71 years) and ATL patients (n= 6) with median of 39 years (18–59 years) were assessed by fluorescence in situ hybridization and flow cytometry (Flow-FISH) and compared with healthy subjects (n = 58) matched for gender and age. We did not detected significant difference the telomere length of the TCD4 and TCD8 lymphocytes between HTLV-I carriers and health subjects. However we showed an accentuated loss in telomere length of the malignant T lymphocyte in ATL disease and values closed to those ageexpected in non-malignant T lymphocyte in ATL disease. We concluded that these results do not support a major role of telomere disfunction as an early event in pathogenesis of the ATL.

Introduction: Immunological monitoring could indirectly measure the suppressive effects of the drugs and provide early guidance on necessary preventive interventions in transplant recipients. Objectives: Our goal was to determine whether mycophenolic acid (MPA) modulates peripheral blood lymphocyte T in kidney transplant recipients. Patients and Methods: We assessed T lymphocytes CD3, CD4 and CD8 in peripheral blood in 30 donors and 35 recipients one day before and 10 days after transplantation using Becton Dickinson’s direct immune fluorescent light. Results: Comparisons showed that the number of T lymphocytes CD3+, CD4+, CD8+ in peripheral blood of transplant recipients were lower than donors (TCD3 was 1690.31±503.45 versus 2280.73± 522.48; TCD4 was 549.51 ±211.72 cell/µL versus 766.37± 341.72 cell/µL and CD8 was 1134.37 ±431.07 cell/µL versus 1523.4± 349.23 cell/µL with P<0.001; P=0.001 and P= 0.0002 respectively). Additionally, post-transplantation lymphocytes TCD4 decreased in 10/35 of recipients and increased in 22/35 of recipients (P=0.036). Conclusion: The T lymphocytes CD3, CD4 and CD8 in peripheral blood should be monitored at multiple post-transplant times to make early predictions of transplant rejection during follow-up treatment.

The aim of this study was to describe the effect of non-adherence on the main laboratory outcomes, TCD4+ lymphocyte count and viral load, routinely used to monitor patients initiating treatment according to three different approaches to measure adherence to antiretroviral therapy. Among 288 participants, 22.9%, 31.9% and 74.3% were considered non-adherent, according to medical charts, self-report and pharmacy records, respectively. Depending on the adherence measures used, the average gain in TCD4+ lymphocyte count ranged from 142.4 to 195.4 cells/mm3 among adherent patients, and from 58.5 to 99.8 lymphocytes TCD4+/mm3 among those non-adherent. The average reduction on viral load ranged from 4.25 to 4.62 log copies/mL among the adherent patients, and from 1.99 to 4.07 log among those non-adherent. Monitoring antiretroviral adherence should be considered a priority in these public AIDS referral centers in order to identify patients at high risk of developing virologic failure. Early interventions are necessary in order to maintain the initial therapeutic regimens for longer periods.

Abstract Abstract 4777 Introduction. Recently, several researchers have showed that mesenchymal stromal cells (MSCs) are useful for preventing and treating the graft versus host disease, however the molecular mechanisms involved in immunomodulation of T lymphocytes by MSCs remains to be explored. Aims. With that in mind, we performed a whole genome microarray study to explore transcriptional differences in activated TCD4 and TCD8 lymphocytes immunomodulated or not by MSCs. Materials and Methods. Peripheral blood CD3+ T lymphocytes from 3 individuals were activated by anti-CD3/CD28 beads and cultured either in the absence or in the presence of MSC (5 to 1 ratio). Following a 5 day period, CD4+ and CD8+ lymphocytes cultivated or not with MSCs were immunomagnetically purified to over 95% purity (flow cytometry) and profiled by whole genome microarrays. Differentially expressed genes were analyzed in search of regulatory networks and levels of selected transcripts were evaluated by RT-PCR. Results. T lymphocytes proliferation was inhibited significantly by MSC (BrdU incorporation assay) and induced the expression of classical regulatory T cell genes (IL10, FOXP3, CTLA-4 and GITR). Our microarray analyses revealed that lymphocytes cultured with MSCs displayed several transcriptional modifications related to distinct signaling pathways. Among them, TCR signaling, cell cycle (G1/S progression), NF-kB pathway, mTOR signaling and in enzymes and factors related with chromatin modification and remodeling. An insignificant number of transcript behaved distinctly (eg. with changes in opposite direction) when TCD4+ and TCD8+ lymphocytes where compared. Conclusion. Our results clearly show that both CD4+ and CD8+ T lymphocytes are immunomodulated by MSCs by similar mechanisms and this effect occurs by means of distinct molecular mechanisms. Supported by FAPESP, CNPq and FINEP. Disclosures: No relevant conflicts of interest to declare.

In our previous study, calcitriol and its analogs PRI-2191 and PRI-2205 stimulated 4T1 mouse mammary gland cancer metastasis. Therefore, we aimed to analyze the inflammatory response in 4T1-bearing mice treated with these compounds. Gene expression analysis of the splenocytes and regional lymph nodes demonstrated prevalence of the T helper lymphocytes (Th2) response with an increased activity of regulatory T (Treg) lymphocytes in mice treated with these compounds. We also observed an increased number of mature granulocytes and B lymphocytes and a decreased number of TCD4+, TCD4+CD25+, and TCD8+, as well as natural killer (NK) CD335+, cells in the blood of mice treated with calcitriol and its analogs. Among the splenocytes, we observed a significant decrease in NK CD335+ cells and an increase in TCD8+ cells. Calcitriol and its analogs decreased the levels of interleukin (IL)-1β and IL-10 and increased the level of interferon gamma (IFN-γ) in the plasma. In the tumor tissue, they caused an increase in the level of IL-10. Gene expression analysis of lung tissue demonstrated an increased level of osteopontin (Spp1) and transforming growth factor β (TGF-β) mRNA. The expression of Spp1 was also elevated in lymph nodes. Calcitriol and its analogs caused prevalence of tumor-conducive changes in the immune system of 4T1 tumor-bearing mice, despite the induction of some tumor-disadvantageous effects.

A variety of mechanisms are involved in the regulation of offspring allergy development through maternal immunization with allergens. The passive transfer of antigens, antibodies, and cytokines, the induction of phenotypic alterations in offspring lymphocytes, and the induction of regulatory populations in offspring have been proposed, but these mechanisms remain incompletely understood. It is likely that maternal immunization could affect the intrathymic maturation of offspring TCD4+, TCD8+,γδT, nTreg, iNKT, and B lymphocytes, although there are currently no human maternal immunization protocols for the regulation of allergic responses in children. Some studies have suggested a direct interaction between the maternal immune status and the offspring intrathymic microenvironment; this interaction could influence the maturation of offspring regulatory cells and must be explored for the development of therapies to control allergy development in children.

Abstract The mechanism by which kinin B1 receptor (B1R) contributes to type 1 diabetes is addressed by determining the impact of its inhibition on diabetes and on its pancreatic expression and cellular localisation on immunocompetent cells and primary sensory C-fibres. Rats were made diabetic with streptozotocin (STZ). On day 4, they were treated daily for 7 days with a B1R antagonist (SSR240612, 10 mg/kg) or its vehicle. The surviving β-cells were measured by immunostaining. The expression of B1R, iNOS, TNF-α, macrophages, TCD4+, CGRP and TRPV1 was measured by Western blotting, qRT-PCR and immunofluorescence. Macrophages and TCD4+ lymphocytes were absent in control, but distributed abundantly in the pancreas of STZ-diabetic rats. B1R was upregulated on these immune cells infiltrating the diabetic rat pancreas while it was not expressed on primary sensory C-fibres even if the expression of TRPV1 and CGRP was enhanced. SSR240612 prevented the infiltration of macrophages and TCD4+ lymphocytes and the upregulation of B1R, iNOS, TNF-α and TRPV1. SSR240612 corrected hyperglycaemia and hypoinsulinaemia by improving the Langerhans islets survival or regeneration. It is concluded that kinin B1R antagonism exerts anti-diabetic action by preventing the infiltration of immune cells in the pancreas and by preserving the integrity of Langerhans islets β-cells.

Paracoccidioidomycosis, a key issue for Brazilian health service, can be aggravated in patients with impaired immunological responses, such as diabetic patients. We evaluated the role of insulin in inflammatory parameters in diabetic and nondiabetic mice using a systemic mycosis Paracoccidioides brasiliensis (Pb) model. Diabetic C57BL-6 mice and controls were infected with Pb18 and treated with insulin for 12 days prior to experiments. After 55 days, infected diabetic mice exhibited fewer leukocytes in both peritoneal lavage fluid (PeLF) and bronchoalveolar lavage fluid and reduced secretion of interleukin- (IL-) 6 in lungs. In addition, diabetic mice presented a reduced influx of TCD4+ cells, TCD8+ cells, B lymphocytes, NK cells, and dendritic cells compared to control infected groups. Insulin treatment restored the leukocyte number in PeLF and restored the presence of B lymphocytes, dendritic cells, and NK cells in lungs of diabetic animals. The data suggest that diabetic mice present impaired immunological response to Pb18 infection and insulin modulates inflammation by reducing IL-6 levels in lung and CINC-1 levels in spleen and liver homogenates, restoring leukocyte concentrations in PeLF and also restoring populations of dendritic cells and B lymphocytes in lungs of diabetic mice, permitting the host to better control the infection.

Introduction:Sézary Syndrome (SS) is a leukemic form of Fungal Mycosis (FM), a rare form of T-cell lymphoma, characterized by erythroderma, generalized lymphadenopathy and infiltration of neoplastic T cells (Sézary cells) with cerebriform nucleus on the skin, lymph nodes and peripheral blood, being observed predominantly in men and individuals over the age of 60 and black. In the diagnosis of SS / FM, at least one of the criteria must be observed: minimum absolute Sézary cells count of 1000/mm3, expansion of TCD4+ cells with a ratio CD4/CD8 &gt;10, loss of at least one mature T cell antigens as CD2, CD3, CD5, CD7 and CD26 in associated with increased lymphocyte count with evidence of a clone of circulating TCD4 cells determined by flow cytometry (CF).Objective:To investigate MF/SS in patients diagnosed with cutaneous lymphoma by CF immunophenotyping. Methodology: Were investigated in samples of peripheral blood (SP) from 11 patients of both sexes with initial history of MF and confection of SS due to the presence of Sézary cells by cytomorphological analysis by CF constituted by a panel of conjugated monoclonal antibodies (AcMo) to fluorochromes and targeted to T lymphocytes: CD1a, CD2, CD3, CD5, CD7, subpopulation T-Helper (CD3+/CD4+) and T-cytotoxic (CD3+/CD8+), in addition to TCR a/b and TCR g/d; Natural Killer cells: CD16-56; B lymphocytes: CD19, CD20, CD21, CD22, CD23, IgM, IgG, IgD anti-kappa and anti-lambda, in addition to CD10, TdT, CD103, CD25, CD38 and CD138, CD45 and CD14. At the same time, a complete blood count with differential white blood cell count and investigation of clinical and demographic data such as age, sex and ethnicity/race were also performed. Results: Of the patients analyzed, 6/11 were male, the age group above 60 years and white individuals were also found in 6/11 patients. The blood count showed lymphocytosis in 9/11 patients with the presence of convoluted cells in all cases. The diagnosis of SS was confirmed by the presence of Sezary cells in PB counting above 1000/mm3, with an immunophenotype confirmed by the predominance of TCD4+ lymphocytes (CD4/CD8 ratio &gt; 10.0), associated with the expression of CD5, CD2, TCR a/b, CD3 weakly expressed. CD7 was absent in 10/11 samples analyzed. Antigens related to B lymphocytes and NK cells were absent in neoplastic cells as well as CD10, TdT and CD1a.Conclusions:SS is a leukemic variant of FM, characterized by exfoliative erythroderma, associated with lymphadenopathy and leukemization of FM with the appearance of Sézary cells in PB. Because it is a rare and essential disease, an accurate diagnosis of these diseases is necessary, and FC is an important diagnostic confirmation tool for SS. Disclosures No relevant conflicts of interest to declare.

A demodicidose é uma importante dermatopatia parasitária em cães. É decorrente da proliferação excessiva de ácaros comensais do gênero Demodex sp no tegumento canino. A manifestação clínica da demodicidose juvenil generalizada têm sido associada à disfunção imune hereditária de linfócitos T específica ao parasita, enquanto que, a demodicidose do aduto pode ser consequente a doenças imunossupressoras. A resposta imune celular é considerada crucial na defesa contra o parasita e, encontra-se comprometida em cães com demodicidose. Com o escôpo de se determinar se linfócitos sanguíneos periféricos, TCD4+, TCD8+ e a razão TCD4+:TCD8+ são bons indicadores da evolução clínica da doença e do estado imune de cães demodicidose generalizada, tais parâmetros foram quantificados em 16 animais com demodicidose generalizada, na pré, trans e pós terapia, e em outros 30 animais hígidos, utilizando-se da técnica de citometria de fluxo. Para as análises estatísticas comparativas, foram padronizados quatro momentos de observação dos animais com demodicidose, a saber: primeira consulta: quando do estabelecimento do diagnóstico; segunda consulta; consulta de obtenção do primeiro exame parasitológico negativo e, finalmente, naquela de estabelecimento da alta clínica, preconizada quando da ausência de evidenciação do ácaro no exame parasitológico cutâneo, por três consultas consecutivas. Os valores absolutos médios de linfócitos totais, linfócitos TCD4+ e TCD8+ nos animais com demodicidose, mostraram-se inferiores àqueles do Grupo Controle em todos os momentos de observação. Somente os valores absolutos de linfócitos TCD4+, apresentaram diminuição significativa, em relação ao Grupo Controle, no momento da primeira consulta. Entre o Grupo Experimental, foi observado elevação signficativa entre os valores absolutos dos linfócitos totais, TCD4+ e TCD8+, entre a primeira consulta e aquela de obtenção do primeiro exame parasitológico negativo, quando estes valores se aproximaram daqueles observados no Grupo Controle. Paralelamente, evidenciou-se alta correlação da elevação dos número absoluto médio dos linfócitos TCD4+ e TCD8+, com a diminuição da contagem de ácaros. A razão TCD4+:TCD8+, não diferiu significativamente entre o Grupo Controle e Experimental. O tratamento da demodicidose não alterou a razão TCD4+: TCD8+. Não foi observado correlação entre o período necessário para o estabelecimento da alta clínica e a razão TCD4+:TCD8+. O comportamento dos linfócitos sanguíneos periféricos TCD4+, TCD8+, e da razão TCD4+:TCD8+, demonstra que a participação de outros mecanismos, que não a franca alteração destas subpopulações, sejam importantes na patogenia da doença. Em linhas gerais, não foram observadas diferenças significativas, nos valores dos linfócitos TCD4+, TCD8+ e a razão TCD4+:TCD8+, entre os animais do Grupo Controle, e os animais com demodicidose generalizada , de forma que, o uso da determinação destes parâmetros, é desaconselhado no monitoramento da evolução clínica e no estabelecimento do prognóstico, nos animais com demodicidose generalizada.
Demodicosis is a serious canine parasitic skin disease. It is caused by the presence of increasead amounts of Demodex mite in the skin. Clinical signs of juvenile-onset generalized demodicosis are associated with specific hereditary dysfunction of T lymphocytes while adult-onset can be induced by immunosuppressive diseases. The cellular immunity is crucial in keeping low numbers of skin mites and it is depressed in dogs with generalized demodicosis. The aim of this study was to verify whether CD4+, CD8+ T-lymphocytes counts and CD4+:CD8+ ratio could be good indicators of disease progression and immune status in canine demodicosis. For this, using the flow cytometry technique, the CD4+, CD8+ T-lymphocytes counts and CD4+:CD8+ ratio of 16 dogs with generalized demodicosis were evaluated at four moments: first and second consultation, first time animal was presented without mites in skin scrapings and finally, on clinical improvement. These values were then compared with those of 30 controls healthy dogs. The absolute numbers of CD4+, CD8+ and total lymphocytes were lower than control healthy dogs at all moments of analysis. Only at the first consultation CD4+ lymphocyte counts was significant lower than control group. Dogs with generalized demodicosis had signicant increased counts of CD4+, CD8+ and total lymphocytes from the first consultation until the first negative skin scraping. At this point lymphocyte counts reached levels closesth to control group ones. CD4 : CD8+ ratio didn´t differ throughout treatment of canine demodicosis neither when average level for ill dogs were compared to with those healthy ones. Furthermore CD4+, CD8+ and CD4:CD8+ ratio didn´t correlated with time taken for successfull treatment completion and so they couldn´t be used as prognosis predictor. A high correlation between increased of CD4+, CD8+ T-lymphocytes counts and decreased mites counts was observed in dogs with generalized demodicosis. Circulating lymphocyte subpopulations are therefore similar in dogs with canine demodicosis and healthy dogs and there is no correlation between clinical status or response to therapy and the lymphocytes subpopulations counts. We can than conclude that CD4+, CD8+ T-lymphocytes counts and CD4+:CD8+ ratio cannot be used as a parameters to predict progression of an individual patient in a clinical context.

Para investigar o papel dos linfócitos T no modelo de Paracoccidioidomicose pulmonar, depletamos in vivo os linfócitos TCD4+ e TCD8+ de camundongos resistentes (A/J) e susceptíveis (B10.A) infectados intratraquealmerite com um milhão de leveduras de isolado virulento do Paracoccidioides brasiliensis. Quando comparados ao grupo não depletado de linfócitos TCD4+, após 4 semanas de infecção, camundongos A/J apresentaram aumento da disseminação de leveduras ao fígado; na 8ª semana, contudo, houve redução na carga fúngica pulmonar destes animais. Ao contrário, a depleção de células TCD4+ não alterou a gravidade da doença de animais B10.A em ambos os períodos de infecção. O tratamento com o AcM anti-CD4, em ambos os tempos de infecção, diminuiu as reações de HTT dos animais A/J, mas não alterou a anergia cutânea dos camundongos B10.A. Em adição, ambas as linhagens depletadas tiveram a produção específica de anticorpos diminuída na 4ª e 8ª semanas. Quanto às citocinas pulmonares, após 4 semanas de infecção, os animais A/J depletados apresentaram redução nos níveis de IL-12 pulmonar e na 8ª semana da infecção, esta linhagem apresentou redução nos níveis de IL-10, IL-4, IL-5,IL-2 e GM-CSF pulmonar, e os animais B10.A mostraram aumento nos níveis de IL-12. Por outro lado, verificamos que ambas as linhagens depletadas com o AcM anti-CD8, apresentaram aumento da gravidade da doença após a semanas de infecção, pois camundongos A/J tratados apresentaram aumento da carga fúngica pulmonar, e animais B10.A apresentaram aumento do crescimento fúngico no pulmão e fígado. Animais B10.A depletados de células TCD8+ apresentaram ainda um incremento nas suas reações de HTT específicas. Esses dados sugerem que na linhagem susceptível há a presença de duas subpopulações celulares de TCD8+, uma subpopulação protetora que controla o crescimento fúngico e outra inibidora de reações de HTT. A depleção dos linfócitos TCD8+ não levou a grandes alterações na produção de anticorpos específicos em ambas as linhagens; no entanto, levou a alterações significativas nos níveis das citocinas pulmonares; os animais A/J apresentaram aumento de IL-4, IL-12, IL-3 e GM-CSF, e diminuição de IL-2. Por outro lado, os animais B10.A apresentaram aumento nos níveis de IL-10, IL-12, IL-3 e IFN-γ. Estes resultados demonstraram que nos animais B10.A as células TCD4+ têm pouca ou nenhuma participação no controle da infecção. No entanto, nos animais A/J há duas subpopulações, uma protetora (4ª semana) e outra exacerbadora (8ª semana) dependendo do estágio da doença. Contudo, em ambas as linhagens a subpopulação TCD8+ apresentou-se importante no controle da doença. Além disso, na PCM experimental, tanto as respostas de HTT como a produção de anticorpos específicos, são mecanismos que dependem de células TCD4+ e na linhagem B10.A há uma subpopulação TCD8+ que regula negativamente as reações de HTT.
To investigate the role of T lymphocytes in the pulmonary model of Paracoccidioidomycosis (PCM), resistant and susceptible mice were in vivo depleted of T CD4+ and T CD8+ cells by intraperitoneal injection of specific monoclonal (mAb) antibodies and infected intratracheally with one million yeast cells of a virulent isolate of Paracoccidioides brasiliensis. When compared with the non-depleted group, at week 4 after infection A/J mice presented increased dissemination of yeasts to liver; however, at week 8 A/J-depleted mice showed decreased fungal loads in the lungs. In contrast, depletion of TCD4+ cells of B10.A mice did not alter the severity of disease at any periods of infection assayed. Treatment with anti-CD4 mAb diminished the delayed-type hypersensitivity reactions (DTH) of resistant mice but the cutaneous anergy of B10.A mice was not modified. In addition, CD4-depleted A/Sn and B10.A mice presented decreased titers of P.brasiliensis specific antibodies at both the 4th and 8th week postinfection. Regarding pulmonary cytokines, at week 4 of infection A/J-depleted mice presented diminished levels of IL-12 but at week 8 IL-10, IL-4, IL-S, IL-2 and GM-CSF appeared in lower levels. Only IL-12 was detected in lower levels in the lungs of B10.A-depleted mice at week 8 after infection. Depletion of CD8+ cells led to a more severe disease in both mouse strains. A/J-treated mice presented increased fungal burdens in the lungs whereas in the B10.A strain increased number of yeast cells was detected in the lungs and liver. Importantly, neutralization of CD8+ cells reverted the DTH anergy of susceptible mice. These data suggest the existence of two T CD8+ subpopulations in B10.A mice, a protective that controls fungal growth and another one that suppresses DTH reactions. The production of P.brasiliensis specific antibodies by resistant and susceptible mice depleted of CD8+ T cells was similar to that of mice given control antibody. Neutralization of CD8+ cells, however, induced significant alterations in the concentrations of pulmonary cytokines. In A/J-treated mice, higher levels of IL-4, IL-12, IL-3 and GM-CSF were concomitant with reduced amounts of IL-2. B10.A mice depleted of CD8+ cells presented higher levels of pulmonary IL-10, IL-12, IL-3 and IFN-γ than their controls. As a whole, our results demonstrate that CD4+ T cells have no influence on the control of disease severity of B10.A mice. Depending on the period of the infection, A/J mice develop two subpopulations of CD4+ T cells: one protective subset which appeared early in the infection, followed by a subpopulation that lead to disease exacerbation. Moreover, in both mouse strains CD8+ T cells are protective and able to control fungal growth. It was also verified that DTH reactions and antibody production in murine PCM are CD4+ T cells mediated. Finally, only in B10.A mice a regulatory CD8+ T cell subpopulation was characterized by its ability to suppress DTH reactions

L’inflammasome NLRP3 est un complexe multiprotéique responsable notamment de la production d’IL-1β, une cytokine inflammatoire. Les effets délétères de l’inflammasome NLRP3 ont été démontrés dans de nombreuses maladies dont le cancer. Ce travail se concentre sur les effets de NLRP3 dans le contexte du cancer.Dans un premier projet, j’ai étudié l’activation de l’inflammasome NLRP3 dans les MSDC après un traitement par chimiothérapie. Deux chimiothérapies, le 5-Fluorouracile et la Gemcitabine, sont capables d’éliminer de façon spécifique les MDSC, population de cellules immunosuppressives dont la taille augmente en cas de cancer. J’ai découvert que le 5-Fluorouracile et la Gemcitabine activaient l’inflammasome NLRP3 dans les MDSC. En effet, le 5-Fluorouracile et la Gemcitabine provoquent la perméabilisation du lysosome des MDSC, permettant la sortie de la cathepsine B, protéine lysosomale, dans le cytoplasme où elle interagit directement avec NLRP3. Cette interaction active l’inflammasome NLRP3 et la production d’IL-1β. Cette IL-1β est responsable du développement d’une nouvelle population immunosuppressive, les Th17.J’ai ensuite étudié le rôle de NLRP3 dans la différenciation des lymphocytes T CD4 Th2. Dans ces cellules, le rôle de NLRP3 s’effectue indépendamment du reste du complexe multiprotéique qui forme l’inflammasome. Après avoir été induit par la cascade de signalisation de l’IL-2, NLRP3 interagit avec IRF4 (interferon regulatory factor) et agit comme un facteur de transcription sur le promoteur du gène de l’IL-4. L’absence de NLRP3 a pour conséquence une production moins importante d’IL-4 par les Th2 qui sont alors moins fonctionnels
The inflammasome NLRP3 (NOD like receptor pyd containing 3) is a multiprotein complex notably responsible for IL-1β (interleukine-1β) production, an inflammatory cytokine. Negative effects have been observed in various diseases including cancer. My thesis focuses on the effects of NLRP3 in cancer.In my first project, I studied the NLRP3 inflammasome activation in MDSC (myeloïd derived suppressor cells) after a chemotherapy treatment. Two chemotherapies, 5-Fluorouracil and Gemcitabine, are selectively able to kill MDSC, an immunosuppressive population growing during cancer evolution. MDSC’s death restores anti-tumor immunity for a while but another immunosuppressive population is established by MDSC produced IL-1β before their disappearance. I discovered that 5-Fluorouracil and Gemcitabine trigger NLRP3 inflammasome activation in MDSC. 5-Fluorouracil and Gemcitabine induce lysosomal permeabilisation, allowing for Cathepsin B release into the cytoplasm where it directly interacts with NLRP3. That interaction activates the inflammasome and induces IL-1β production which is responsible for the development of another immunosuppressive population, called Th17 cells.I then studied the role of NLRP3 during Th2 differentiation. Here, NLRP3 actions are done independently of the other inflammasome forming proteins. After being induced by IL-2 signalization pathway, NLRP3 interacts with IRF4 (interferon regulatory factor 4) and acts as a transcription factor on the IL-4 promoter gene. Lack of NLRP3 leads to a smaller IL-4 production by Th2 cells which are consequently less functional

INTRODUÇÃO: As infecções causadas por bactérias ou vírus são frequentes em pacientes com imunodeficiência comum variável (ICV) devido à deficiência de anticorpos e associação com alteração da função das células T. OBJETIVOS: Avaliar o efeito da ativação de receptores Toll-like (TLR) utilizando ligantes de TLRs em células T monofuncionais ou polifuncionais em pacientes com ICV. MÉTODOS: Foram selecionados 16 pacientes com ICV do Ambulatório de Manifestações Dermatológicas das Imunodeficiências Primárias ADEE3003 HC-FMUSP e 16 controles saudáveis. Os métodos utilizados de citometria de fluxo foram: a) análise em sangue periférico de linfócitos B, linfócitos T quanto ao perfil de ativação/maturação, linfócitos T foliculares (Tfh) e células T reguladoras (Treg); b) dosagem de citocinas e quimiocinas em amostras de soro e em sobrenadante de culturas de células mononucleares do sangue periférico (CMNs) estimuladas com agonistas de TLRs; c) avaliação das células TCD4+ mono e polifuncionais secretoras de IL-17a, IL-22, TNF, IFN- e IL-10, e expressão de marcador de ativação crônica de CD38 estimuladas por agonistas de TLR2, TLR3 e TLR7/8 e estímulos policlonais como enterotoxina B de Staphylococcus aureus (SEB) e acetato miristato de forbol e ionomicina (PMA/IONO); d) células Th22 e Tc22 estimuladas com TLR e SEB. RESULTADOS: Na ICV, os linfócitos B do sangue periférico mostram diminuída frequência, sendo em maior frequência de linfócitos B naïve (CD19+IgD+CD27-), e ausência de células B de memória. Além disto, um aumento na expressão do marcador de exaustão PD-1 foi observado nas células TCD4+ de memória efetora (CD45RA-CCR7-) e na expressão de CD38, em células TCD8+ terminalmente diferenciadas (CD45RA + CCR7-). Em contraste, houve diminuição na frequência de células T reguladoras naïve nos pacientes com ICV. Nos indivíduos com ICV foi observado aumento na frequência de células TCD4+ TNF+ sob estímulo TLR2 e TLR7/8 comparado ao grupo controle, enquanto que sob estímulo com PMA/IONO houve menor frequência de células TCD4+ e TCD8+ secretoras de IFN-y IL-17a, IL-22 ou TNF. Já em células TCD8+ houve importante redução na ativação via TLR3 na resposta de IL-22, IFN-y e IL-17a. Contudo, os estímulos com TLR7/8 e SEB foram capazes de aumentar a frequência de células Th22 e Tc22 nos pacientes com ICV. Em geral, as células TCD4+, que secretam simultaneamente 4 a 5 citocinas induzidas por TLR foram preservadas em ICV. Embora as células TCD4+ polifuncionais secretoras de 3 citocinas, foram capazes de responder a estímulos via TLR2 e TLR7/8, as células TCD8+ não responderam para qualquer estímulo via TLRs. Além disso, as células T que expressam CD38 mostraram menor polifuncionalidade aos estímulos via TLRs e PMA/IONO. O perfil inflamatório nos pacientes com ICV foi observado pela elevação sérica de IL-6, CCL-2, CCL-5, CXCL8, CXCL-9, CXCL-10. Alteração na resposta aos agonistas de TLRs em ICV pode ser observada com a ativação dos agonistas de TLRs em CMNs, que mostrou maior produção de TNF e diminuição de CCL2 e CXCL8 após ativação via TLR4. Em contraste, o agonista de TLR7/8, teve ação oposta induzindo CXCL10 e reduzindo os níveis de CXCL9. Chama atenção no ICV, à reduzida secreção de IFN-alfa induzida por TLR7/8, que não foi observada com a ativação via TLR9. CONCLUSÕES: Até o momento, os achados em ICV mostram alterações nas células T, seja quanto à baixa frequência de células T reguladoras naïve e a reduzida resposta efetora, em especial das células TCD8+. Contudo, enfatiza o potencial de adjuvante dos agonistas de TLR7/8 na ativação das células T
INTRODUCTION: Infections caused by bacteria or viruses are common in patients with Common Variable Immunodeficiency (CVID), due to antibody deficiency and association with altered function of T cells. OBJECTIVES: To evaluate the effect of Toll-like receptors (TLR) activation using TLR agonists on the monofunctional or polyfunctional T cells in patients with CVID. METHODS: We selected 16 patients with ICV from the Dermatologic Manifestations of Primary Immunodeficiencies Clinic ADEE3003 HC-FMUSP and 16 healthy controls. The methods used for flow cytometry were: a) analysis of peripheral blood B lymphocytes, T lymphocytes were assessed by the activation/maturation profile, follicular T cells (Tfh) and regulatory T cells (Treg); b)evaluation of cytokines and chemokines serum levels and in supernatants of mononuclear cell cultures from peripheral blood (PBMC) stimulated with TLR agonists; c) evaluation of mono and polyfunctional CD4+ T cells secreting IL-17a, IL-22, TNF, IFN-y and IL-10, and expression chronic activation marker of CD38 stimulated by agonists of TLR2, TLR3 and TLR7/8 and polyclonal stimuli such as Staphylococcus aureus enterotoxin B (SEB) and phorbol myristate acetate and ionomycin (PMA / IONO); d) analysis of Tc22 and Th22 cells stimulated with TLR and SEB. RESULTS: In the CVID, the peripheral blood B cells show decreased frequency, being higher frequency of naïve B cells (IgD+ CD19+ CD27-) and lack of memory B cells. Moreover, an increased expression of PD-1, an exhaustion marker, was detected in the CD4+ T cell effector memory (CD45RA- CCR7-) and expression of CD38 on CD8+ T terminally differentiated cells (CD45RA+ CCR7-). In contrast, a decreased frequency of naïve regulatory T cells was detected in the patients with CVID. In CVID patients it was observed increased frequency of T CD4+ TNF+ cells upon TLR2 and TLR7/8 agonists compared to the control group, while under stimulation with PMA /IONO there was a lower frequency of CD4+ and CD8+T cells secreting IFN-y, IL-17a, IL-22 or TNF. The CD8+T cells showed a significant reduction of in the IL-22 response, IFN-? and IL-17a induced by TLR3 activation. However, stimulation with TLR7/8 and SEB were able to increase the frequency of Th22 and TC22 cells in the patients with CVID. In CVID patients it was observed increased frequency of T CD4+ TNF+ cells upon TLR2 and TLR7/8 agonists compared to the control group, while under stimulation with PMA /IONO there was a lower frequency of CD4+ and CD8+ T cells secreting IFN-y, IL-17a, IL-22 or TNF. The CD8+ T cells showed a significant reduction of in the IL-22 response, IFN-y and IL-17a induced by TLR3 activation. However, stimulation with TLR7/8 and SEB were able to increase the frequency of Th22 and TC22 cells in the patients with CVID. In general, CD4+ T cells that secrete simultaneously 4 to 5 cytokines induced by TLR were preserved in CVID. Although polyfunctional CD4+ T cells secreting 3 cytokines were able to respond to TLR2 and TLR7/8 agonists, the CD8+ T cells did not respond to any stimuli. In addition, T cells expressing CD38, showed lower polyfunctionality to the stimuli via TLRs and PMA/IONO. Furthermore, the inflammatory status in the patients with CVID was observed by the increased serum levels of IL-6, CCL-2, CCL-5, CXCL8, CXCL-9, CXCL-10. In contrast, the agonist of TLR7/8 had opposite action inducing CXCL10 and reducing the CXCL9 levels. Noteworthy in CVID, that the reduced secretion of IFN-alfa induced by TLR7/8 was not observed with TLR9 activation. CONCLUSIONS: To date, the CVID findings shows alterations in the T cells, as the low frequency of naïve regulatory T cells and reduced effector response, mainly of CD8+ T cells. However, it emphasizes the adjuvant potential of the TLR7/8 agonist in the T cells activation

L'allogreffe hématopoïétique permet de traiter des maladies cancéreuses de nature hématologique. Mais les lymphocytes T allogéniques présents dans le greffon hématopoïétique sont responsables d'une complication majeure : la réaction du greffon contre l'hôte (GVH). Le développement de nouvelles approches thérapeutiques pour moduler l'alloréactivité des lymphocytes T du greffon est donc nécessaire pour prévenir la GVH. Ce travail montre que la molécule CD45RC exprimée par les lymphocytes T chez le rat et chez l'homme, permet de distinguer des sous populations T ayant des propriétés alloréactives différentes. Les cellules T CD45RChigh contiennent des cellules alloréactives responsables de l'apparition de la GVH, tandis que les cellules T CD45RClow contiennent des cellules régulatrices capables de la contrôler. Ce travail suggère que la molécule CD45RC pourrait être un marqueur prédictif de l'évolution de la GVH chez l'homme, améliorant l'efficacité des allogreffes hématopoïétiques
Haematopoietic allograft is the most powerful treatment against haematological malignancies. But allogeneic mature T lymphocytes present in the haematopoietic transplant are responsible for a major complication: the graft against the host (GVHD). The development of new strategies to modulate the alloreactivity of T cells in the transplant is thus necessary to prevent GVHD. This work shows that CD45RC expressed by T lymphocytes in the rat and in the humans, allows to distinguish distinct T cell subsets with different alloreactive functions. The CD45RChigh subset contains alloreactive lymphocytes responsible for GVHD, while the CD45RClow subset contains regulatory T lymphocytes capable of controlling this disease. This work suggests that D45RC could be a predictive marker of the GVHD evolution, improving the efficiency of this treatment

La réponse immunitaire anti-CMVH est protectrice et implique les différents effecteurs de la réponse immunitaire cellulaire. Nous avons, dans un but vaccinal, potentialisé la réponse TCD4+ dirigée contre la protéine IE1 grâce à l'utilisation d'une protéine de localisation cytoplasmique et de petite taille. Cette réponse est due à une présentation endogène de cette protéine suivie d'une présentation exogène à des temps tardifs d'infection. Enfin, cette présentation est dépendante du protéasome et des compartiments acides. Nous avons également montré que les clones de lymphocytes TCD4+ anti-IE1 pouvaient exercer une fonction cytotoxique sur des cibles sensibilisées par un peptide mais pas sur des cellules infectées. De plus, la réponse cytotoxique contre le peptide est inhibée par l'infection, ce qui suggère un mécanisme actif d'échappement à cette réponse par le CMVH. Il existe donc un équilibre entre l'immunité anti-CMVH et son inhibition au cours de l'infection
Human cytomegalovirus (HCMV) infects 50-100 % of the world population without overt symptoms but is pathogenic in immunosuppressed individuals. Cellular immunity plays a major role in the control of HCMV infection. Our data show potentiation of the CD4+ T cell response to a specific epitope of IE1 protein through shortening and relocation of an otherwise nuclear protein and suggest applications in vaccination. This response was due to endogenous presentation followed by exogenous presentation at later time points. Presentation was dependent on both proteasome and acidic compartments. We have also shown that although IE1-specific CD4+T cell clones were cytotoxic against peptide-pused targets, their cytotoxicity was undetectable on infected targets. The main reason for this lack of cytotocixity was found to be the selective inhibition of cytotoxicity of infected targets whereas IFN-g production was not impaired. Our data describe for the first the inhibition of cytotoxicity of CD4+ T lymphocytes against infected targets

Au cours de ma these, je me suis attachee a etudier le role des lymphocytes t auxiliaires chez des souris balb/c infectees par l. Major. Dans un premier temps, j'ai montre que l'antigene parasitaire lack constituait une des cibles preferentielles de la reponse immunitaire chez les souris infectees. J'ai ensuite demontre que les lymphocytes t anti-lack jouaient un role determinant dans la susceptibilite au parasite. Ainsi, le simple fait de rendre des souris balb/c tolerantes pour lack est suffisant pour permettre a ces souris d'eliminer le parasite. Dans un deuxieme temps, j'ai essaye de comprendre ce qui differencie les lymphocytes t anti-lack de ceux reagissant avec d'autres antigenes parasitaires. Je me suis egalement interessee aux mecanismes d'action de ces cellules et aux raisons pour lesquelles elles jouent un role determinant dans la susceptibilite a l. Major. Parallelement au groupe de jacques louis, j'ai montre que les lymphocytes t anti-lack produisent tres rapidement de l'il-4 chez les souris balb/c infectees par l. Major. Cette reponse acceleree est due a l'existence d'un phenomene de mimetisme moleculaire entre lack et des antigenes de la flore intestinale. En effet, les lymphocytes t anti-lack reagissent avec des extraits bacteriens, et l. Major n'induit pas la production rapide d'il-4 chez des souris balb/c depourvues de germes. Toutefois, la production rapide d'il-4 par les lymphocytes t anti-lack ne permet pas d'expliquer le role determinant de ces cellules dans la susceptibilite au parasite. En effet, des souris balb/c depourvues de germes restent susceptibles a l. Major, et les lymphocytes t anti-lack produisent rapidement de l'il-4 chez les souris b10. D2 qui sont resistantes.

Au cours d'une greffe de cellules souches hématopoïétiques allogéniques, le greffon contient des lymphocytes T (LyT) qui exercent des effets bénéfiques mais peuvent aussi induire la maladie du greffon contre l'hôte (GVH). Le traitement de la GVH repose sur un régime immunosuppresseur immunologiquement non spécifique et partiellement efficace. Le laboratoire s'est intéressé aux LyT régulateurs (Treg) et a développé des Treg spécifiques du receveur (rsTreg) qui contrôlent la GVH tout en favorisant la reconstitution immunitaire. Dans ces modèles, les souris ayant un thymus fonctionnel, la reconstitution immunitaire observée peut dériver de la thymopoïèse ou de la prolifération périphérique des LyT du donneur présents dans le greffon. Chez l'Homme adulte, du fait de l'involution thymique, la reconstitution immunitaire repose principalement sur la prolifération périphérique des LyT du donneur, dans les premiers temps qui suivent la greffe. La fonctionnalité de ces LyT du donneur est donc essentielle à la survie des patients. Avant toute utilisation clinique des rsTreg, il était donc important de déterminer leur impact sur les LyT du donneur. Nous avons utilisé un modèle de GVH contrôlé par les rsTreg, dans lequel les LyT du donneur sont la seule source de reconstitution T et nous avons montré que chez les souris protégées de la GVH par les rsTreg, la reconstitution immunitaire dérivée des LyT du donneur reste limitée en nombre mais fonctiohnelle. Les LyT sont, en effet, capables de répondre à des antigènes viraux ou des alloantigènes. Ces résultats suggèrent que les rsTreg peuvent être considérés comme une option thérapeutique sûre pour le traitement de la GVH
During allogeneic hematopoietic stem cell transplantation, the graft contains donor T cells that have beneficial effects but can also induce graft versus host disease (GVHD). The treatment for GVHD consists in an immunosuppressive regimen that remains immunologically non-specific and is only partially effective. We have proposed that regulatory T cells (Treg) could be used for GVHD prophylaxis and developed recipient-specific Treg (rsTreg) that are able to control GVHD while favoring immune reconstitution. In these models, grafted mice having a functional thymus, immune reconstitution could either be derived from thymic differentiation or alternatively from the peripheral expansion of donor T cells from the graft. In human adults, due to thymic involution, the immune reconstitution mainly relies on the peripheral expansion of the mature donor T cells present in the graft, during the first time post-HSCT. Thus, the functionality of these donor T cells is very important for the survival of the patient. Importantly, before any clinical use of rsTreg, it was essential to determine the impact of these therapeutic cells on the remaining donor T cells. To answer this question, we used a GVHD model in which donor T cells present in the graft are the sole source for T cell reconstitution and where GVHD is controlled by rsTreg. In this study, we show that in mice protected by rsTreg,;the immune reconstitution derived from the donor T cells initially present in the graft, remained limited in number but functional because T cells were capable to respond to viral or allogeneic antigens. These results suggest that rsTreg may be a safe therapeutic option for the treatment of GVHD

Les lymphocytes TCD4 (LTCD4) auxiliaires ont un rôle essentiel dans la coordination de la réponse immunitaire alors que les lymphocytes T régulateurs (Treg) par leurs capacités immunosuppressives en assurent le contrôle ou la régulation. Les propriétés de ces différentes sous-populations de LTCD4 ont été exploitées dans des protocoles d’immunothérapie adoptive montrant des résultats prometteurs. Actuellement, la principale limite de ces stratégies thérapeutiques est le manque de méthodes permettant d’expandre à large échelle des LTCD4 spécifiques d’antigène (Ag). Dans ce contexte, notre équipe a mis au point à partir de fibroblastes murins, un modèle de cellules présentatrices d’antigène artificielles (CPAA) exprimant les molécules HLA de classe II ainsi que les molécules humaines de costimulation B7. 1 et d’adhérence ICAM-1 et LFA-3, essentielles à l'activation lymphocytaire. Nos CPAA HLA-DR s’avèrent efficaces pour présenter des Ag sous forme peptidique ou protéique. En culture primaire la stimulation par les CPAA HLA-DR de LTCD4 de sujets sains induit des LTCD4 spécifiques de l’Ag d’intérêt (l'hémagglutinine). Nous observons également un fort contingent de LTCD4 non spécifiques reconnaissant probablement des peptides provenant d'Ag murins. Toutefois, les CPAA HLA-DR sont plus efficaces que des CPA autologues pour restimuler des LTCD4 mémoires de type Th1. Notre protocole d’expansion par les CPAA HLA-DR, d’effecteurs TCD4 initialement stimulés in vitro par des CPA autologues, permet de générer un nombre de LTCD4 spécifiques compatible pour une immunothérapie adoptive, environ 10 esposant 9 cellules à partir d’une poche de sang. En conclusion, bien que la présentation des Ag par les CPAA HLA-DR reste à optimiser, notre modèle représente un outil intéressant pour identifier des épitopes d’intérêt et expandre des LTCD4 spécifiques. Ce modèle de CPAA HLA-DR constitue une plateforme pouvant être adapté à la stimulation de différentes sous-populations de LTCD4, notamment les Treg, au travers de modifications par transfert de gènes codant d’autres molécules de costimulation ou des cytokines spécifiques
Helper CD4 T cells have a key role in coordinating the immune response while regulatory T cells (Tregs) control or regulate it by their immunosuppressive abilities. These different CD4 T cell subpopulations are of interest for adoptive immunotherapy protocols that show promising results. Currently, the main limitation of these therapeutic strategies is the lack of reliable methods for large-scale expansion of antigen (Ag) specific CD4 T cells. In this context, our team has developed a model of artificial antigen presenting cells (AAPC) derived from mouse fibroblasts, expressing HLA class II molecules but also human costimulatory B7. 1 and adhesion ICAM-1 and LFA-3 molecules, essential for lymphocyte activation. Our AAPC HLA-DR are effective to present AG in peptide or protein form. In primary culture, stimulation by AAPC HLA-DR of CD4 T cells isolated from healthy donors induces specific CD4 T cells of Ag of interest (hemagglutinin). We also observe a strong population of non-specific CD4 T cells probably recognizing peptides from murine origin. However, AAPC HLA-DR were more effective than autologous APC to re-stimulate memory CD4 T cells with Th1 profile. Our expansion protocol of specific CD4 T cells using amplification by AAPC HLA-DR of CD4 T cell primed by autologous APC, was able to generate a number of cells compatible with adoptive immunotherapy, around 10 exposant 9 cells from a blood bag. In conclusion, although the Ag presentation by AAPC HLA-DR remains to be optimized, our model represents a useful tool to identify epitopes and to expand specific CD4 T cells. This AAPC HLA-DR model is customizable for the stimulation of different subpopulations of CD4 T cells, including Tregs, through gene transfer of adapted co-stimulatory molecules or specific cytokines