Academic literature on the topic 'Heterologous Immunity'

Heterologous immunity, or protection by one invading organism against another across phylogenetic divides, has been recognised for decades. It was initially thought to operate largely through enhancement of phagocytosis, but this explanation became untenable when it was realised it worked extremely well against intraerythrocytic protozoa and killed them while they were free in the circulation. Clearly a soluble mediator was called for. This review summarises the logic that arose from this observation, which led to a wider appreciation of the roles of pro-inflammatory cytokines, and then nitric oxide, in the host's response against invaders, as well as the ability of these mediators to harm the host itself if they are generated too enthusiastically. This has led to a discernable pattern across heterologous immunity as a whole, and its lessons influence a range of areas, including vaccine development.

Clinical manifestations of viral infections are highly variable, both in type and severity, among individual patients. Differences in host genetics and in dose and route of infection contribute to this variability but do not fully explain it. New studies now show that each subject's history of past infections individualizes the memory T cell pool. Private T cell receptor specificities of these preexisting memory T cell populations influence both disease severity and outcome of subsequent, unrelated virus infections.

The dynamics of T cell responses have been extensively studied during single virus infection of naïve mice. During a viral infection, viral antigen is presented in the context of MHC class I molecules on the surface of infected cells. Activated CD8 T cells that recognized viral antigens mediate clearance of virus through lysis of these infected cells. We hypothesize that the balance between the replicating speed of the virus and the efficiency at which the T cell response clears the virus is key in determining the disease outcome of the host. Lower T cell efficiency and delayed viral clearance can lead to extensive T cellmediated immunopathology and death in some circumstances. To examine how the efficiency of the immune response would impact immunopathology we studied several viral infection models where T cell responses were predicted to be less than optimal: 1. a model of co-infection with two viruses that contain a crossreactive epitope, 2. a viral infection model where a high dose infection is known to induce clonal exhaustion of the CD8 T cell response, 3. a neonatal virus infection model where the immune system is immature and 4. A model of beneficial heterologous immunity and T cell crossreactivity where mice are immunized as neonates when the T cell pool is still developing. Model 1. Simultaneous co-infections are common and can occur from mosquito bites, contaminated needle sticks, combination vaccines and the simultaneous administration of multiple vaccines. Using two distantly related arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV), we questioned if immunological T cell memory and subsequent protection would be altered following a simultaneous co-infection, where two immune responses are generated within the same host at the same time. Coinfection with these two viruses, which require CD8 T cell responses to clear, resulted in decreased immune protection and enhanced immunopathology after challenge with either virus. After primary co-infection, each virus-specific immune response impacted the other as they competed within the same host and resulted in several significant differences in the CD8 T cell responses compared to mice infected with a single virus. Co-infected mice had a dramatic decrease in the overall size of the LCMV-specific CD8 T cell response and variability in which virus-specific response dominated, along with skewing in the immunodominance hierarchies from the normal responses found in single virus infected mice. The reduction in the number of LCMV-specific CD8 memory T cells, specifically cells with an effector memory-like phenotype, was associated with higher viral loads and increased liver pathology in co-infected mice upon LCMV challenge. The variability in the immunodominance hierarchies of co-infected mice resulted in an enhanced cross-reactive response in some mice that mediated enhanced immune-mediated fat pad pathology during PICV challenge. In both viral challenge models, an ineffective memory T cell response in co-infected mice facilitated increased viral replication, possibly leading to enhanced and prolonged accumulation of secondary effector T cells in the tissues, thereby leading to increased immune pathology. Thus, the magnitude and character of memory CD8 T cell responses in simultaneous co-infections differed substantially from those induced by single immunization. This has implications for the design of combination vaccines and scheduling of simultaneous immunizations. Model 2. The balance between protective immunity and immunopathology often determines the fate of the virus-infected host. Several human viruses have been shown to induce a wide range of severity of disease. Patients with hepatitis B virus (HBV), for example, show disease progression ranging from acute resolving infection to a persistent infection and fulminant hepatitis. Certain rapidly replicating viruses have the ability to clonally exhaust the T cell response, such as HBV and hepatitis C virus (HCV) in humans and the clone 13 strain of LCMV in mice. How rapidly virus is cleared is a function of initial viral load, viral replication rate, and efficiency of antigen-specific T cells. By infecting mice with three different inocula of LCMV clone 13, we questioned how the race between virus replication and T cell responses could result in different disease outcomes. A low dose of LCMV generated efficient CD8 T effector cells, which cleared the virus with minimal lung and liver pathology. A high dose of LCMV resulted in clonal exhaustion of T cell responses, viral persistence and little immunopathology. An intermediate dose only partially exhausted the CD8 T cell responses and was associated with significant mortality, and the surviving mice developed viral persistence and massive immunopathology, including necrosis of the lungs and liver. This was a T cell-mediated disease as T cell-deficient mice had no pathology and became persistently infected like mice infected with a high dose of LCMV clone 13. This suggests that for non-cytopathic viruses like LCMV, HCV and HBV, clonal exhaustion may be a protective mechanism preventing severe immunopathology and death. Model 3. Newborns are more susceptible to infections due to their lack of immunological memory and under-developed immune systems. Passive maternal immunity helps protect neonates until their immune systems have matured. We questioned if a noncytolytic virus that produces strong T cell responses in adult mice would also induce an equally effective response in neonatal mice. Neonates were infected with very low doses of LCMV Armstrong and surprisingly the majority succumbed to infection between days 7-11, which is the peak of the T cell response in adult mice infected with LCMV. Death was caused by T cell-dependent pathology and not viral load as 100% of T cell deficient neonates survived with minimal lung and liver pathology. This is similar to the adult model of medium dose LCMV clone 13, but T cell responses in neonates were not partially clonal exhausted. Furthermore, surviving neonates were not persistently infected, clearing virus by day 14 post infection. In adult mice direct intracranial infection leads to LCMV replication and CD8 T cell infiltration in the central nervous system (CNS), causing CD8 T cell-mediated death. However, this does not occur in adults during LCMV intraperitoneal (ip) infections. We questioned if unlike adults LCMV could be gaining access to the CNS in neonates following ip infection. Replicating LCMV was found in the brain of neonates after day 5 post infection along with virus-specific CD8 T cells producing IFNγ at day 9 post infection. Neonates lacking perforin had complete survival when followed until day 14 post infection, suggesting perforin-mediated T cell-dependent immunopathology within the CNS of neonates was causing death after LCMV infection. Passive immunity from LCMV-immune mothers also protected 100% of pups from death by helping control viral load early in infection. We believe that the maternal antibody compensates for the immature innate immune response of neonates and controls viral replication early so the neonatal T cell response induced less immunopathology. Neonates are commonly thought to have less functional immune systems, but these results show that neonates are capable of producing strong T cell responses that contribute to increased mortality. Model 4. Due to their enhanced susceptibility to infection neonatal and infant humans receive multiple vaccines. Several non-specific effects from immunizations have been observed, for example, measles or Bacillus Calmette- Guerin (BCG) vaccines have been linked to decreased death of children from infections other than measles virus or tuberculosis. These studies mirror the concepts of beneficial heterologous immunity, where previous immunization with an unrelated pathogen can result in faster viral clearance. LCMV-immune mice challenged with vaccinia virus (VV) have lower viral loads then naïve mice and survive lethal infections, but some mice do develop fat pad immunopathology in the form of panniculitis or acute fatty necrosis (AFN). We questioned how immunological T cell memory formed during the immature neonatal period would compare to memory generated in fully mature adults during a heterologous viral challenge. Mice immunized as neonates had comparable reduction in VV load and induction of AFN, indicating that heterologous immunity is established during viral infections early in life. Interestingly, the LCMV-specific memory populations that expanded in mice immunized as neonates differed from that of mice immunized as adults. In adult mice 50% of the mice have an expansion of LCMVNP205- specific CD8 T cells while the majority of neonates expanded the LCMVGP34- specific CD8 T cell pool. This alteration in dominant crossreactivities may be due to the limited T cell receptor repertoire of neonatal mice. In naïve neonatal mice we found altered Vβ repertoires within the whole CD8 T cell pool. Furthermore, there was altered Vβ usage within virus-specific responses compared to adult mice and a wide degree of variability between individual neonates, suggesting enhanced private specificity of the TCR repertoire. Beneficial heterologous immunity is maintained in neonates, but there was altered usage of crossreactive responses. As neonatal mice were found to be so sensitive to LCMV infection we questioned if neonates could control another arena virus that did not replicate as efficiently in mice, PICV. Unlike LCMV infection, neonatal mice survived infection with PICV even with adult-like doses. However, viral clearance was protracted in neonates compared to adults, but was cleared from fat pad and kidney by day 11 post infection. The peak of the CD8 T cell response was similarly delayed. PICV infected neonates showed dose-dependent PICV-specific CD8 T cell responses, which were similar to adult responses by frequency, but not total number. As with LCMV infection there were changes in immunodominance hierarchies in neonates. Examination of the immunodominance hierarchies of PICV-infected neonates showed that there were adult-like responses to the dominant NP38- specific response, but a loss of the NP122-specific response. Six weeks post neonatal infection mice were challenged with LCMV Armstrong and there was a strong skewing of the PICV immunodominance hierarchy to the crossreactive NP205-specific response. These data further support the hypothesis that heterologous immunity and crossreactivity develop following neonatal immunization, much as occurs in adults, although TCR repertoire and crossreactive patterns may differ. Changing the balance between T cell efficiency and viral load was found to altered the severity of the developing immunopathology after viral infection.

The dynamics of T cell responses have been extensively studied during single virus infection of naïve mice. During a viral infection, viral antigen is presented in the context of MHC class I molecules on the surface of infected cells. Activated CD8 T cells that recognized viral antigens mediate clearance of virus through lysis of these infected cells. We hypothesize that the balance between the replicating speed of the virus and the efficiency at which the T cell response clears the virus is key in determining the disease outcome of the host. Lower T cell efficiency and delayed viral clearance can lead to extensive T cellmediated immunopathology and death in some circumstances. To examine how the efficiency of the immune response would impact immunopathology we studied several viral infection models where T cell responses were predicted to be less than optimal: 1. a model of co-infection with two viruses that contain a crossreactive epitope, 2. a viral infection model where a high dose infection is known to induce clonal exhaustion of the CD8 T cell response, 3. a neonatal virus infection model where the immune system is immature and 4. A model of beneficial heterologous immunity and T cell crossreactivity where mice are immunized as neonates when the T cell pool is still developing. Model 1. Simultaneous co-infections are common and can occur from mosquito bites, contaminated needle sticks, combination vaccines and the simultaneous administration of multiple vaccines. Using two distantly related arenaviruses, lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PICV), we questioned if immunological T cell memory and subsequent protection would be altered following a simultaneous co-infection, where two immune responses are generated within the same host at the same time. Coinfection with these two viruses, which require CD8 T cell responses to clear, resulted in decreased immune protection and enhanced immunopathology after challenge with either virus. After primary co-infection, each virus-specific immune response impacted the other as they competed within the same host and resulted in several significant differences in the CD8 T cell responses compared to mice infected with a single virus. Co-infected mice had a dramatic decrease in the overall size of the LCMV-specific CD8 T cell response and variability in which virus-specific response dominated, along with skewing in the immunodominance hierarchies from the normal responses found in single virus infected mice. The reduction in the number of LCMV-specific CD8 memory T cells, specifically cells with an effector memory-like phenotype, was associated with higher viral loads and increased liver pathology in co-infected mice upon LCMV challenge. The variability in the immunodominance hierarchies of co-infected mice resulted in an enhanced cross-reactive response in some mice that mediated enhanced immune-mediated fat pad pathology during PICV challenge. In both viral challenge models, an ineffective memory T cell response in co-infected mice facilitated increased viral replication, possibly leading to enhanced and prolonged accumulation of secondary effector T cells in the tissues, thereby leading to increased immune pathology. Thus, the magnitude and character of memory CD8 T cell responses in simultaneous co-infections differed substantially from those induced by single immunization. This has implications for the design of combination vaccines and scheduling of simultaneous immunizations. Model 2. The balance between protective immunity and immunopathology often determines the fate of the virus-infected host. Several human viruses have been shown to induce a wide range of severity of disease. Patients with hepatitis B virus (HBV), for example, show disease progression ranging from acute resolving infection to a persistent infection and fulminant hepatitis. Certain rapidly replicating viruses have the ability to clonally exhaust the T cell response, such as HBV and hepatitis C virus (HCV) in humans and the clone 13 strain of LCMV in mice. How rapidly virus is cleared is a function of initial viral load, viral replication rate, and efficiency of antigen-specific T cells. By infecting mice with three different inocula of LCMV clone 13, we questioned how the race between virus replication and T cell responses could result in different disease outcomes. A low dose of LCMV generated efficient CD8 T effector cells, which cleared the virus with minimal lung and liver pathology. A high dose of LCMV resulted in clonal exhaustion of T cell responses, viral persistence and little immunopathology. An intermediate dose only partially exhausted the CD8 T cell responses and was associated with significant mortality, and the surviving mice developed viral persistence and massive immunopathology, including necrosis of the lungs and liver. This was a T cell-mediated disease as T cell-deficient mice had no pathology and became persistently infected like mice infected with a high dose of LCMV clone 13. This suggests that for non-cytopathic viruses like LCMV, HCV and HBV, clonal exhaustion may be a protective mechanism preventing severe immunopathology and death. Model 3. Newborns are more susceptible to infections due to their lack of immunological memory and under-developed immune systems. Passive maternal immunity helps protect neonates until their immune systems have matured. We questioned if a noncytolytic virus that produces strong T cell responses in adult mice would also induce an equally effective response in neonatal mice. Neonates were infected with very low doses of LCMV Armstrong and surprisingly the majority succumbed to infection between days 7-11, which is the peak of the T cell response in adult mice infected with LCMV. Death was caused by T cell-dependent pathology and not viral load as 100% of T cell deficient neonates survived with minimal lung and liver pathology. This is similar to the adult model of medium dose LCMV clone 13, but T cell responses in neonates were not partially clonal exhausted. Furthermore, surviving neonates were not persistently infected, clearing virus by day 14 post infection. In adult mice direct intracranial infection leads to LCMV replication and CD8 T cell infiltration in the central nervous system (CNS), causing CD8 T cell-mediated death. However, this does not occur in adults during LCMV intraperitoneal (ip) infections. We questioned if unlike adults LCMV could be gaining access to the CNS in neonates following ip infection. Replicating LCMV was found in the brain of neonates after day 5 post infection along with virus-specific CD8 T cells producing IFNγ at day 9 post infection. Neonates lacking perforin had complete survival when followed until day 14 post infection, suggesting perforin-mediated T cell-dependent immunopathology within the CNS of neonates was causing death after LCMV infection. Passive immunity from LCMV-immune mothers also protected 100% of pups from death by helping control viral load early in infection. We believe that the maternal antibody compensates for the immature innate immune response of neonates and controls viral replication early so the neonatal T cell response induced less immunopathology. Neonates are commonly thought to have less functional immune systems, but these results show that neonates are capable of producing strong T cell responses that contribute to increased mortality. Model 4. Due to their enhanced susceptibility to infection neonatal and infant humans receive multiple vaccines. Several non-specific effects from immunizations have been observed, for example, measles or Bacillus Calmette- Guerin (BCG) vaccines have been linked to decreased death of children from infections other than measles virus or tuberculosis. These studies mirror the concepts of beneficial heterologous immunity, where previous immunization with an unrelated pathogen can result in faster viral clearance. LCMV-immune mice challenged with vaccinia virus (VV) have lower viral loads then naïve mice and survive lethal infections, but some mice do develop fat pad immunopathology in the form of panniculitis or acute fatty necrosis (AFN). We questioned how immunological T cell memory formed during the immature neonatal period would compare to memory generated in fully mature adults during a heterologous viral challenge. Mice immunized as neonates had comparable reduction in VV load and induction of AFN, indicating that heterologous immunity is established during viral infections early in life. Interestingly, the LCMV-specific memory populations that expanded in mice immunized as neonates differed from that of mice immunized as adults. In adult mice 50% of the mice have an expansion of LCMVNP205- specific CD8 T cells while the majority of neonates expanded the LCMVGP34- specific CD8 T cell pool. This alteration in dominant crossreactivities may be due to the limited T cell receptor repertoire of neonatal mice. In naïve neonatal mice we found altered Vβ repertoires within the whole CD8 T cell pool. Furthermore, there was altered Vβ usage within virus-specific responses compared to adult mice and a wide degree of variability between individual neonates, suggesting enhanced private specificity of the TCR repertoire. Beneficial heterologous immunity is maintained in neonates, but there was altered usage of crossreactive responses. As neonatal mice were found to be so sensitive to LCMV infection we questioned if neonates could control another arena virus that did not replicate as efficiently in mice, PICV. Unlike LCMV infection, neonatal mice survived infection with PICV even with adult-like doses. However, viral clearance was protracted in neonates compared to adults, but was cleared from fat pad and kidney by day 11 post infection. The peak of the CD8 T cell response was similarly delayed. PICV infected neonates showed dose-dependent PICV-specific CD8 T cell responses, which were similar to adult responses by frequency, but not total number. As with LCMV infection there were changes in immunodominance hierarchies in neonates. Examination of the immunodominance hierarchies of PICV-infected neonates showed that there were adult-like responses to the dominant NP38- specific response, but a loss of the NP122-specific response. Six weeks post neonatal infection mice were challenged with LCMV Armstrong and there was a strong skewing of the PICV immunodominance hierarchy to the crossreactive NP205-specific response. These data further support the hypothesis that heterologous immunity and crossreactivity develop following neonatal immunization, much as occurs in adults, although TCR repertoire and crossreactive patterns may differ. Changing the balance between T cell efficiency and viral load was found to altered the severity of the developing immunopathology after viral infection.

The immune response to an infection is determined by a number of factors, which also affect the generation of memory T cells afterwards. The immune response can also affect the stability of the pre-existing memory populations. The memory developed after an infection can influence the response to subsequent infections with unrelated pathogens. This heterologous immunity may deviate the course of disease and alter the disease outcome. The generation and stability of memory CD8 T cells and the influence of the history of infections on subsequent heterologous infections are studied in this thesis using different viral infection sequences. Previous studies using mice lacking individual immunoproteasome catalytic subunits showed only modest alterations in the CD8 T cell response to lymphocytic choriomeningitis virus (LCMV). In this study, I found that the CD8 T cell response to LCMV was severely impaired in mice lacking all three catalytic subunits of the immunoproteasome, altering the immunodominance hierarchy of the CD8 T cell response and CD8 T cell memory. Adoptive transfer experiments suggested that both inefficient antigen presentation and altered T cell repertoire contribute to the reduction of the CD8 T cell response in the immunoproteasome knockout mice. Immune responses generated during infections can reduce pre-existing memory T cell populations. Memory CD8 T cells have been shown to be reduced by subsequent heterologous infections. In this study, I re-examined the phenomenon using immune mice infected with LCMV, murine cytomegalovirus (MCMV) and vaccinia virus (VACV) in different infection sequences. I confirmed that memory CD8 T cells were reduced by heterologous infections, and showed that LCMV-specific memory CD4 T cells were also reduced by heterologous infections. Reduction of the memory CD8 T cells is thought to be the result of apoptosis of memory CD8 T cells associated with the peak of type I interferon early during infection. I showed that memory CD4 T cells were similarly driven to apoptosis early during infection; however, Foxp3+ CD4+ regulatory T cells were relatively resistant to virus infection-induced apoptosis, and were stably maintained during LCMV infection. The stability of Treg cells during viral infections may explain the relatively low incidence of autoimmunity associated with infections. The history of infections can deviate the course of disease and affect the disease outcome, but this heterologous immunity is not necessarily reciprocal. Previous studies have shown the effects of heterologous immunity during acute infections. In this thesis, I showed that the history of LCMV infection led to higher viral titers during persistent MCMV infection, caused more severe immunopathology at the beginning of infection, and reduced the number of MCMV-specific inflationary memory CD8 T cells after the period of memory inflation. In a different context of infection, the history of LCMV infection can be beneficial. LCMV-immune mice have been shown to have lower viral titers after VACV infection, but VACV-immune mice are not protected during LCMV infection. I found that memory CD8 T cells generated from LCMV and VACV infections were phenotypically different, but the differences could not explain the nonreciprocity of heterologous immunoprotection. By increasing the number of crossreactive VACV A11R198-205-specific memory CD8 T cells, however, I showed that some VACV-immune mice displayed reduced viral titers upon LCMV challenge, suggesting that the low number of potentially cross-reactive CD8 T cells in VACV-immune mice may be part of the reasons for the non-reciprocity of immunoprotection between LCMV and VACV. Further analysis deduced that both number of potentially cross-reactive memory CD8 T cells and the private specificity of memory CD8 T cell repertoire played a part in determining the outcome of heterologous infections.

The immune response to an infection is determined by a number of factors, which also affect the generation of memory T cells afterwards. The immune response can also affect the stability of the pre-existing memory populations. The memory developed after an infection can influence the response to subsequent infections with unrelated pathogens. This heterologous immunity may deviate the course of disease and alter the disease outcome. The generation and stability of memory CD8 T cells and the influence of the history of infections on subsequent heterologous infections are studied in this thesis using different viral infection sequences. Previous studies using mice lacking individual immunoproteasome catalytic subunits showed only modest alterations in the CD8 T cell response to lymphocytic choriomeningitis virus (LCMV). In this study, I found that the CD8 T cell response to LCMV was severely impaired in mice lacking all three catalytic subunits of the immunoproteasome, altering the immunodominance hierarchy of the CD8 T cell response and CD8 T cell memory. Adoptive transfer experiments suggested that both inefficient antigen presentation and altered T cell repertoire contribute to the reduction of the CD8 T cell response in the immunoproteasome knockout mice. Immune responses generated during infections can reduce pre-existing memory T cell populations. Memory CD8 T cells have been shown to be reduced by subsequent heterologous infections. In this study, I re-examined the phenomenon using immune mice infected with LCMV, murine cytomegalovirus (MCMV) and vaccinia virus (VACV) in different infection sequences. I confirmed that memory CD8 T cells were reduced by heterologous infections, and showed that LCMV-specific memory CD4 T cells were also reduced by heterologous infections. Reduction of the memory CD8 T cells is thought to be the result of apoptosis of memory CD8 T cells associated with the peak of type I interferon early during infection. I showed that memory CD4 T cells were similarly driven to apoptosis early during infection; however, Foxp3+ CD4+ regulatory T cells were relatively resistant to virus infection-induced apoptosis, and were stably maintained during LCMV infection. The stability of Treg cells during viral infections may explain the relatively low incidence of autoimmunity associated with infections. The history of infections can deviate the course of disease and affect the disease outcome, but this heterologous immunity is not necessarily reciprocal. Previous studies have shown the effects of heterologous immunity during acute infections. In this thesis, I showed that the history of LCMV infection led to higher viral titers during persistent MCMV infection, caused more severe immunopathology at the beginning of infection, and reduced the number of MCMV-specific inflationary memory CD8 T cells after the period of memory inflation. In a different context of infection, the history of LCMV infection can be beneficial. LCMV-immune mice have been shown to have lower viral titers after VACV infection, but VACV-immune mice are not protected during LCMV infection. I found that memory CD8 T cells generated from LCMV and VACV infections were phenotypically different, but the differences could not explain the nonreciprocity of heterologous immunoprotection. By increasing the number of crossreactive VACV A11R198-205-specific memory CD8 T cells, however, I showed that some VACV-immune mice displayed reduced viral titers upon LCMV challenge, suggesting that the low number of potentially cross-reactive CD8 T cells in VACV-immune mice may be part of the reasons for the non-reciprocity of immunoprotection between LCMV and VACV. Further analysis deduced that both number of potentially cross-reactive memory CD8 T cells and the private specificity of memory CD8 T cell repertoire played a part in determining the outcome of heterologous infections.

Prior immunity to a related or unrelated pathogen greatly influences the host’s immune response to a subsequent infection and can cause a dramatic difference in disease course, a phenomenon known as heterologous immunity. Heterologous immunity can influence protective immunity, immunopathology and/or immune deviation of cytokine-producing T cell subsets. Examples of heterologous immunity have been well documented in mouse models, as well as during human infections. For example, prior immunity to lymphocytic choriomeningitis virus (LCMV) provides partial protection against vaccinia virus (VV), as LCMV-immune mice show reduced VV titers and increased survival upon lethal dose VV infection. Heterologous protection against VV challenge, as a result of LCMV immunity, is mediated by LCMV-specific CD4 and CD8 T cells, as transfer of LCMV-specific memory T cells can mediate this protective effect in naïve mice. The recognition of a single TCR with more than one MHC-peptide complex is referred to as T cell cross-reactivity. A VV Kb-restricted epitope a11r198 was identified to be able to induce cross-reactive responses from LCMV-specific CD8 T cells. During VV infection, LCMV-specific memory T cells that are cross-reactive to VV epitopes produce IFN-γ early in VV infection. IFN-γ is essential for mediating the protection against VV in LCMV-immune mice, as this heterologous protection is absent in IFN-γR-/-and IFN-γ blocking antibody-treated LCMV-immune mice. In addition to protective immunity, cross-reactive LCMV-specific memory T cells and IFN-γ also induce an altered immunopathology during heterologous VV challenge. LCMV-immune mice show moderate to severe levels of inflammation of the fat tissue, known as panniculitis, in the visceral fat pads upon VV challenge. In humans, panniculitis is a painful condition, most commonly presenting as erythema nodosum. Erythema nodosum is a disease of unknown etiology with no known treatment. It may occur following intracellular bacterial and viral infections, and occasionally happens after vaccination with VV for smallpox. During infections there can be a delicate balance between the ability of immune responses to provide protective immunity, and the tendency to induce immunopathology. By using the mouse model of heterologous immunity between LCMV and VV, we tried to understand how the immunity to LCMV biased the balance between the protective immunity and immunopathology, and what effector molecules were responsible for the pathogenesis of panniculitis in this system. TNF is a pleiotropic cytokine, which is required for normal innate and adaptive immune responses. Its functions range from inducing proliferative responses including cell survival, to destructive responses such as promoting apoptosis and programmed necrosis. In response to inflammatory stimuli, activated macrophages/ monocytes produce large amounts of TNF, and upon activation, T cells, B cells and NK cells also produce TNF. In vitro and in vivo studies have shown that TNF in synergy with IFN-γ plays an important role in mediating host defense against pathogens, such as Listeria monocytogenesand poxviruses in mice and hepatitis B virus and human immunodeficiency virus in humans. However, inappropriate expression of TNF often results in tissue damage. Considering the important role TNF plays in both host defense and mediating autoimmune diseases, we hypothesized that TNF was required for mediating both protective and pathogenic effects in the heterologous immunity between LCMV and VV. We first examined whether TNF was involved in mediating protective heterologous immunity. LCMV-immune mice, that were TNF-deficient as a consequence of genetic deletion (TNF-/-) or receptor blockade by treatment with etanercept (TNFR2: Fc fusion protein), were challenged with VV. These TNF-deficient mice showed normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells. They also exhibited efficient clearance of VV similar to LCMV-immune mice with normal TNF function. Thus, we concluded that neither TNF nor lymphotoxin (LT), which uses the same receptors as TNF, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV could completely compensate for the role of TNF in protection of naïve mice against VV infection, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of humans with etanercept is reasonably well tolerated with relatively few infectious complications. One of the histological characteristics of panniculitis is necrosis of adipose tissue. It is known that three members in the TNF superfamily, i.e. TNF/LT, FasL and TRAIL are able to induce necrosis of a target cell. It is also known that TNF is able to induce VV-infected cells to go through necrosis, when apoptosis is blocked in these cells by VV protein. Furthermore, TNF and FasL have already been shown to be associated with some skin and fat pathology. Thus, we hypothesized that TNF, FasL and TRAIL were involved in the pathogenesis of panniculitis in VV infected LCMV-immune mice. By using blocking antibodies or genetically deficient mice, we demonstrated that both TNF/LT and FasL were crucial for inducing panniculitis. Although TNFR1 has been reported to induce programmed necrosis, our data indicated that TNFR2, not TNFR1, was involved in mediating tissue damage in the fat pads of LCMV-immune mice infected with VV. We also found that TNF signaled through TNFR2 to up-regulate the expression of Fas on adipocytes. Thus, the engagement of Fas on the adipocytes with FasL expressed on activated VV-specific and cross-reactive LCMV-specific CD8 T cells in the fat pads could lead to panniculitis. Thus, our data may identify a potential mechanism in the pathogenesis of human panniculitis, and may suggest a possible treatment for this painful disease. Recent reports suggest that heterologous immunity may contribute to the tremendous variation in symptoms between individuals, from subclinical to death, upon viral infection. Even in genetically identical mice, variations in immunopathology from none to life-threatening levels of pathology are observed in LCMV-immune mice during VV infection. By adoptive transfer of splenocytes from a single LCMV-immune donor into two recipients, we showed that similar levels of pathology were generated in mice receiving the same splenocytes. However, the level of pathology varied among recipients receiving splenocytes from different LCMV-immune donors. The difference in levels of VV-induced pathology observed in individual LCMV-immune mice was a reflection of the private specificity of the T cell repertoire, which is a unique characteristic of each individual immune host. The goal of this doctoral thesis is to understand how heterologous immunity contributes to the pathogenesis of panniculitis. Our data demonstrate that TNF/LT and FasL directly contribute to development of panniculitis in LCMV-immune mice during VV infection, and suggest that anti-TNF treatment might be a useful treatment for diseases, such as erythema nodosum and lupus-induced acute fatty necrosis in humans.

Heterologous immunity is a mechanism by which immunological memory within an individual, developed in response to a previous infection, plays a role in the immune response to a subsequent unrelated infection. In murine studies, heterologous immunity facilitated by cross-reactive CD8 T-cell responses can mediate either beneficial (protective immunity) or detrimental effects (e.g. enhanced lung and adipose immunopathology and enhanced viral titers) (Selin et al., 1998; Chen et al., 2001; Welsh and Selin, 2002; Nie et al., 2010; Welsh et al., 2010). Protective heterologous immunity results in enhanced clearance of virus during a subsequent infection with an unrelated pathogen. Such is the case when mice are immunized with lymphocytic choriomeningitis virus (LCMV) and subsequently challenged with Pichinde virus (PV) or vaccinia virus (VACV) (Selin et al., 1998). However, heterologous immunity may also mediate enhanced immunopathology as mice immunized with influenza A virus (IAV) and challenged with LCMV show increased viral titers and enhanced lung immunopathology (Chen et al., 2003). The role heterologous immunity plays during infection is not limited to the murine system. In fact, there have now been several reports of enhanced immunopathology due to heterologous immunity during human infections, involving viruses such as IAV, Epstein-Barr Virus (EBV), hepatitis C virus (HCV), and dengue virus (DENV) (Mathew et al., 1998; Wedemeyer et al., 2001; Acierno et al., 2003; Nilges et al., 2003; Clute et al., 2005; Urbani et al., 2005). Interestingly, in all reported cases in humans, heterologous immunity mediated enhanced immunopathology. Upon infection with EBV the clinical presentation can range from asymptomatic to severe, occasionally fatal, acute infectious mononucleosis (AIM) (Crawford et al., 2006b; Luzuriaga and Sullivan, 2010) which is marked by a massive CD8 lymphocytosis. This lympho-proliferative effect in AIM was shown to be partially mediated by reactivation of cross-reactive IAV-M1 58-66 (IAV-GIL) specific CD8 memory T-cells in HLA-A2 patients reacting to the EBV-BMLF1 280 (EBV-GLC) epitope (Clute et al., 2005). Interestingly, EBV infects ~90% of individuals globally by the third decade of life, establishing a life-long infection (Henle et al., 1969). However, it is unknown why 5-10% of adults remain EBV-sero-negative (EBV-SN), despite the fact that the virus infects the vast majority of the population and is actively shed at high titers even during chronic infection (Hadinoto et al., 2009). Here, we show that EBV-SN HLA-A2+ adults possess cross-reactive IAV-GIL/EBV-GLC memory CD8 T-cells that show highly unique properties. These IAV-GIL cross-reactive memory CD8 T-cells preferentially expand and produce cytokines to EBV antigens at high functional avidity. Additionally, they are capable of lysing EBV-infected targets and show the potential to enter the mucosal epithelial tissue, where infection is thought to initiate, by CD103 expression. This protective capacity of these cross-reactive memory CD8 T-cells may be explained by a unique T-cell receptor (TCR) repertoire that differs by both organization and CDR3 usage from that in EBV-seropositive (EBV-SP) donors. The composition of the CD8 T-cell repertoire is a dynamic process that begins during the stochastic positive selection of the T-cell pool during development in the thymus. Thus, upon egress to the periphery a naïve T-cell pool, or repertoire, is formed that is variable even between genetically identical individuals. This T-cell repertoire is not static, as each new infection leaves its mark on the repertoire once again by stochastically selecting and expanding best-fit effectors and memory populations to battle each new infection while at the same time deleting older memory CD8 T-cells to make room for the new memory cells (Selin et al., 1999). These events induce an altered repertoire that is unique to each individual at each infection. It is this dynamic and variable organization of the T-cell repertoire that leads to private specificity even between genetically identical individuals upon infection with the same pathogens and thus a different fate (Kim et al., 2005; Cornberg et al., 2006a; Nie et al., 2010). It is this private specificity of the TCR repertoire that helps explain why individuals with the same epitope specific cross-reactive response, but composed of different cross-reactive T-cell clones, can either develop AIM or never become infected with EBV. Our results suggest that heterologous immunity may protect EBV-SN adults against the establishment of productive EBV infection, and potentially be the first demonstration of protective T-cell heterologous immunity between unrelated pathogens in humans. Our results also suggest that CD8 T-cell immunity can be sterilizing and that an individual’s TCR repertoire ultimately determines their fate during infection. To conclusively show that heterologous immunity is actively protecting EBV-SN adults from the establishment of a productive EBV infection, one would have to deliberately expose an individual to the virus. Clearly, this is not an acceptable risk, and it could endanger the health of an individual. A humanized mouse model could allow one to address this question. However, before we can even attempt to address the question of heterologous immunity mediating protection from EBV infection in humanized mice, we must first determine whether these mice can be infected with, and build an immune response to the two viruses we are studying, EBV and IAV. We show here that these mice can indeed be infected with and also mount an immune response to EBV. Additionally, these mice can also be infected with IAV. However, at this time the immune responses that are made to these viruses in our established humanized mouse model are not substantial enough to fully mimic a human immune response capable of testing our hypothesis of heterologous immunity mediating protection from EBV infection. Although the immune response in these mice to EBV and IAV infection is not suitable for the testing of our model the data are promising, as the humanized mouse model is constantly improving. Hopefully, with constant improvements being made there will be a model that will duplicate a human immune system in its entirety. This thesis will be divided into 5 major chapters. The first chapter will provide an introduction to both general T-cell biology and also to the role of heterologous immunity in viral infection. The second chapter will provide the details of the experimental procedures that were performed to test our hypothesis. The third chapter will describe the main scientific investigation of the role of heterologous immunity in providing natural resistance to infection in human subjects. This chapter will also consist of the data that will be compiled into a manuscript for publication in a peer-reviewed journal. The fourth chapter will consist of work performed pertaining to the establishment of a humanized mouse model of EBV and IAV infection. The establishment of this model is important for us to be able to show causation for protection from EBV infection mediated by heterologous immunity.

A person is exposed to many pathogens throughout their lifetime, and with the resolution of each infection, there remains a pool of pathogen-specific immune cells that protect that person from re-infection with the same pathogen. However, there is a great deal of evidence to suggest that the pool of pathogen-specific memory cells can also participate in the immune response to future infections with unrelated pathogens. Many believe T cells to be cross-reactive in nature because of their interaction with self antigens during development in the thymus and their interaction with foreign antigens once in the periphery. There are many features of the interaction between a T cell and its ligand that facilitate this cross-reactive nature. Based on solved crystal structures, relatively few contacts are required for a stable interaction, and that interaction is often mediated by the flexible CDR3 loops of the T cell receptor that accommodate ligands of various structure. There is also evidence in the murine and human systems that subsets of virus-specific memory CD8 T cells take on an activated phenotype upon infection with an unrelated virus. In murine models, these memory T cell subsets could kill target cells, secrete several cytokines, and proliferate in response to a cross-reactive stimulation, suggesting that a cross-reactive T cell response could impact the outcome of a viral infection. In fact, upon heterologous infection, mice immune to a previous virus were often protected, having lower titers of the second unrelated virus, their epitope-specific and T cell receptor repertoires were often skewed, and they were more prone to immune-mediated pathologies. All of these observations coincided with the presence of cross-reactive T cell responses. Thus, we define heterologous immunity as changes in viral replication and the disease pathology associated with that viral infection as a result of the host's history of infection, and this can be mediated, in part, by cross-reactive CD8 T cell responses. Since many human viral infections are associated with a wide range of disease states, we questioned whether cross-reactive CD8 T cell responses occurred as commonly as they appeared to occur in the murine models and whether they influenced the outcome of such infections. Epstein-Barr virus (EBV) infects over 90% of the U. S. population and has a large genome with the capacity to encode a multitude of T cell epitopes. The first part of this thesis research focuses on the identification of cross-reactive CD8 T cell responses with specificity for known epitopes derived from EBV, a common human virus. We directed our study to HLA-A2-restricted responses because of the common expression of this MHC Class I allele in the U. S. population. This study resulted in the detection of cross-reactive responses with five different specificities that involved either the immunodominant lytic EBV-BMLF1280 epitope or the latent EBNA 3A596epitope. Three of the cross-reactive responses had specificity for epitopes derived from another unrelated, but common, human virus, influenza A virus (IV). Each of these cross- reactive responses had the potential to participate in the collective immune response to acute EBV infection. EBV is also well-suited as a model system to study heterologous immunity in humans, as infection at an early age is frequently asymptomatic, while the same infection during adolescence often results in an immune-mediated syndrome, infectious mononucleosis (IM). Since older individuals have presumably been exposed to more pathogens in their lifetime and, therefore, would have memory CD8 T cell pools with more extensive specificities, we hypothesized that acute EBV infection activated cross-reactive memory CD8 T cell responses that promoted the development of IM. In order to determine if the cross-reactive responses we identified above contributed to the immune response to acute EBV infection, we first screened the blood of IM patients for cross-reactive T cells with specificity for EBV-BMLFl280 and IV-M158. The total number of M1-specific T cells of 5 of 8 patients was increased at presentation with IM, which was suggestive of their specific activation during the EBV infection since a bystander mechanism would have resulted in 8 out of 8 patients having increased numbers of M1-specific T cells. Our hypothesis was further supported by the fact that we clearly detected cross-reactive T cells capable of recognizing both BMLF1 and M1 epitopes in the blood of 2 of the 5 IM patients with an augmented M1-specific T cell frequency. Furthermore, the M1-specific TCR repertoires of those two patients were dramatically skewed, which was an indication of cross-reactive M1-specific T cell expansions and, therefore, participation in the lymphoproliferation characteristic of IM. In addition, T cell lines derived from 3 out of 8 healthy donors with previous exposure to both viruses contained a subset of T cells that responded to both BMLF1 and M1 epitopes, suggesting that these cross-reactive cells are often maintained in memory. These cross-reactive T cells were cytotoxic and produced MIP-1β, IFNγ, and TNFα, functions which could potentially promote the symptoms of IM and, indeed, may have been contributed to the severe case of IM noted in one patient. The final part of this thesis research focused on defining the structure of the cross-reactive TCR that recognized both BMLF1 and M1 epitopes, which have only 33% sequence similarity. In addition, we examined the cross-reactive TCR repertoire organization of multiple individuals to determine the breath and, therefore, the likelihood that this cross-reactive T cell response will occur. These studies revealed that a wide range of Vα and Vβ families can mediate interaction with both epitopes and that the cross-reactive TCR repertoire was unique to each individual, relying heavily on the T cell clones present in that individual's private BMLF1- and M1-specific repertoires. We also observed an increased frequency of TCRs with longer CDR3 regions within the cross-reactive repertoire, which were often extended by non-bulky amino acid residues that could provide these TCRs with more flexibility in order. to accommodate the two different epitope structures. Given that we detected a cross-reactive T cell response with specificity for two immunodominant epitopes derived from two of the most common human viruses among people that share one of the most common MHC Class I alleles in the U. S. population, we predict that cross-reactive T cells are common components of human immune responses. The variability in the magnitude and specificity of each cross-reactive T cell response is dependent on each individual's unique history of infection and th,eir unique TCR repertoire, and such responses likely represent one of many factors that could explain the individual variability in disease severity associated with EBV and many other human viral infections.

Each individual experiences many sequential infections throughout the lifetime. An increasing body of work indicates that prior exposure to unrelated pathogens can greatly alter the disease course during a later infection. This can be a consequence of a phenomenon known as heterologous immunity. Most viruses invade the host through the mucosa of a variety of organs and tissues. Using the intranasal mucosal route of infection, the thesis focused on studying modulation of lymphocytic choriomeningitis virus (LCMV)-specific memory CD8 T cells upon respiratory vaccinia virus (VV) infection and the role of these memory CD8 T cells in heterologous immunity against VV and altered immunopathology in the lung. The VV infection had a profound impact on memory T cells specific for LCMV. The impact included the up-regulation of CD69 expression on LCMV-specific CD8 memory T cells and the activation of their in vivoIFN-γ production and cytotoxic function. Some of these antigen-specific memory T cells selectively expanded in number, resulting in modulation of the original LCMV-specific T cell repertoire. In addition, there was a selective organ-dependent redistribution of these LCMV-specific memory T cell populations in secondary lymphoid tissue (the mediastinal lymph node and spleen) and the non-lymphoid peripheral (the lung) organs. The presence of these LCMV-specific memory T cells correlated with IFN-γ-dependent enhanced VV clearance, decreased mortality and marked changes in lung immunopathology. Thus, the participation of pre-existing memory T cells specific for unrelated agents can alter the dynamics of mucosal immunity. This is associated with an altered disease course in response to a pathogen. The roles for T cell cross-reactivity and cytokines in the modulation of memory CD8 T cells during heterologous memory CD8 T cell-mediated immunity and immunopathology were investigated. Upon VV challenge, there were preferential expansions of several LCMV-specific memory CD8 T cell populations. This selectivity suggested that cross-reactive responses played a role in this expansion. Moreover, a VV peptide, partially homologous to LCMV NP 205, stimulated LCMV-NP205 specific CD8 T cells, suggesting that NP205 may be a cross-reactive epitope. Poly I:C treatment of LCMV-immune mice resulted in a transient increase but no repertoire alteration of LCMV-epitope-specific CD8 T cells. These T cells did not produce IFN-γ in vivo. These results imply that poly I:C, presumably through its induced cytokines, was assisting in initial recruitment of LCMV-specific memory CD8 T cells in a nonspecific manner. VV challenge of LCMV-immune IL-12KO mice resulted in activation and slightly decreased accumulation of LCMV-specific CD8 T cells. Moreover, there was a dramatic reduction of in vivoIFN-γ production by LCMV-specific IL-12KO CD8 T cells in the lung. I interpreted this to mean that IL-12 was important to augment IFN-γ production by memory CD8 T cells upon TCR engagement by antigens and to induce further accumulation of activated memory CD8 T cells during the heterologous viral infection. This thesis also systematically examined what effect the sequence of two heterologous virus challenges had on viral clearance, early cytokine profiles and immunopathology in the lung after infecting mice immune to one virus with another unrelated viruses. Four unrelated viruses, [LCMV, VV, influenza A virus or murine cytomegalovirus (MCMV)], were used. There were many common changes observed in the acute response to VV as a consequence of prior immunity to any of three viruses, LCMV, MCMV or influenza A virus. These included the enhanced clearance of VV in the lung, associated with enhanced TH1 type responses with increased IFN-γ and suppressed pro-inflammatory responses. However, immunity to the three different viruses resulted in unique pathologies in the VV-infected lungs, but with one common feature, the substitution of lymphocytic and chronic mononuclear infiltrates for the usual acute polymorphonuclear response seen in non-immune mice. Immunity to influenza A virus appeared to influence the outcome of subsequent acute infections with any of the three viruses, VV, LCMV and MCMV. Most notably, influenza A virus-immunity protected against VV but it actually enhanced LCMV and MCMV titers. This enhanced MCMV replication was associated with enhanced TH1 type response and pro-inflammatory cytokine responses. Immunity to influenza A virus appeared to dramatically enhance the mild lymphocytic and chronic mononuclear response usually observed during acute infection with either LCMV or MCMV in non-immune mice, but LCMV infection and MCM infection of influenza A virus-immune mice each had its own unique features. Thus, the specific sequence of virus infections controls the outcome of disease.

Previously, our lab demonstrated that heterologous immunity could result in either gain or loss of protective immunity and alteration in immune pathology following infection by a second un-related pathogen. One of the prototypical models to study T cell-mediated heterologous immunity involves two distantly related arenaviruses, namely lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV). Each virus encodes a cross-reactive CD8 epitope that has six out of eight in amino acid (aa) similarity with respect to its counterpart at the position 205-212 of the nucleoprotein (NP205). Heterologous challenge between LCMV and PV results in 1) expansion of the cross-reactive NP205-specific CD8 T cell responses and alteration of the immunodominance hierarchy and 2) partial protective immunity (heterologous immunity). Our lab showed that cross-reactive NP205-specific CD8 T cell receptor (TCR) repertoires become extremely narrowed following a heterologous challenge between LCMV and PV. Therefore, I questioned if LCMV NP205 epitope escape variants could be isolated during a dominant but narrowed crossVI reactive NP205-specific CTL response. In the first part of my thesis, I describe the isolation of a LCMV NP-V207A CTL escape variant in vivo using PV-immune animals challenged with LCMV clone 13. The LCMV NP-V207A variant contains a point mutation, which results in the switching of valine to alanine at the third non-anchoring residue of the LCMV NP205 CD8 epitope. Immunization of mice with the LCMV NP-V207A variant results in a significantly diminished cross-reactive NP205-specific CD8 T cell response. This suggests that the point mutation is responsible for the loss in the immunogenicity of the LCMV NP205 CD8 epitope. In addition, an in vitrorescued(r) recombinant LCMV variant (r/V207A) that encodes the original mutation also induces a highly diminished cross-reactive NP205-specific CD8 T cell response in mice. In agreement with the result obtained from the intracellular cytokine assays (ICS), MHC-Ig dimers loaded with the LCMV NP205 (V-A) peptide could only detect a minute population of cross-reactive NP205-specific CD8 T cells in mice infected with r/V207A variant virus. All the data indicate that the point mutation results in a significant loss in immunogenicity of the LCMV NP205 CD8 epitope. So far, no direct link between the cross-reactive NP205-specific CD8 T cells and heterologous immunity had been established in this system. Therefore, we immunized mice with either LCMV WT or the LCMV NP-V207A variant virus and showed that a significant loss of heterologous immunity is associated with the group immunized with LCMV NP-V207A variant virus. Again, r/V207Aimmune animals also displayed a significant loss in heterologous immunity following PV challenge. This suggests that the cross-reactive NP205-specific CD8 T cells mediate the majority of heterologous immunity between LCMV and PV in vivo. In comparison to the PV-immune control group, PV clearance kinetics mediated by the cross-reactive NP205-specific CD8 T cells were significantly delayed. Finally, these data also suggest that bystander activation plays very little role in heterologous immunity between LCMV and PV. Many studies in murine systems and humans suggest that cross-reactive T cells are often associated with immune pathology. We showed that in mice that were sequentially immunized with PV and LCMV (PV+LCMV WT double immune mice), there was a development of a high incidence and high level of immune pathology known as acute fatty necrosis (AFN) following a final PV challenge. The data suggest that these cross-reactive NP205-specific CD8 T cells might play an important role in immune pathogenesis. Therefore, we asked if the cross-reactive NP205-specific CD8 T cells play a role in immune pathogenesis by comparing the incidence of AFN between the (PV+LCMV WT) and the (PV+LCMV NP-V207A) double immune mice following a final PV challenge. In agreement with our hypothesis, the result showed the (PV+LCMV NP-V207A) double immune mice developed a significantly lower incidence of AFN compared to the (PV+LCMV WT) double immune mice. However, linear correlation studies comparing the frequency of different antigen-specific CD8 T cell populations within the (PV+LCMV WT) double immune mice before challenge and the severity of AFN following the PV challenge suggest that two opposing antigen-specific CD8 T cell populations are involved in determining the final outcome of the immune pathology. The PV NP38-45-specific CD8 T cell response (PV NP38) appears to be more protective than the cross-reactive NP205-specific CD8 T cell response. In addition, a positive linear correlation between the ratio of cross-reactive NP205 to PV NP38 and the severity of AFN seem to suggest that these cross-reactive populations are important contributors to immune pathogenesis. Peptide titration studies examining the functional avidities to different antigenic specificities suggest that both populations consist of high avidity TCR and peptide MHC (TCR:pMHC) interactions. However, skewing within the cross-reactive NP205 specific CD8 T cell response towards the LCMV NP205 epitope response in one of the (PV+LCMV WT) double immune mice suggests that cross-reactive NP205 specific CD8 T cells could constitute a sub-optimal response to a PV challenge. In summary, I questioned what might be some of the immunological consequences of heterologous immunity in this model. First of all, we have established a direct link between the cross-reactive NP205-specific CD8 T cell response and heterologous immunity in LCMV and PV. Second of all, I demonstrated that a LCMV NP205 epitope escape variant could be selected in vivo under the conditions of heterologous immunity. In addition, I showed that PV clearance kinetic was significantly delayed in cross-reactive NP205-mediated heterologous immunity as compared to homologous challenge. Finally, we demonstrated that cross-reactive NP205-specific CD8 T cells could play an important role in immune pathogenesis in this model. However, correlation data indicate that two opposing antigen-specific CD8 T cell populations could ultimately decide the outcome and magnitude of immune pathology in each individual mouse. All the data presented above strongly suggest that the cross-reactive NP205 CD8 T cells play a crucial role in immune pathology in this model system by 1) interfering with the regular establishment of immunodominance hierarchy orders, or 2) exhibiting a sub-optimal protective immunity due to the nature of the cross-reactive epitope.