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PD-L1 decreases anti-viral CD8+ T cell motility and PD-1 blockade restores motility in the presence of high viral loads., Immune responses to persistent viral infections and cancer often fail because of intense regulation of antigen-specific T cells—a process referred to as immune exhaustion. The mechanisms that underlie the induction of exhaustion are not completely understood. To gain novel insights into this process, we simultaneously examined the dynamics of virus-specific CD8+ and CD4+ T cells in the living spleen by two-photon microscopy (TPM) during the establishment of an acute or persistent viral infection. We demonstrate that immune exhaustion during viral persistence maps anatomically to the splenic marginal zone/red pulp and is defined by prolonged motility paralysis of virus-specific CD8+ and CD4+ T cells. Unexpectedly, therapeutic blockade of PD-1–PD-L1 restored CD8+ T cell motility within 30 min, despite the presence of high viral loads. This result was supported by planar bilayer data showing that PD-L1 localizes to the central supramolecular activation cluster, decreases antiviral CD8+ T cell motility, and promotes stable immunological synapse formation. Restoration of T cell motility in vivo was followed by recovery of cell signaling and effector functions, which gave rise to a fatal disease mediated by IFN-γ. We conclude that motility paralysis is a manifestation of immune exhaustion induced by PD-1 that prevents antiviral CD8+ T cells from performing their effector functions and subjects them to prolonged states of negative immune regulation.

Viral infections of central nervous system (CNS) often trigger inflammatory responses that give rise to a wide range of pathological outcomes. The CNS is equipped with an elaborate network of innate immune sentinels (e.g. microglia, macrophages, dendritic cells) that routinely serve as first responders to these infections. The mechanisms that underlie the dynamic programming of these cells following CNS viral infection remain undefined. To gain insights into this programming, we utilized a combination of genomic and two-photon imaging approaches to study a pure innate immune response to a noncytopathic virus (lymphocytic choriomeningitis virus) as it established persistence in the brain. This enabled us to evaluate how global gene expression patterns were translated into myeloid cell dynamics following infection. Two-photon imaging studies revealed that innate myeloid cells mounted a vigorous early response to viral infection characterized by enhanced vascular patrolling and a complete morphological transformation. Interestingly, innate immune activity subsided over time and returned to a quasi-normal state as the virus established widespread persistence in the brain. At the genomic level, early myeloid cell dynamics were associated with massive changes in CNS gene expression, most of which declined over time and were linked to type I interferon signaling (IFN-I). Surprisingly, in the absence of IFN-I signaling, almost no differential gene expression was observed in the nervous system despite increased viral loads. In addition, two-photon imaging studies revealed that IFN-I receptor deficient myeloid cells were unresponsive to viral infection and remained in a naïve state. These data demonstrate that IFN-I engages non-redundant programming responsible for nearly all innate immune activity in the brain following a noncytopathic viral infection. This Achilles' heel could explain why so many neurotropic viruses have acquired strategies to suppress IFN-I., Author Summary The central nervous system is equipped with innate immune cells that serve as first responders to sterile injuries and infections. The mechanisms that program the movement and morphological transformations of these cells following infection remain undefined. Here, we utilized a combination of genomic and in vivo imaging approaches to define pathways that program the motion of innate immune cells responding to a noncytopathic virus as it established persistence in the brain. In vivo imaging studies performed in the living brain revealed that innate myeloid cells mounted a vigorous early response that returned to a “naïve” state during persistence. This was associated at the genomic level with robust changes in gene expression that were mostly quenched over time. Analysis of the gene expression pattern revealed a prominent type I interferon (IFN-I) signature only at the early stage of infection. Surprisingly, in the absence of type I interferon (IFN-I) signaling, almost no genes were differentially expressed in the virally infected nervous system and all innate myeloid cells were unresponsive. These data indicate IFN-I programs all innate myeloid activity in the nervous system following a noncytopathic viral infection. This non-redundant anti-viral program represents an Achilles' heel that can be exploited by neurotropic viruses.

Neuronal death is a prominent, but poorly understood, pathological hallmark of prion disease. Notably, in the absence of the cellular prion protein (PrP C ), the disease-associated isoform, PrP Sc , appears not to be intrinsically neurotoxic, suggesting that PrP C itself may participate directly in the prion neurodegenerative cascade. Here, cross-linking PrP C in vivo with specific monoclonal antibodies was found to trigger rapid and extensive apoptosis in hippocampal and cerebellar neurons. These findings suggest that PrP C functions in the control of neuronal survival and provides a model to explore whether cross-linking of PrP C by oligomeric PrP Sc can promote neuronal loss during prion infection.

Type I interferons participate in the host defense against most viral infections through induction of innate antiviral proteins like tetherin (BST-2). In vitro studies have shown that tetherin can physically sequester viruses by linking them to infected cells, which in turn reduces virion release. It is not currently understood how this antiviral protein functions in vivo to control an immune response mounted against a persistent viral infection. We sought novel mechanistic insights into the function of tetherin by studying its role in the establishment of a persistent lymphocytic choriomeningitis virus (LCMV) infection. Following viral infection, tetherin was significantly upregulated in B cells, plasmacytoid dendritic cells (DCs), conventional DCs, and brain-resident microglia. Genetic deletion of tetherin resulted in an early failure to contain a persistence-prone strain of LCMV in the splenic marginal zone. This resulted in a rapid re-distribution of LCMV into the red and white pulp at early time points post-infection. This also interfered with the early priming of antiviral T cells by causing them to localize preferentially to the splenic red pulp where they proliferated less and acquired an impaired phenotype characterized by upregulation of exhaustion markers (PD-1, LAG-3, TIGIT), loss of cytokine production, and decreased motility. Importantly, these early alterations impeded the ability of the host to control persistence-prone LCMV. Viral control was delayed in peripheral tissues, and lifelong persistence was established in the brain. These data demonstrate that tetherin plays a critical in vivo role in shaping the anatomical distribution and adaptive immune response against a persistent viral infection.

Macrophage activation syndrome (MAS) is a life-threatening sepsis-like hyperinflammatory state arising in certain infectious and rheumatic diseases. Previous genetic associations have implicated genes critical for perforin-mediated cytotoxicity. However, we recently identified that activating mutations in inflammasome component NLRC4 lead to MAS and early-onset enterocolitis. Patients with NLRC4-MAS also have chronically and extraordinarily elevated serum IL-18. Notably, disease-causing mutations in other inflammasomes do not cause MAS or chronic IL-18 elevation. In vitro, IL-1b production by THP1 cells expressing various NLRC4 patient mutations correlated with disease severity and identified pathogenic clusters in the molecular structure. To further study the function of NLRC4, we created a mouse bearing the T337S point mutation. Like NLRC4-MAS patients, NLRC4-T337S mice demonstrate elevated serum IL-18 in the absence of systemic inflammation. However, macrophages from NLRC4-T337S mice did not demonstrate enhanced IL-1β or IL-18 production. Correspondingly, bone marrow chimeras demonstrated that IL-18 hyperproduction was derived from non-hematopoietic cells. NLRC4-T337S mice did not demonstrate more severe disease in response to repeated systemic stimulation through TLR9 or TLR4, nor upon LCMV infection, but developed colitis with IL-10 receptor blockade. These results suggest intestinal epithelia, which express both NLRC4 and proIL-18, may be a source of pathogenic IL-18 in this model. Future work will clarify the source(s) of excessive IL-18 and identify how chronic IL-18 effects on gut homeostasis and T and NK cell responses may promote systemic hyperinflammation.

Th1 cells and T follicular helper cells (Tfh cells), are generated early after viral infection. However, the molecular pathways that drive the differentiation of these two distinct subsets remain unclear. Here, we demonstrate that a molecular circuit involving TCF1, Blimp1, and IL-2 regulates the differentiation of viral-specific Tfh and Th1 cells. Tcf7 gene (encodes TCF1) is actively transcribed in naïve and Tfh cells but strongly repressed in Th1 cells. TCF1 expression was negatively correlated with CD25 and Blimp1 expression, and was suppressed by IL2 signaling and Blimp1. Notably, loss of Tcf7 in T cells led to defective Tfh-cell and GC responses after viral infection. Tcf7-deficient Tfh cells had reduced Tfh transcription signatures and increased Th1 transcription signatures. Moreover, Tcf7-deficient Tfh cells had lower mitochondrial mass and membrane potential, and showed reduced expression of genes related to oxidative metabolism. Il2ra and Prdm1 (encodes Blimp1) were ectopically expressed in Tcf7-deficient Tfh cells and were bound by TCF1, suggesting the existence of a negative feedback loop linking TCF1 to IL-2 and Blimp1. Over-expression of Bcl6 rescued Tfh defects caused by Tcf7 deficiency, demonstrating that TCF1 is upstream of the Bcl6-Blimp1 axis. Finally, we found that Tcf7-deficiency did not cause evident defect in Tfh responses in Th2 immunization models. Therefore, TCF1 specifically regulates antiviral Tfh responses.

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. Pathological studies of CM victims reveal vascular hemorrhaging and edema within the brain, yet the underlying mechanisms that lead to this fatal condition are unknown. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the factors that drive the pathogenesis of CM. We found that ECM is associated with a large influx of immune cells into the central nervous system (CNS) at the peak of infection. By depleting individual innate and adaptive immune cell populations, we discovered that CD8 T cells alone were capable of mediating the fatal pathology associated with ECM, including breakdown of the blood brain barrier, edema, and extensive neuronal death in the brainstem. Using two-photon microscopy, we observed parasite-specific CD8 T cells slowly crawling and arresting along the CNS vasculature in vivo during ECM. This behavior was facilitated by the IFN-γ-dependent upregulation of antigen presentation and adhesion molecules on CNS endothelial cells (ECs). Antibody therapy against the adhesion molecules VLA-4 and LFA-1 disrupted CD8 T cell interactions with the CNS vasculature and prevented disease. Importantly, infected mice rendered genetically unable to present antigen on ECs had reduced CD8 T cell-brain EC interactions and were resistant to ECM. These results demonstrate that parasite-specific CD8 T cells interact with brain vascular ECs in an antigen-dependent manner to induce fatal vascular and neuronal pathology during ECM.

The immune system is highly evolved and can respond to infection throughout the body. Pathogen-specific immune cells are usually generated in secondary lymphoid tissues (e.g., spleen, lymph nodes) and then migrate to sites of infection where their functionality is shaped by the local milieu. Because immune cells are so heavily influenced by the infected tissue in which they reside, it is important that their interactions and dynamics be studied in vivo. Two-photon microscopy is a powerful approach to study host-immune interactions in living tissues, and recent technical advances in the field have enabled researchers to capture movies of immune cells and infectious agents operating in real time. These studies have shed light on pathogen entry and spread through intact tissues as well as the mechanisms by which innate and adaptive immune cells participate in thwarting infections. This review focuses on how two-photon microscopy can be used to study tissue-specific immune responses in vivo, and how this approach has advanced our understanding of host-immune interactions following infection.

We quantified CD8 T cells needed to cause type 1 diabetes and studied the anatomy of the CD8 T cell/beta (β) cell interaction at the immunologic synapse. We used a transgenic model, in situ tetramer staining to distinguish antigen specific CD8 T cells from total T cells infiltrating islets and a variety of viral mutants selected for functional deletion(s) of various CD8 T cell epitopes. Twenty percent of CD8 T cells in the spleen were specific for all immunodominant and subdominant viral glycoprotein (GP) epitopes. CTLs to the immunodominant LCMV GP33-41 epitope accounted for 63% of the total (12.5% of tetramers). In situ hybridization analysis demonstrated only 1 to 2% of total infiltrating CD8 T cells were specific for GP33 CD8 T cell epitope, yet diabetes occurred in 94% of mice. The immunologic synapse between GP33 CD8 CTL and β cell contained LFA-1 and perforin. Silencing both immunodominant epitopes (GP33, GP276–286) in the infecting virus led to a four-fold reduction in viral specific CD8 CTL responses, negligible lymphocyte infiltration into islets and absence of diabetes., Author Summary Insulin-dependent type 1 diabetes (T1D) is characterized by elevated blood sugar, lymphocytic infiltration into the islets of Langerhans and T cell destruction of beta (β) cells. β cells produce insulin whose function is to maintain and regulate glucose hemostasis. However, in vivo, the numbers of antigen specific T cells that migrate to the islets to cause T1D, the engagement of such T cells with β cells at the immunologic synapse and the molecules expressed at the synapse are not clear. Using a transgenic model of virus induced T1D, a panel of viruses with CD8 T cell epitope mutations and in situ tetramer hybridization, we note of the total CD8 T cells infiltrating the islets, only 1–2% are antigen specific recognizing the immunodominant virus CD8 T cell epitope expressed on β cells. Immunohistochemical analysis of the synapse found between antigen specific CD8 T cells and β cells displays attachment by LFA-1 and presence of perforin, the molecule indicative of lytic activity.

Persistent virus infection drives follicular T helper cell differentiation., CD4 T cell responses are crucial to prevent and control viral infection; however, virus-specific CD4 T cell activity is considered to be rapidly lost during many persistent viral infections. This is largely caused by the fact that during viral persistence CD4 T cells do not produce the classical Th1 cytokines associated with control of acute viral infections. Considering that CD4 T cell help is critical for both CD8 T cell and B cell functions, it is unclear how CD4 T cells can lose responsiveness but continue to sustain long-term control of persistent viral replication. We now demonstrate that CD4 T cell function is not extinguished as a result of viral persistence. Instead, viral persistence and prolonged T cell receptor stimulation progressively redirects CD4 T cell development away from the Th1 response induced during an acute infection toward T follicular helper cells. Importantly, this sustained CD4 T cell functionality is critical to maintain immunity and ultimately aid in the control of persistent viral infection.

Viral infections are a major cause of human disease. Although most viruses replicate in peripheral tissues, some have developed unique strategies to move into the nervous system, where they establish acute or persistent infections. Viral infections in the central nervous system (CNS) can alter homeostasis, induce neurological dysfunction and result in serious, potentially life-threatening inflammatory diseases. This Review focuses on the strategies used by neurotropic viruses to cross the barrier systems of the CNS and on how the immune system detects and responds to viral infections in the CNS. A special emphasis is placed on immune surveillance of persistent and latent viral infections and on recent insights gained from imaging both protective and pathogenic antiviral immune responses.

Despite decades of intense research, persistent viral infections still continue to outsmart investigators and maintain an active presence in the human population. Recent estimates suggest that ∼8–12 chronic infections can be found in each of us and comprise what is referred to as our ‘virome’ (Virgin et al., 2009). For better or for worse, persistent viral infections do influence human physiology. At one end of the spectrum, persistent infections can heighten immune awareness, helping to ward off other pathogenic invaders seeking a new home in which to replicate. This is considered advantageous, as occupation by a benign infection is better than invasion by a pathogenic one. Pathogenic persistent viral infections lie at the other end of the spectrum and are, for good reason, the focus of major investigations within the research community. Viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), papilloma virus, and adenovirus can negatively impact human health by causing well-described symptoms and chronic diseases. It is because of these diseases that researchers are determined to understand every facet of their biology as well as how our immune system senses and responds to these pathogens. The fact that these pathogens persist signifies that they have already achieved the upper hand, and thus researchers face the challenge of how to tip the balance back in favor of our immune system. This is particularly difficult when dealing with pathogens that establish latency such as HIV, EBV, CMV and papilloma virus. These viruses have the ability to lie dormant, avoiding detection by the immune system. A strategy commonly used in the research community to gain novel insights into our anti-viral defenses is to study murine models of persistent viral infection. Although mouse and human are by no means identical, enough similarities exist such that fundamental immunological discoveries which apply to humans are routinely uncovered in murine systems. One of the most widely used murine viral model systems relies on a pathogen known as lymphocytic choriomeningitis virus (LCMV). Some of the most seminal findings in viral immunology, such as the mapping of MHC I restriction, were discovered using this model system. A major advantage of working with the LCMV model is the availability of advanced tools and well-characterized strains of the virus that yield unique outcomes upon inoculation. Two commonly used paradigms in the field involve the use of LCMV Armstrong/Clone 13 (Ahmed et al., 1984) and LCMV Aggressive/Docile (both derived from LCMV WE) (Pfau et al., 1982). Injection of high-dose (2 × 106 PFU) Clone 13 or Docile results in a protracted phase of viral clearance, whereas LCMV Armstrong and Aggressive injected in a similar manner are purged acutely (i.e. 7–10 days). The Armstrong/Clone 13 comparison has been particularly useful in identifying factors associated with protracted clearance. For example, it is well described that intravenous inoculation with high-dose Clone 13 is associated with functional exhaustion/deletion of LCMV-specific CD8+ (Zajac et al., 1998) and CD4+ T cells (Brooks et al., 2005), which is not observed in mice infected with Armstrong. Exhaustion is defined as a reduced ability (or inability) of anti-viral T cells to produce certain cytokines and effector molecules. Importantly, similar exhausted anti-viral T cells have been found in patients persistently infected with HIV and HCV, suggesting an association between viruses that persist and T cell exhaustion. Because of this association, it was surmised that reversal of the exhaustive state might promote viral clearance. In the Clone 13 model, three molecules [PD-1 (Barber et al., 2006), IL-10 (Brooks et al., 2006) and LAG-3 (Blackburn et al., 2009)] were recently shown to be up-regulated during the chronic stage of infection, and blockade (or genetic deletion) of these molecules facilitates viral clearance. Blockade of PD-1 also improved anti-viral responses in primates infected with simian immunodeficiency virus (Velu et al., 2009), suggesting that interference with inhibitory molecules might prove useful for the treatment of persistent viral infections in humans such as HIV infection. The LCMV model has undoubtedly proven useful for the identification of factors associated with persistence that apply to chronic viral infections in humans. Another fundamental feature of the LCMV model is the requirement for anti-viral CD4+ T cells in the maintenance of functional CD8+ T cells and the eventual clearance of the virus (Matloubian et al., 1994). Over a period of months, LCMV Clone 13 is eventually controlled in immunocompetent hosts; however, in the absence of CD4+ T cells, the virus persists at high titers indefinitely. It is presently not known how anti-viral CD4+ T cells sustain the activities of CD8+ T cells in the LCMV model, but three papers recently published in Science have shed light on this important question by identifying a cytokine (IL-21) produced by anti-viral CD4+ T cells that acts on CD8+ T cells and is required for the clearance of immunosuppressive LCMV strains (i.e. Clone 13 and Docile) (Elsaesser et al., 2009; Frohlich et al., 2009; Yi et al., 2009). IL-21 is a member of a family of cytokines that bind to the common cytokine receptor γ chain (Spolski and Leonard, 2008). This family includes IL-2, IL-4, IL-7, IL-9 and IL-15. The receptor for IL-21 is expressed on T cells, B cells, NK cells, dendritic cells, macrophages, epithelial cells and keratinocytes, and the cytokine itself is produced predominantly by CD4 T cells (Th1, Th2 and Th17 lineages). Fitting with its diverse receptor expression pattern, IL-21 is thought to play a role in CD4+ T cell differentiation/function, NK cell cytotoxicity/proliferation, plasma cell differentiation, immunoglobulin production and cytotoxic lymphocyte function/proliferation/survival. In fact, IL-21 is presently being tested in clinical trials for the treatment of certain types of cancer. The diversity of immune functions associated with IL-21, and its production predominantly by CD4+ T cells, made it an interesting candidate to consider during a persistent viral infection. In these three recently published studies, investigators used the LCMV Clone 13 (Elsaesser et al., 2009; Yi et al., 2009) and Docile (Frohlich et al., 2009) models of chronic infection to define the importance of IL-21 in facilitating anti-viral immunity. IL-21 was found to be produced predominantly by LCMV-specific CD4 T cells at both early and late time points post-infection. Interestingly, infection of IL-21 receptor knockout mice with the immunosuppressive variants of LCMV resulted in more pronounced CD8+ T cell exhaustion/deletion and an inability to eventually control the infection. In contrast, IL-21 receptor-knockout mice had no difficulty clearing LCMV Armstrong or WE, which are purged acutely following intravenous inoculation. Virus-specific T cells expanded normally, and a memory T cell pool emerged following LCMV clearance. It was postulated in all three studies that IL-21 acts directly on anti-viral CD8+ T cells during chronic infection, and this was based on two lines of investigation. First, infection of bone marrow chimeras containing wild-type and IL-21 receptor knockout bone marrow revealed that wild-type LCMV-specific CD8+ T cells numerically out-competed their receptor knockout counterparts. Second, direct injection of IL-21 into CD4+ T cell knockout mice, which normally show heightened exhaustion following Clone 13 infection, restored some function to anti-viral CD8+ T cells and reduced viral loads. Unfortunately, treatment was also associated with 70% mortality, suggesting that direct injection of IL-21 promotes intolerable levels of immunopathology during chronic infection. This result needs to be taken into consideration when considering IL-21 as a therapy for persistently infected humans. In addition to the T cell defects, a significant reduction in LCMV-specific antibodies was also observed in IL-21 receptor knockout mice following infection with immunosuppressive LCMV strains, consistent with the importance of IL-21 in the generation of plasma cells and production of immunoglobulins (Spolski and Leonard, 2008). Although the anti-viral B cell response was not a major focus of these studies, it is important to consider the collective impact of IL-21 on adaptive immunity in the Clone 13 and Docile models. Recent studies have shown that an antibody response is important for controlling chronic LCMV infections (Bergthaler et al., 2009), and IL-21 receptor deficiency on B cells could strain CD8 T cell responses by impeding antibody production and consequently elevating viral titers. CD8 T cells exposed to higher viral loads would be expected to exhaust more quickly, which is the phenotype observed in IL-21 receptor knockout mice. Additional studies are required to tease out the exact role played by IL-21 on anti-viral B cells versus CD8+ T cells during chronic viral infection. Given the complexity and diversity of persistent viral infections, it is important to understand all of the factors that decide the outcome of a viral infection. Following infection, many factors can dictate whether a virus transitions into a state of persistence. These recent studies indicate that IL-21, a cytokine produced by anti-viral CD4 T cells, is one such factor that orchestrates successful viral clearance following LCMV infection. Because IL-21 has the capacity to impact multiple arms of the immune system simultaneously, an important next step will be to define how IL-21 influences each immune cell that expresses its receptor during chronic infection. It will also be important to extend these findings to persistent viral infections in humans.

Persistent viruses, such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, cause major health problems worldwide and are extraordinarily difficult to clear following the establishment of persistence. Given the challenges associated with clearing persistent infections, it is important to develop and mechanistically understand therapeutic strategies that successfully achieve viral eradication without inducing permanent damage in the host. Studies using the lymphocytic choriomeningitis virus (LCMV) model system have convincingly demonstrated that a systemic persistent viral infection can be completely purged from a murine host by using a therapeutic approach referred to as adoptive immunotherapy (1, 15, 22, 29, 30). Remarkably, total body control of multiple persistent viral infections in both the mouse (1, 15, 22, 29, 30) and humans (8, 14, 24, 26, 31) can be achieved using adoptive immunotherapy. When mice are persistently infected at birth or in utero with LCMV (referred to as carrier mice), the virus establishes systemic persistence (6). Adult LCMV carrier mice are tolerant to the virus at the T-cell level and thus are unable to eradicate the pathogen (23), which provides an excellent model to study immunotherapeutic regimens. Immunocytotherapy relies on the adoptive transfer of virus-specific memory CD8 and CD4 T cells from LCMV-immune donor mice into recipient carrier mice (1, 15, 22, 29, 30). Following the therapeutic administration of memory cells, LCMV is purged from most peripheral tissues of carrier mice in 14 days, whereas more than 100 days are required to clear virus from the central nervous system (CNS) and kidneys (1, 15, 22). Furthermore, successful viral clearance requires antiviral “memory” but not “effector” T cells (11). Thus, in addition to its proven therapeutic relevance, this model also provides a paradigm to understand factors that regulate memory T cells following secondary exposure to pathogens in vivo. The mechanisms leading to activation of naive T cells have been well described and involve recognition of major histocompatibility complex (MHC) peptide through the T-cell receptor (TCR) as well as costimulation (e.g., CD80 and CD86 interactions) (4, 25, 27). On the other hand, the factors that govern the activation and secondary expansion of memory CD8+ and CD4+ T cells are less clearly defined, particularly in an in vivo therapeutic setting. When memory T cells reencounter cognate antigen, they respond rapidly by producing cytokines and dividing. Previous studies indicated that there was no role for dendritic cells or costimulation (4, 27) in the reactivation of memory T cells; however, three recent studies have shown that dendritic cells (DCs) stimulate memory T-cell activity upon antigen rechallenge (2, 33) and during adoptive immunotherapy (15). Because MHC class I antigen (MHC-I) is expressed on nearly all cell types but costimulatory molecules are not, these three studies strongly suggested that DCs were influencing memory T cells with costimulatory pathways thought only to be required during priming. Indeed, when the issue was reexamined, it was revealed that memory CD8+ and CD4+ T cells require CD28-CD80/CD86 costimulation to be fully reactivated upon secondary exposure to antigen (3, 7, 21). Because therapeutically administered memory T cells require effective interactions with the host hematopoietic system (10), in particular dendritic cells (15), to achieve successful viral clearance, we set out to address several unanswered questions. First, is costimulation required for the immunotherapeutic clearance of an established persistent viral infection? This is a particularly important question because the requirements imposed on therapeutically administered memory T cells, which encounter immediate and overwhelmingly high levels of virus, heightened antigenic stimulation, and a unique inflammatory milieu, are likely to be different than those faced by endogenous memory T cells following pathogen rechallenge in an otherwise-quiescent environment. The second question we set out to address in this study was whether costimulation blockade could modulate the activities of an immunotherapeutic regimen consisting of memory T cells. This question is of great importance in a clinical setting where pathogen-specific memory T cells can induce severe tissue pathology through the release of effector molecules (12). Thus, it is critical to have a strategy to limit the magnitude of an undesirable response without impeding viral clearance.

Several arenaviruses cause hemorrhagic fever disease in humans for which no licensed vaccines are available and current therapeutic intervention is limited to the off-label use of the wide-spectrum antiviral ribavirin. However, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) has proven to be a Rosetta stone for the investigation of virus–host interactions. Arenaviruses have a bisegmented negative-strand RNA genome. The S segment encodes for the virus nucleoprotein and glycoprotein, whereas the L segment encodes for the virus polymerase (L) and Z protein. The ability to generate recombinant LCMV (rLCMV) expressing additional foreign genes of interest would open novel avenues for the study of virus–host interactions and the development of novel vaccine strategies and high-throughput screens to identify antiarenaviral molecules. To this end, we have developed a trisegmented (1L + 2S) rLCMV-based approach (r3LCMV). Each of the two S segments in r3LCMV was altered to replace one of the viral genes by a gene of interest. All r3LCMVs examined expressing different reported genes were stable both genetically and phenotypically and exhibited wild-type growth properties in cultured cells. Reporter gene expression in r3LCMV-infected cells provided an accurate surrogate of levels of virus multiplication. Notably, some r3LCMVs displayed highly attenuated virulence in mice but induced protective immunity against a subsequent lethal challenge with wild-type LCMV, supporting the potential development of r3LCMV-based vaccines.

After infection with RML murine scrapie agent, transgenic (tg) mice expressing prion protein (PrP) without its glycophosphatidylinositol (GPI) membrane anchor (GPI −/− PrP tg mice) continue to make abundant amounts of the abnormally folded disease-associated PrPres but have a normal life span. In contrast, all age-, sex-, and genetically matched mice with a GPI-anchored PrP become moribund and die due to a chronic progressive neurodegenerative disease by 160 days after RML scrapie agent infection. We report here that infected GPI −/− PrP tg mice, although free from progressive neurodegenerative disease of the cerebellum and extrapyramidal and pyramidal systems, nevertheless suffer defects in learning and memory, long-term potentiation, and neuronal excitability. Such dysfunction increases over time and is associated with an increase in gamma aminobutyric acid (GABA) inhibition but not loss of excitatory glutamate/ N -methyl- d -aspartic acid. Enhanced deposition of abnormally folded infectious PrP (PrPsc or PrPres) in the central nervous system (CNS) localizes with GABAA receptors. This occurs with minimal evidence of CNS spongiosis or apoptosis of neurons. The use of monoclonal antibodies reveals an association of PrPres with GABAA receptors. Thus, the clinical defects of learning and memory loss in vivo in GPI −/− PrP tg mice infected with scrapie agent may likely involve the GABAergic pathway.

Once a virus infection establishes persistence in the central nervous system (CNS), it is especially difficult to eliminate from this specialized compartment. Therefore, it is of the utmost importance to fully understand scenarios during which a persisting virus is ultimately purged from the CNS by the adaptive immune system. Such a scenario can be found following infection of adult mice with an immunosuppressive variant of lymphocytic choriomeningitis virus (LCMV) referred to as clone 13. In this study we demonstrate that following intravenous inoculation, clone 13 rapidly infected peripheral tissues within one week, but more slowly inundated the entire brain parenchyma over the course of a month. During the establishment of persistence, we observed that genetically tagged LCMV-specific cytotoxic T lymphocytes (CTL) progressively lost function; however, the severity of this loss in the CNS was never as substantial as that observed in the periphery. One of the most impressive features of this model system is that the peripheral T cell response eventually regains functionality at ~60–80 days post-infection, and this was associated with a rapid decline in virus from the periphery. Coincident with this "reanimation phase" was a massive influx of CD4 T and B cells into the CNS and a dramatic reduction in viral distribution. In fact, olfactory bulb neurons served as the last refuge for the persisting virus, which was ultimately purged from the CNS within 200 days post-infection. These data indicate that a functionally revived immune response can prevail over a virus that establishes widespread presence both in the periphery and brain parenchyma, and that therapeutic enhancement of an existing response could serve as an effective means to thwart long term CNS persistence.

Glioblastoma Multiforme (GBM), a grade IV astrocytoma is the most aggressive primary brain tumor. GBM patients have the worst prognoses with median overall survival of less than 15 months from the time of diagnosis. The standard of care consists of maximal surgical resection followed by chemotherapy, given concomitantly with and following radiotherapy. Despite these measures, disease progression or recurrence is virtually inevitable and occurs at a median time of approximately 7 months. Among the more promising experimental treatments for recurrent GBM is the “vaccination” of patients with dendritic cells to elicit anti-tumor immune responses. The safety of this approach has been well demonstrated, but the clinical response rate of patients has ranged from 8%-30%. To improve upon these responses, we investigated the ability of microglia; the resident immune cells of the CNS, to better activate cytotoxic T lymphocytes within the tumor microenvironment. Using a murine syngeneic intracranial tumor model, we found that combined vaccination with dendritic cells and microglia is more effective than either alone. Combination treated mice had sustained regression of tumors over a four-week imaging period and the median survival of the animals was greater than one year. In contrast, the median survivals of the various control groups were ≤ 52 days. Translation of our findings to the clinical setting requires a source of patient-specific microglia. Here, we report the sequential differentiation of human induced pluripotent stem cells (hiPSC) into hematopoietic progenitor-like cells, and then into cells with a phenotype and gene expression profile resembling that of primary brain-isolated microglia. Similarly, human iPSC-derived microglia (hiPS-MG) exhibit phagocytic activity; produce inflammatory cytokines and reactive oxygen species. We have also differentiated mouse induced pluripotent stem cells into microglia (miPS-MG) with gene expression profile and phenotype similar to primary neonatal microglia. Mice treated with a combination therapy of tumor lysate-pulsed miPS-MG and dendritic cells show median survival greater than one year, whereas the survival for the control group was less than 50 days. For direct clinical translation of our findings, the ability to readily differentiate hiPSC into microglia in vitro will allow for the generation of patient-specific cells for use in a GBM immunotherapy protocol. hiPSC-derived microglia may also be useful as modulatory cells for a variety of CNS diseases and serve as gene and protein delivery vehicles to the CNS. Acknowledgement: This research was supported by the Intramural Research Program of the NIH [NINDS]. Citation Format: Hetal Pandya, David M. Ichikawa, Michael J. Shen, Andrea B. Sedlock, Kory R. Johnson, Dragan Maric, Dorian B. McGavern, Harry L. Malech, John K. Park. Microglia differentiated from induced pluripotent stem cells treat glioma tumors in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5029. doi:10.1158/1538-7445.AM2015-5029

Doni et al. use four tissue damage models in mice and find that the fluid phase pattern recognition molecule pentraxin 3 (PTX3) plays a role in tissue remodeling and repair. PTX3 binds fibrinogen/fibrin and plasminogen at an acidic pH within tissues. Mice deficient in PTX3 present defects in fibrin deposition, clot formation, collagen deposition, and macrophage-mediated fibrinolysis., Pentraxin 3 (PTX3) is a fluid-phase pattern recognition molecule and a key component of the humoral arm of innate immunity. In four different models of tissue damage in mice, PTX3 deficiency was associated with increased fibrin deposition and persistence, and thicker clots, followed by increased collagen deposition, when compared with controls. Ptx3-deficient macrophages showed defective pericellular fibrinolysis in vitro. PTX3-bound fibrinogen/fibrin and plasminogen at acidic pH and increased plasmin-mediated fibrinolysis. The second exon-encoded N-terminal domain of PTX3 recapitulated the activity of the intact molecule. Thus, a prototypic component of humoral innate immunity, PTX3, plays a nonredundant role in the orchestration of tissue repair and remodeling. Tissue acidification resulting from metabolic adaptation during tissue repair sets PTX3 in a tissue remodeling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity.

Persistent infections and amyloid disorders afflict a significant number of people worldwide. It would appear at first glance that the treatment of these afflictions should be entirely unrelated; however, in both cases components of the adaptive immune system have been harnessed in an attempt to provide some therapeutic relief. Given that the ability of a pathogen to establish persistence often depends in part on a shortcoming of the adaptive immune response, it seems logical to devise immunotherapies with the intention of supplementing (or replacing) the insufficient immunologic element. A case in point is an intervention referred as immunocytotherapy, which relies upon the adoptive transfer of pathogen-specific T lymphocytes into a persistently infected host. Remarkably, the adoptively transferred T lymphocytes not only have the capacity to clear the persistent infection, but can also provide the recipient with protection against subsequent rechallenge (i.e., immunologic memory). Treatment of amyloid disorders (e.g., Alzheimer disease, sporadic inclusion-body myositis) with a similar therapeutic approach is complicated by the fact that the aberrant protein accumulations are self-derived. Focusing the adaptive response on these aberrant self-proteins has the potential to result in autoimmune pathology. This review critically evaluates the importance of immunotherapeutic approaches for the treatment of persistent infections and amyloid disorders, and attempts to delineate the interventions that are most likely to succeed in an exceedingly complex disorder such as sporadic inclusion-body myositis.

It is generally accepted that both self- and pathogen-specific T lymphocytes have the potential to mediate immunopathogenesis and contribute to a variety of human ailments. Despite this unfortunate tendency to induce tissue injury, these cells are guided by interactions with peptide-loaded major histocompatibility complexes (MHC) and adhere appropriately to a vital evolutionary constraint imposed by the host: specificity. More recently, a series of studies have demonstrated that bystander T cells of an irrelevant specificity can bypass peptide/MHC restriction and become active participants in immunopathology. This review critically evaluates the role of bystander T cells in immunopathogenesis and pathogen clearance in the periphery as well as the central nervous system and attempts to establish the likelihood of their participation in human disease.

Failure to clear persistent viral infections results from the early loss of T cell activity. A pertinent question is whether the immune response is programmed to fail or if nonresponsive T cells can specifically be fixed to eliminate infection. Although evidence indicates that T cell expansion is permanently programmed during the initial priming events, the mechanisms that determine the acquisition of T cell function are less clear. Herein we show that in contrast to expansion, the functional programming of T cell effector and memory responses in vivo in mice is not hardwired during priming but is alterable and responsive to continuous instruction from the antigenic environment. As a direct consequence, dysfunctional T cells can be functionally reactivated during persistent infection even after an initial program of inactivation has been instituted. We also show that early therapeutic reductions in viral replication facilitate the preservation of antiviral CD4+ T cell activity, enabling the long-term control of viral replication. Thus, dysfunctional antiviral T cells can regain activity, providing a basis for future therapeutic strategies to treat persistent viral infections.

Inhibition of tumor angiogenesis is a promising approach for cancer therapy. As an endothelial cell–specific receptor kinase expressed almost exclusively on the surface of vascular endothelium, Tie-2 has an important role in tumor angiogenesis. To explore the therapeutic potential of blocking Tie-2 receptor-interaction pathway, an adenoviral vector was used to deliver a recombinant single-chain antibody fragment rabbit intrabody (pAd-2S03) capable of inhibition of both mouse and human Tie-2 surface expression. pAd-2S03 was given to mice with well-established primary tumors, either a human Kaposi's sarcoma (SLK) or a human colon carcinoma (SW1222). The intrabody significantly inhibited growth of both tumors (75% and 63%, respectively) when compared with pAd-GFP control-treated tumors (P < 0.01). Histopathologic analysis of cryosections taken from mice treated with pAd-2S03 revealed a marked decrease in vessel density, which was reduced by >87% in both tumor models when compared with control-treated tumors (P < 0.01). In contrast, human Tie-2-monospecific pAd-1S05 intrabody did not affect the growth of tumors, indicating that the antitumor effect of pAd-2S03 was due to the inhibition of tumor angiogenesis in these murine models. Our results show that the Tie-2 receptor pathway is essential for both SLK sarcoma and SW1222 colon carcinoma xenograft growth. The present study shows the potential utility of antiangiogenic agents that target the endothelium-specific receptor Tie-2 for down-regulation or genetic deletion.

Two subsets of dendritic cell (DCs), plasmacytoid (p) and myeloid (m) DCs, have been described in humans and mice. These subsets are known to have divergent roles during an immune response, but their developmental course is unclear. Here we report that virus infection induces bone marrow pDCs to differentiate into mDCs, thereby undergoing profound phenotypic and functional changes including the acquisition of enhanced antigen-presenting capacity and the ability to recognize different microbial structures through Toll-like receptor 4. The conversion of pDCs into mDCs is also induced by the injection of double-stranded RNA and requires type I interferons. Our results establish a precursor-product developmental relationship between these two DC subsets and highlight unexpected plasticity of bone marrow pDCs.

Although both self- and pathogen-specific T cells can participate in tissue destruction, recent studies have proposed that after viral infection, bystander T cells of an irrelevant specificity can bypass peptide-MHC restriction and contribute to undesired immunopathological consequences. To evaluate the importance of this mechanism of immunopathogenesis, we determined the relative contributions of Ag-specific and bystander CD8+ T cells to the development of CNS disease. Using lymphocytic choriomeningitis virus (LCMV) as a stimulus for T cell recruitment into the CNS, we demonstrate that bystander CD8+ T cells with an activated surface phenotype can indeed be recruited into the CNS over a chronic time window. These cells become anatomically positioned in the CNS parenchyma, and a fraction aberrantly acquires the capacity to produce the effector cytokine, IFN-γ. However, when directly compared with their virus-specific counterparts, the contribution of bystander T cells to CNS damage was insignificant in nature (even when specifically activated). Although bystander T cells alone failed to cause tissue injury, transferring as few as 1000 naive LCMV-specific CD8+ T cells into a restricted repertoire containing only bystander T cells was sufficient to induce immune-mediated pathology and reconstitute a fatal CNS disease. These studies underscore the importance of specific T cells in the development of immunopathology and subsequent disease. Because of highly restrictive constraints imposed by the host, it is more likely that specific, rather than nonspecific, bystander T cells are the active participants in T cell-mediated diseases that afflict humans.

Devic's disease [neuromyelitis optica (NMO)] is an idiopathic inflammatory demyelinating disease of the CNS, characterized by attacks of optic neuritis and myelitis. The mechanisms that result in selective localization of inflammatory demyelinating lesions to the optic nerves and spinal cord are unknown. Serological and clinical evidence of B cell autoimmunity has been observed in a high proportion of patients with NMO. The purpose of this study was to investigate the importance of humoral mechanisms, including complement activation, in producing the necrotizing demyelination seen in the spinal cord and optic nerves. Eighty-two lesions were examined from nine autopsy cases of clinically confirmed Devic's disease. Demyelinating activity in the lesions was immunocytochemically classified as early active (21 lesions), late active (18 lesions), inactive (35 lesions) or remyelinating (eight lesions) by examining the antigenic profile of myelin degradation products within macrophages. The pathology of the lesions was analysed using a broad spectrum of immunological and neurobiological markers, and lesions were defined on the basis of myelin protein loss, the geography and extension of plaques, the patterns of oligodendrocyte destruction and the immunopathological evidence of complement activation. The pathology was identical in all nine patients. Extensive demyelination was present across multiple spinal cord levels, associated with cavitation, necrosis and acute axonal pathology (spheroids), in both grey and white matter. There was a pronounced loss of oligodendrocytes within the lesions. The inflammatory infiltrates in active lesions were characterized by extensive macrophage infiltration associated with large numbers of perivascular granulocytes and eosinophils and rare CD3(+) and CD8(+) T cells. There was a pronounced perivascular deposition of immunoglobulins (mainly IgM) and complement C9neo antigen in active lesions associated with prominent vascular fibrosis and hyalinization in both active and inactive lesions. The extent of complement activation, eosinophilic infiltration and vascular fibrosis observed in the Devic NMO cases is more prominent compared with that in prototypic multiple sclerosis, and supports a role for humoral immunity in the pathogenesis of NMO. Based on this study, future therapeutic strategies designed to limit the deleterious effects of complement activation, eosinophil degranulation and neutrophil/macrophage/microglial activation are worthy of further investigation.

Antigen-specific CD8(+) T cells are required for the clearance of most viral infections and several cancers. However, it is not clear in vivo whether CD8(+) T cells can engage multiple targets simultaneously, engagement results in the formation of an immunologic synapse or molecules involved in CD8 function are redistributed to the synapse. We used here high-resolution microscopy to visualize interactions between virus-specific effectors and target cells in vivo. Using either in situ tetramer staining or green fluorescent protein-labeled virus-specific T cells, we have shown that a single CD8(+) T cell can engage two or three targets, a synapse occurs at the site of engagement and molecules involved in attachment (lymphocyte function-associated antigen 1), signaling (Lck) and lytic activity (perforin) are differentially positioned on the T cell. In addition, we have established an in vivo approach for assessing the intricacies of antigen-specific T cell activation, migration, engagement, memory and other defining elements of adaptive immunity.

IFN-gamma is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-gamma, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler's virus model of multiple sclerosis. During viral infections, IFN-gamma is produced by natural killer (NK) cells, CD4(+) and CD8(+) T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-gamma-mediated protection. To determine the lymphocyte subsets that produce IFN-gamma to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-gamma-production. We demonstrate that IFN-gamma production by both CD4(+) and CD8(+) T cell subsets is critical for resistance to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4(+) T cells make a greater contribution to IFN-gamma-mediated protection. To determine the cellular targets of IFN-gamma-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-gamma receptor. We demonstrate that IFN-gamma receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination.

Spinal cord pathology, such as demyelination and axonal loss, is a common feature in multiple models of central nervous system (CNS) injury and disease. Development of methods to quantify spinal cord pathology objectively would aid studies designed to establish mechanisms of damage, correlate pathology with neurologic function, and assess therapeutic interventions. In this study, we describe sensitive methods to objectively quantify spinal cord demyelination, remyelination, atrophy, and axonal loss following the initiation of a progressive inflammatory demyelinating disease with Theiler's murine encephalomyelitis virus (TMEV). Spinal cord demyelination, remyelination, and atrophy were quantified from representative 1-μm-thick cross sections embedded in Araldite plastic using interactive image analysis. In addition, this study demonstrates novel, automated methodology to quantify axonal loss from areas of normal-appearing white matter, as a measure of secondary axonal injury following demyelination. These morphologic methods, which are applicable to various models of CNS injury, provide an innovative way to assess the benefits of therapeutic agents, to determine mechanisms of spinal cord damage, or to establish a correlation with sensitive measures of neurologic function. J. Neurosci Res 58:492–504. © 1999 Wiley-Liss, Inc.

We previously showed that Theiler's murine encephalomyelitis virus (TMEV)-infected major histocompatibility complex (MHC) class II-deficient mice develop both demyelination and neurologic deficits, whereas MHC class I-deficient mice develop demyelination but no neurologic deficits. The absence of neurologic deficits in the class I-deficient mice was associated with preserved sodium channel densities in demyelinated lesions, a relative preservation of axons, and extensive spontaneous remyelination. In this study, we investigated whether TMEV-infected class II-deficient mice, which have an identical genetic background (C57BL/6 x 129) as the class I-deficient mice, have preserved axons and spontaneous myelin repair following chronic TMEV-infection. Both class I- and class II-deficient mice showed similar extents of demyelination of the spinal cord white matter 4 months after TMEV infection. However, the class I-deficient mice demonstrated remyelination by oligodendrocytes, whereas class II-deficient mice showed minimal if any myelin repair. Demyelinated lesions, characterized by inflammatory infiltrates in both mutants, revealed disruption of axons in class II- but not class I-deficient mice. Further characterization revealed that even though class II-deficient mice lacked TMEV-specific IgG, they had virus-specific IgM, which, however, did not neutralize TMEV in vitro. In addition, class II-deficient mice developed TMEV-specific cytotoxic T-lymphocytes in the CNS during the acute (7 days) disease, but these cytotoxic lymphocytes were not present in the chronic stage of disease, despite a high titer of infectious virus throughout the disease. We envision that the presence of demyelination, high virus titer, absence of remyelination, and axonal disruption in chronically infected class II-deficient mice contributes to the development of paralytic disease.

In this study we demonstrate perforin-mediated cytotoxic effector function is necessary for viral clearance and may directly contribute to the development of neurologic deficits after demyelination in the Theiler’s murine encephalomyelitis virus (TMEV) model of multiple sclerosis. We previously demonstrated major histocompatability complex (MHC) class I-deficient (β2m-deficient) mice with an otherwise resistant genotype develop severe demyelination with minimal neurologic disease when chronically infected with TMEV. These studies implicate CD8+T cells as the pathogenic cell in the induction of neurologic disease after demyelination. To determine which effector mechanisms of CD8+T cells, granule exocytosis or Fas ligand expression, play a role in the development of demyelination and clinical disease, we infected perforin-deficient,lpr(Fas mutation), andgld(Fas ligand mutation) mice with TMEV. Perforin-deficient mice showed viral persistence in the CNS, chronic brain pathology, and demyelination in the spinal cord white matter. Perforin-deficient mice demonstrated severely impaired MHC class I-restricted cytotoxicity against viral epitopes, but normal MHC class II-restricted delayed-type hypersensitivity responses to virus antigen. Despite demyelination, virus-infected perforin-deficient mice showed only minimal neurologic deficits as indicated by clinical disease score, activity monitoring, and footprint analysis. Perforin- and MHC class II-deficient mice (with functional CD8+T cells and perforin molecules and an H-2bhaplotype) had comparable demyelination and genotype, however, only the latter showed severe clinical disease.Gldandlprmice demonstrated normal TMEV-specific cytotoxicity and maintained resistance to TMEV-induced demyelinating disease. These studies implicate perforin release by CD8+T cells as a potential mechanism by which neurologic deficits are induced after demyelination.

Demyelination alone has been considered sufficient for development of neurological deficits following central nervous system (CNS) disease. However, extensive demyelination is not always associated with clinical deficits in patients with multiple sclerosis (MS), the most common primary demyelinating disease in humans. We used the Theiler's murine encephalomyelitis virus model of demyelination to investigate the role of major histocompatibility complex (MHC) class I and class II gene products in the development of functional and neurophysiological deficits following demyelination. We measured spontaneous clinical activity by two independent assays and recorded hind-limb motor-evoked potentials in infected class I-deficient and class II-deficient mice of an identical genetic background as well as in highly susceptible SJL/J mice. The results show that despite a similar distribution and extent of demyelinated lesions in all mice, only class I-deficient mice were functionally normal. We propose that the mechanism by which demyelinated class I-deficient mice maintain neurologic function results from increased sodium channel densities and the relative preservation of axons. These findings are the first to implicate a role for MHC class I in the development of neurological deficits following demyelination.

By definition persistent viral infections are accompanied by immune evasion, and most notably the failure to sustain robust T cell responses, responses that are essential to control of these infections. Identifying and understanding the immunoregulatory mechanisms that direct productive versus abortive T cell responses to viral infections is critical, but currently unclear. Using the model of persistent lymphocytic choriomeningitis virus (LCMV) infection, we found that upregulation of interleukin (IL)-10 early during infection induces T cell exhaustion and directly leads to viral persistence. Blockade of IL-10 activity induced sustained T cell function, the rapid elimination of an otherwise persistent virus infection and development of functional memory T cells capable of preventing re-infection. Antibody blockade of the IL-10 receptor during the chronic phase of infection restored T cell activity and eliminated an established persistent viral infection. Further, by manipulating this immunoregulatory molecule we significantly enhance immune responses to vaccination, indicating a potential “adjuvant” role to anti-IL10 immunotherapy. Thus, we propose a novel therapy to treat persistent viral infection by targeting host immunoregulatory molecules and reactivating antiviral T cells.

Adoptive immunotherapy relies on transfer of exogenously derived pathogen-specific (memory) T cells and is a highly successful approach to purge an established persistent viral infection. For example, administration of adoptive immunotherapy to mice persistently infected with lymphocytic choriomeningitis virus (LCMV) (carrier mice) results in systemic eradication of the virus, including noncytopathic clearance of persistently infected CNS neurons. By visualizing GFP-tagged LCMV-specific memory CD8+ T cells on brain reconstructions, it was revealed that cytotoxic lymphocytes (CTL) distributed throughout the CNS within 8 days after immunotherapy. Flow cytometric and microscopic analyses revealed that conventional dendritic cells arrived in the CNS concurrent with the memory CTL and induced anti-viral cytokine production (e.g., TNF-alpha). When adoptive immunotherapy was performed in carrier mice depleted of DCs or with memory T cells deficient in TNF-alpha, the adoptively administered memory T cells failed to purge the persistent infection. We further demonstrated by employing CTLA-4-Fc blockade and CCR2 deficient recipients that memory CTL rely on costimulation and DC migration, respectively, for optimal secondary expansion and efficient viral clearance. These studies underscore the importance of DC mobility and memory CTL-DC interactions in the clearance of a persistent viral infection.

IFN-gamma is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-gamma, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler's virus model of multiple sclerosis. During viral infections, IFN-gamma is produced by natural killer (NK) cells, CD4(+) and CD8(+) T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-gamma-mediated protection. To determine the lymphocyte subsets that produce IFN-gamma to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-gamma-production. We demonstrate that IFN-gamma production by both CD4(+) and CD8(+) T cell subsets is critical for resistance to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4(+) T cells make a greater contribution to IFN-gamma-mediated protection. To determine the cellular targets of IFN-gamma-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-gamma receptor. We demonstrate that IFN-gamma receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination.

A major question in neurobiology is whether myelin repair can restore neurological function following the course of a severe, progressive CNS demyelinating disease that induces axonal loss. In this study we used Theiler's murine encephalomyelitis virus (TMEV) to induce a chronic progressive CNS demyelinating disease in mice that was immune-mediated and pathologically similar to human multiple sclerosis. Because immunosuppression of chronically TMEV-infected mice has been shown to enhance myelin repair, we first addressed the potential roles of CD4(+) and CD8(+) T cells in the inhibition of CNS remyelination during chronic disease. TMEV infection of susceptible PL/J mice deficient in CD4(+) but not CD8(+) T cells demonstrated a significant increase in severity of pathogenesis when compared with wild-type controls. This was characterized by enhanced demyelination, spinal cord atrophy, neurological deficits, and mortality. Interestingly, the PL/J CD4(-/-) mice that survived to the chronic stage of the disease had nearly complete spontaneous myelin repair mediated by both oligodendrocytes and infiltrating Schwann cells. Therefore, we next addressed whether this spontaneous myelin repair was associated with improved neurological function despite the increased pathology. Of interest, all surviving PL/J CD4(-/-) mice showed partial restoration of motor coordination and gait that coincided temporally with spontaneous myelin repair. Furthermore, functional recovery of motor coordination correlated strongly with the percentage of myelin repair mediated by Schwann cells, whereas restoration of hindlimb gait correlated with oligodendrocyte-mediated myelin repair. This is the first study to demonstrate that spontaneous remyelination correlates with partial restoration of neurological function during the course of a progressive, immune-mediated CNS demyelinating disease. Of greater importance, functional recovery occurred despite previous severe demyelination and spinal cord atrophy.

Dendritic cells (DCs) comprise the major antigen-presenting cells (APCs) of the host, uniquely programmed to stimulate immunologically naïve T lymphocytes. Viruses that can target and disorder the function of these cells enjoy a selective advantage. The cellular receptor for lymphocytic choriomeningitis virus (LCMV), Lassa fever virus (LFV), and several other arenaviruses is alpha-dystroglycan (alpha-DG). Among cells of the immune system, CD11c+ and DEC-205+ DCs primarily and preferentially express alpha-DG. By selection, strains and variants of LCMV generated as quasi-species that bind alpha-DG with high affinity replicate in the majority of CD11c+ and DEC-205+ (75%) DCs, causing a generalized immunosuppression, and establish a persistent infection. In contrast, viral strains and variants that bind with low affinity to alpha-DG display minimal replication in CD11c+ and DEC-205+ DCs (10%), rarely replicate in the white pulp, and generate a robust anti-LCMV CTL response that clears the virus infection. Hence, receptor-virus interaction on DCs in vivo is an essential step in the initiation of virus-induced immunosuppression and viral persistence. Investigation into the mechanism of how virus-infected DCs cause immunosuppression reveals loss of MHC class II surface expression and costimulatory molecules on surface of such DCs. As a consequence DCs are unable to act as APCs, initiate immune responses, and have a defect in migration into the T cell area. These data indicate that LCMV infection influences DC maturation and migration, leading to decreased T cell stimulatory capacity of DCs, events essential for the initiation of immune responses. Because several other viruses known to cause immunosuppression (HIV, measles) interact with DCs, the observations noted here are likely a common selective mechanism by which viruses also are able to evade the host's immune system.