Tissue-Specific Differences in PD-1 and PD-L1 Expression during Chronic Viral Infection: Implications for CD8 T-Cell Exhaustion

The PD-1/PD-L pathway plays a major role in regulating T-cell exhaustion during chronic viral infections in animal models, as well as in humans, and blockade of this pathway can revive exhausted CD8⁺ T cells. We examined the expression of PD-1 and its ligands, PD-L1 and PD-L2, in multiple tissues during the course of chronic viral infection and determined how the amount of PD-1 expressed, as well as the anatomical location, influenced the function of exhausted CD8 T cells. The amount of PD-1 on exhausted CD8 T cells from different anatomical locations did not always correlate with infectious virus but did reflect viral antigen in some tissues. Moreover, lower expression of PD-L1 in some locations, such as the bone marrow, favored the survival of PD-1^Hi exhausted CD8 T cells, suggesting that some anatomical sites might provide a survival niche for subpopulations of exhausted CD8 T cells. Tissue-specific differences in the function of exhausted CD8 T cells were also observed. However, while cytokine production did not strictly correlate with the amount of PD-1 expressed by exhausted CD8 T cells from different tissues, the ability to degranulate and kill were tightly linked to PD-1 expression regardless of the anatomical location. These observations have implications for human chronic infections and for therapeutic interventions based on blockade of the PD-1 pathway.Chronic viral infections are often associated with CD8⁺ T-cell dysfunction (30). This dysfunction, termed exhaustion, includes defects in the ability to produce antiviral cytokines, poor cytotoxicity, a loss of antigen-independent self-renewal, and the inability to vigorously re-expand following antigen exposure (30). These functional deficiencies contrast with the highly functional memory CD8⁺ T cells that are generated after acute infection and maintained via interleukin-7 (IL-7)- and IL-15-mediated homeostatic proliferation (30). During chronic viral infections, T-cell exhaustion often correlates with poor control of viral replication (3, 8, 38, 39). Thus, there is considerable interest in developing strategies to reverse exhaustion and restore function in virus-specific CD8⁺ T cells during chronic infections.Recent studies have revealed an important role for the negative regulatory molecule PD-1 in CD8 T-cell exhaustion during chronic viral infections (29). PD-1, a member of the CD28/CTLA-4 family of costimulatory/coinhibitory receptors, contains both ITIM and ITSM motifs in the intracellular tail and can deliver negative signals, at least partly via recruitment of the phosphatase Shp-2 (29). A role for PD-1 in regulating T-cell responses to chronic viral infections was first observed using lymphocytic choriomeningitis virus (LCMV) infection of mice, where PD-1 was found to be highly expressed on exhausted CD8⁺ T cells from chronically infected animals but not on functional memory CD8⁺ T cells from mice that had cleared an acute strain of the virus (3). In vivo blockade of the PD-1 pathway led to a dramatic increase in the number of virus-specific CD8⁺ T cells, improved functionality of these cells, and enhanced control of viral replication (3). These observations were extended to human chronic viral infections, and a series of studies have demonstrated that human immunodeficiency virus (HIV)-, hepatitis C virus (HCV)-, and HBV-specific CD8⁺ T cells upregulate PD-1 in humans compared to CD8⁺ T cells specific for nonpersisting viruses such as influenza virus or vaccinia virus (6-8, 24, 26, 32, 33, 42). Increasing PD-1 expression also correlates with disease status during HIV infection (8, 42). In vitro blockade of PD-1-PD-L interactions can reinvigorate exhausted virus-specific T-cell responses in humans and appears to have a prominent impact on proliferative expansion and/or prevention of apoptosis in these cases (9, 24, 32). Finally, recent results from in vivo blockade in the macaque simian immunodeficiency virus (SIV) infection model demonstrated the effectiveness of blocking PD-1 in primates during chronic viral infection (36). In these studies, PD-1 blockade enhanced virus-specific T and B-cell responses, lowered viral load, and improved the survival of chronically infected animals. Thus, PD-1 has emerged as not only a major regulator of T-cell exhaustion and viral control during chronic infection but also as an important potential therapeutic target.Despite these important studies and the clear impact of PD-1 blockade on the reversal of T-cell exhaustion, important questions remain. For example, previous work has demonstrated that PD-1 expression is not uniform on subsets of exhausted CD8 T cells (4). However, the expression of PD-1 on exhausted CD8 T cells in multiple tissues, and the relationship between PD-1 expression in these tissues to viral load, the PD-1 ligands and function has not been examined. Given the nonlymphoid accumulation of virus-specific CD8 T cells during chronic viral infections (11, 39) and the predilection of many important chronic infections for replicating in anatomically restricted locations (e.g., HCV and the liver, HIV and mucosal tissues, etc.), the dynamics of PD-1 expression by exhausted CD8 T cells outside the blood and spleen could have important therapeutic implications.In the present study we examined these issues using the mouse model of LCMV infection. Our results demonstrate that exhausted CD8 T cells have a wide range of PD-1 expression in different tissues of chronically infected mice. Virus-specific CD8 T cells in some anatomical locations such as the liver, brain, and bone marrow (BM) expressed high PD-1 for substantially longer than virus-specific CD8⁺ T cells from the spleens or blood of the same mice. Although PD-1 expression in the spleen correlated well with reduced gamma interferon (IFN-γ) and tumor necrosis factor (TNF) production, the PD-1^Hi virus-specific CD8⁺ T cells from the BM remained capable of producing antiviral cytokines ex vivo. In contrast, a strong negative correlation between PD-1 expression and cytotoxicity existed for exhausted CD8 T cells from all tissues tested. PD-L1 expression was high in the spleen, whereas in the BM antigen-presenting cell (APC) populations expressed lower amounts of PD-L1. Survival of PD-1^Hi CD8⁺ T cells from the BM was decreased in the presence of splenic APCs, suggesting that different tissue microenvironments in vivo could selectively support the persistence of PD-1^Hi exhausted CD8 T cells. Since PD-1 expression differs by anatomical location, these observations suggest that PD-1 blockade in vivo will have varying impacts on exhausted CD8 T cells from different tissues or anatomical locations. These observations have implications for human chronic infections such as HBV, HCV, and HIV.     

Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Responses during Primary Infection Are Major Determinants of the Viral Set Point and Loss of CD4+ T Cells

Primary HIV-1 infection (PHI) is marked by a flu-like syndrome and high levels of viremia that decrease to a viral set point with the first emergence of virus-specific CD8⁺ T-cell responses. Here, we investigated in a large cohort of 527 subjects the immunodominance pattern of the first virus-specific cytotoxic T-lymphocyte (CTL) responses developed during PHI in comparison to CTL responses in chronic infection and demonstrated a distinct relationship between the early virus-specific CTL responses and the viral set point, as well as the slope of CD4⁺ T-cell decline. CTL responses during PHI followed clear hierarchical immunodominance patterns that were lost during the transition to chronic infection. Importantly, the immunodominance patterns of human immunodeficiency virus type 1 (HIV-1)-specific CTL responses detected in primary, but not in chronic, HIV-1 infection were significantly associated with the subsequent set point of viral replication. Moreover, the preservation of the initial CD8⁺ T-cell immunodominance patterns from the acute into the chronic phase of infection was significantly associated with slower CD4⁺ T-cell decline. Taken together, these data show that the specificity of the initial CTL response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.In the first weeks after human immunodeficiency virus type 1 (HIV-1) acquisition, viral loads peak at high levels, accompanied by a flu-like syndrome (15). A rapid depletion of the CD4⁺ T-cell population occurs during this acute infection, in particular, within the gastrointestinal tract-associated lymphoid tissue (6, 19, 20), marking a nonrecoverable scar on the immune system. With the resolution of the clinical syndromes, viral loads decrease to a set point, which persists at this level for months to years until progressive CD4⁺ T-cell decline results in the onset of AIDS. It has been shown that the initial viral set point following primary infection is a very strong predictor of the disease-free period until the onset of AIDS (18, 21, 22).The initial decrease in the viral load during primary HIV-1 infection (PHI) is temporally associated with the first emergence of virus-specific CD8⁺ T-cell responses, and several studies have provided strong evidence that HIV-1-specific CD8⁺ T-cell responses are capable of controlling viral replication (5, 16, 24, 25, 27, 31, 33). However, significant numbers of virus-specific CD8⁺ T cells are detectable both in chronically infected individuals who progress rapidly to AIDS and in those who do not experience HIV-1 disease progression for decades (1, 11), and the characteristics that define a protective HIV-1-specific CD8⁺ T-cell response are not known. In particular, the level of control over viral replication is not predicted by the overall breadth, magnitude, or function of virus-specific CD8⁺ T-cell responses in chronic HIV-1 infection (1, 4, 11, 26, 28).Here, we demonstrate in a large cohort of individuals identified during PHI that immunodominance patterns of virus-specific CD8⁺ T-cell responses detected in PHI, but not in chronic HIV-1 infection, are strongly associated with the subsequent set point of viral replication. These data show that the specificity of the initial CD8⁺ T-cell response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.     

Mechanism of SUMOylation-Mediated Regulation of Type I IFN Expression

Type I interferons (IFN) are cytokines that bridge the innate and adaptive immune response, and thus play central roles in human health, including vaccine efficacy, immune response to cancer and pathogen infection, and autoimmune disorders. Post-translational protein modifications by the small ubiquitin-like modifiers (SUMO) have recently emerged as an important regulator of type I IFN expression as shown by studies using murine and cellular models and recent human clinical trials. However, the mechanism regarding how SUMOylation regulates type I IFN expression remains poorly understood. In this study, we show that SUMOylation inhibition does not activate IFNB1 gene promoter that is regulated by known canonical pathways including cytosolic DNA. Instead, we identified a binding site for the chromatin modification enzyme, the SET Domain Bifurcated Histone Lysine Methyltransferase 1 (SETDB1), located between the IFNB1 promoter and a previously identified enhancer. We found that SETDB1 regulates IFNB1 expression and SUMOylation of SETDB1 is required for its binding and enhancing the H3K9me3 heterochromatin signal in this region. Heterochromatin, a tightly packed form of DNA, has been documented to suppress gene expression through suppressing enhancer function. Taken together, our study identified a novel mechanism of regulation of type I IFN expression, at least in part, through SUMOylation of a chromatin modification enzyme.     

Type I IFN negatively regulates CD8+ T cell responses through IL-10-producing CD4+ T regulatory 1 cells

Pleiotropic, immunomodulatory effects of type I IFN on T cell responses are emerging. We used vaccine-induced, antiviral CD8(+) T cell responses in IFN-beta (IFN-beta(-/-))- or type I IFN receptor (IFNAR(-/-))-deficient mice to study immunomodulating effects of type I IFN that are not complicated by the interference of a concomitant virus infection. Compared with normal B6 mice, IFNAR(-/-) or IFN-beta(-/-) mice have normal numbers of CD4(+) and CD8(+) T cells, and CD25(+)FoxP3(+) T regulatory (T(R)) cells in liver and spleen. Twice as many CD8(+) T cells specific for different class I-restricted epitopes develop in IFNAR(-/-) or IFN-beta(-/-) mice than in normal animals after peptide- or DNA-based vaccination. IFN-gamma and TNF-alpha production and clonal expansion of specific CD8(+) T cells from normal and knockout mice are similar. CD25(+)FoxP3(+) T(R) cells down-modulate vaccine-primed CD8(+) T cell responses in normal, IFNAR(-/-), or IFN-beta(-/-) mice to a comparable extent. Low IFN-alpha or IFN-beta doses (500-10(3) U/mouse) down-modulate CD8(+) T cells priming in vivo. IFNAR- and IFN-beta-deficient mice generate 2- to 3-fold lower numbers of IL-10-producing CD4(+) T cells after polyclonal or specific stimulation in vitro or in vivo. CD8(+) T cell responses are thus subjected to negative control by both CD25(+)FoxP3(+) T(R) cells and CD4(+)IL-10(+) T(R1) cells, but only development of the latter T(R) cells depends on type I IFN.     

Negative selection of Tcra-V8+CD8+ T cells by MHC class I molecules

Tcrb-V-specific positive and negative selection of T cells has been well documented. In contrast, nothing is known about Tcra-V-specific selection. Using Tcra-V8-specific KT50 antibody Tcra-V8-specific selection of T cells has been examined. The CD8⁺ T cell subpopulation bearing Tcra-V8 are shown to be negatively selected by major histocompatibility complex (MHC) class I H-2K^d and H-2D^d/L^d molecules. Furthermore, percentages of these T cells are also influenced by Tcra-V haplotypes. Involvement of non-H-2 self (super)antigens in this MHC class I restricted negative selection, however, remains to be determined.     

Distinct APC Subtypes Drive Spatially Segregated CD4+ and CD8+ T-Cell Effector Activity during Skin Infection with HSV-1

Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4⁺ and CD8⁺ T-cells during skin infection with HSV-1. IFN-γ-producing CD4⁺ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8⁺ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4⁺ T cells, CD8⁺ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8⁺ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4⁺ and CD8⁺ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC).     

Differential Targeting of Viral Components by CD4+ versus CD8+ T Lymphocytes in Dengue Virus Infection

Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4⁺ and CD8⁺ T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4⁺ and 21 are CD8⁺ T cell epitopes. We observe that whereas CD8⁺ T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4⁺ epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4⁺ T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4⁺ and CD8⁺ T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.     

Mathematical Models of Memory CD8+ T-Cell Repertoire Dynamics in Response to Viral Infections

Immunity to diseases is conferred by pathogen-specific memory cells that prevent disease reoccurrences. A broad repertoire of memory T-cells must be developed and maintained to effectively protect against viral invasions; yet, the total number of memory T-cells is constrained between infections. Thus, creating memory to new infections can require attrition of some existing memory cells. Furthermore, some viruses induce memory T-cell death early in an infection, after which surviving cells proliferate to refill the memory compartment. We develop mathematical models of cellular attrition and proliferation in order to examine how new viral infections impact existing immunity. With these probabilistic models, we qualitatively and quantitatively predict how the composition and diversity of the memory repertoire changes as a result of viral infections. In addition, we calculate how often immunity to prior diseases is lost due to new infections. Comparing our results across multiple general infection types allows us to draw conclusions about, which types of viral effects most drastically alter existing immunity. We find that early memory attrition does not permanently alter the repertoire composition, while infections that spark substantial new memory generation drastically shift the repertoire and hasten the decline of existing immunity.     

CD4+ and CD8+ T-Cell Responses to Latent Antigen EBNA-1 and Lytic Antigen BZLF-1 during Persistent Lymphocryptovirus Infection of Rhesus Macaques

Epstein-Barr virus (EBV) infection leads to lifelong viral persistence through its latency in B cells. EBV-specific T cells control reactivations and prevent the development of EBV-associated malignancies in most healthy carriers, but infection can sometimes cause chronic disease and malignant transformation. Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein consistently expressed during all forms of latency and in all EBV-associated malignancies and is a promising target for a therapeutic vaccine. Here, we studied the EBNA-1-specific immune response using the EBV-homologous rhesus lymphocryptovirus (rhLCV) infection in rhesus macaques. We assessed the frequency, phenotype, and cytokine production profiles of rhLCV EBNA-1 (rhEBNA-1)-specific T cells in 15 rhesus macaques and compared them to the lytic antigen of rhLCV BZLF-1 (rhBZLF-1). We were able to detect rhEBNA-1-specific CD4⁺ and/or CD8⁺ T cells in 14 of the 15 animals screened. In comparison, all 15 animals had detectable rhBZLF-1 responses. Most peptide-specific CD4⁺ T cells exhibited a resting phenotype of central memory (TCM), while peptide-specific CD8⁺ T cells showed a more activated phenotype, belonging mainly to the effector cell subset. By comparing our results to the human EBV immune response, we demonstrate that the rhLCV model is a valid system for studying chronic EBV infection and for the preclinical development of therapeutic vaccines.     

CD4+ T-Cell Help Is Required for Effective CD8+ T Cell-Mediated Resolution of Acute Viral Hepatitis in Mice

Cytotoxic CD8+ T cells are essential for the control of viral liver infections, such as those caused by HBV or HCV. It is not entirely clear whether CD4+ T-cell help is necessary for establishing anti-viral CD8+ T cell responses that successfully control liver infection. To address the role of CD4+ T cells in acute viral hepatitis, we infected mice with Lymphocytic Choriomeningitis Virus (LCMV) of the strain WE; LCMV-WE causes acute hepatitis in mice and is cleared from the liver by CD8+ T cells within about two weeks. The role of CD4+ T-cell help was studied in CD4+ T cell-lymphopenic mice, which were either induced by genetic deficiency of the major histocompatibility (MHC) class II transactivator (CIITA) in CIITA−/− mice, or by antibody-mediated CD4+ cell depletion. We found that CD4+ T cell-lymphopenic mice developed protracted viral liver infection, which seemed to be a consequence of reduced virus-specific CD8+ T-cell numbers in the liver. Moreover, the anti-viral effector functions of the liver-infiltrating CD8+ T cells in response to stimulation with LCMV peptide, notably the IFN-γ production and degranulation capacity were impaired in CIITA−/− mice. The impaired CD8+ T-cell function in CIITA−/− mice was not associated with increased expression of the exhaustion marker PD-1. Our findings indicate that CD4+ T-cell help is required to establish an effective antiviral CD8+ T-cell response in the liver during acute viral infection. Insufficient virus control and protracted viral hepatitis may be consequences of impaired initial CD4+ T-cell help.     

CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression

Expression of co-inhibitory molecules is generally associated with T-cell dysfunction in chronic viral infections such as HIV or HCV. However, their relative contribution in the T-cell impairment remains unclear. In the present study, we have evaluated the impact of the expression of co-inhibitory molecules such as 2B4, PD-1 and CD160 on the functions of CD8 T-cells specific to influenza, EBV and CMV. We show that CD8 T-cell populations expressing CD160, but not PD-1, had reduced proliferation capacity and perforin expression, thus indicating that the functional impairment in CD160⁺ CD8 T cells may be independent of PD-1 expression. The blockade of CD160/CD160-ligand interaction restored CD8 T-cell proliferation capacity, and the extent of restoration directly correlated with the ex vivo proportion of CD160⁺ CD8 T cells suggesting that CD160 negatively regulates TCR-mediated signaling. Furthermore, CD160 expression was not up-regulated upon T-cell activation or proliferation as compared to PD-1. Taken together, these results provide evidence that CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression.     

Enhanced PD-1 Expression by T Cells in Cerebrospinal Fluid Does Not Reflect Functional Exhaustion during Chronic Human Immunodeficiency Virus Type 1 Infection

During chronic viral infections, T cells are exhausted due to constant antigen exposure and are associated with enhanced programmed death 1 (PD-1) expression. Deficiencies in the PD-1/programmed death-ligand 1 (PD-L1) pathway are associated with autoimmune diseases, including those of the central nervous system (CNS). To understand the role of PD-1 expression in regulating T-cell immunity in the CNS during chronic infection, we characterized PD-1 expression in cerebrospinal fluid (CSF) and blood of individuals with chronic human immunodeficiency virus type 1 (HIV-1) infection. PD-1 expression was higher on HIV-specific CD8⁺ T cells than on total CD8⁺ T cells in both CSF and blood. PD-1 expression on CSF T cells correlated positively with CSF HIV-1 RNA and inversely with blood CD4⁺ T-cell counts, suggesting that HIV-1 infection drives higher PD-1 expression on CSF T cells. However, in every HIV-positive individual, PD-1 expression was higher on T cells in CSF than on those in blood, despite HIV-1 RNA levels being lower. Among healthy HIV-negative controls, PD-1 expression was higher in CSF than in blood. Furthermore, frequencies of the senescence marker CD57 were lower on CSF T cells than on blood T cells, consistent with our prior observation of enhanced ex vivo functional capacity of CSF T cells. The higher PD-1 expression level on CSF T cells therefore does not reflect cellular exhaustion but may be a mechanism to downregulate immune-mediated tissue damage in the CNS. As inhibition of the PD-1/PD-L1 pathway is pursued as a therapeutic option for viral infections, potential effects of such a blockade on development of autoimmune responses in the CNS should be considered.Programmed death 1 (PD-1; also called CD279) and its ligands, PD-L1 (also called B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD-273), regulate T-cell activation, peripheral tolerance, and autoimmunity (22, 43). PD-1 can be expressed on CD8⁺ and CD4⁺ T cells, B cells, natural killer T cells, and activated monocytes. PD-L1 is expressed on various cells, including T and B cells, dendritic cells, macrophages, mast cells, nonhematopoietic cell types (including vascular endothelial cells, pancreatic islet cells, astrocytes, keratinocytes, and microglial cells), and cells in immune privileged sites, including the placenta and the eye (22). PD-L2 expression is inducible and is restricted to dendritic cells, monocytes, macrophages, and mast cells (22). During chronic infections, the PD-1/PD-L1 pathway inhibits antigen-specific T-cell responses (7, 8, 35, 46). In human immunodeficiency virus type 1 (HIV-1)-infected individuals, PD-1 expression on HIV-specific T cells in peripheral blood is upregulated and correlates positively with plasma viremia and inversely with CD4⁺ T-cell counts (7, 46). PD-1 expression on HIV-specific T cells is also associated with T-cell exhaustion, as defined by a reduced ability to proliferate and produce cytokines (7, 46). Inhibition of the PD-1/PD-L1 pathway augments HIV-specific CD8⁺ and CD4⁺ T-cell function, and antiretroviral therapy is associated with a significant reduction of PD-1 expression on HIV-specific T cells in peripheral blood (8).The PD-1/PD-L1 pathway also limits immune-mediated tissue damage that may be caused by overreactive peripheral T cells, especially in immune privileged sites such as the central nervous system (CNS). In 1999, the importance of PD-1 for peripheral tolerance was first suggested by studies which showed that PD1^−/− mice develop lupus-like autoimmune diseases (32). In humans, polymorphisms in the PDCD1 gene, which encodes PD-1, have been associated with autoimmune diseases, including lupus, diabetes, rheumatoid arthritis, and multiple sclerosis (20, 21, 25). Upregulation of PD-L1 in multiple sclerosis lesions from human brain tissue suggests a role for the PD-1/PD-L1 pathway in regulating T-cell activation and controlling immunopathological damage (33).The CNS is involved by HIV-1 early during primary infection (6, 13), and approximately 40% of patients who develop advanced AIDS without receiving antiretroviral therapy develop cognitive impairment (6, 13, 38). While HIV-1 proteins gp120 (3, 16) and Tat (30) are directly neurotoxic and may contribute to HIV-associated dementia, detrimental neuropathogenic effects have also been postulated for inflammatory and innate immune cells, especially monocytes/macrophages and T cells (11, 19, 49, 50). Immune responses cause neuropathogenesis during other viral infections, and cytotoxic T lymphocytes can worsen the disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (14). However, we recently described higher frequencies of functional HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) than in blood among asymptomatic HIV-positive individuals with little or no HIV-1 RNA in CSF, suggesting that HIV-1-specific CD8⁺ T cells help to control intrathecal viral replication (40).To understand the role of the PD-1/PD-L1 pathway in regulating T-cell responses during viral infection of the CNS, we characterized PD-1 expression on T cells in CSF and peripheral blood among asymptomatic HIV-positive individuals. We hypothesized that T-cell PD1 expression would be lower in CSF than in blood, since HIV-1 RNA concentrations are lower in CSF than in plasma and the magnitude and breadth of IFN-γ-secreting HIV-specific T cells are greater in CSF than in blood (40). We show that, in CSF, HIV-1 RNA correlates directly with PD-1 expression on CD4⁺, CD8⁺, and HIV-specific CD8⁺ T cells. Unexpectedly, PD-1 expression on all T cells is higher in CSF than in blood in HIV-positive patients and healthy HIV-negative controls. In contrast, expression of the senescence marker CD57 is lower in CSF than in blood. These data suggest that higher PD-1 expression on T cells in CSF may be a mechanism to regulate T-cell immunity in the CNS, rather than indicating T-cell exhaustion, and that this regulation is increased by HIV-1 replication.     

Dendritic Cell Migration Limits the Duration of CD8+ T-Cell Priming to Peripheral Viral Antigen

CD8⁺ T cells (T_CD8⁺) play a crucial role in immunity to viruses. Antiviral T_CD8⁺ are initially activated by recognition of major histocompatibility complex (MHC) class I-peptide complexes on the surface of professional antigen-presenting cells (pAPC). Migration of pAPC from the site of infection to secondary lymphoid organs is likely required during a natural infection. Migrating pAPC can be directly infected with virus or may internalize antigen derived from virus-infected cells. The use of experimental virus infections to assess the requirement for pAPC migration in initiation of T_CD8⁺ responses has proven difficult to interpret because injected virus can readily drain to secondary lymphoid organs without the need for cell-mediated transport. To overcome this ambiguity, we examined the generation of antigen-specific T_CD8⁺ after immunization with recombinant adenoviruses that express antigen driven by skin-specific or ubiquitous promoters. We show that the induction of T_CD8⁺ in response to tissue-targeted antigen is less efficient than the response to ubiquitously expressed antigen and that the resulting T_CD8⁺ fail to clear all target cells pulsed with the antigenic peptide. This failure to prime a fully functional T_CD8⁺ response results from a reduced period of priming to peripherally expressed antigen versus ubiquitously expressed antigen and correlated with a brief burst of pAPC migration from the skin, a requirement for induction of the response to peripheral antigen. These results indicate that a reduced duration of pAPC migration after virus infection likely reduces the amplitude of the T_CD8⁺ response, allowing persistence of the peripheral virus.The induction of effector CD8⁺ T cells (T_CD8⁺) is a vital step in the eradication or control of many viral infections. The induction of antiviral T_CD8⁺ requires the presentation of virally derived peptides in complex with major histocompatibility complex (MHC) class I on the surface of specialized professional antigen-presenting cells (pAPC), most commonly a subset of dendritic cells (DC) that bear the CD8α chain (1, 29). The CD8α⁺ DC reside only in secondary lymphoid organs and not in the tissues, implying that cell-mediated transport or drainage of virus particles to a lymph node is required for initiation of a T_CD8⁺ response. Partial inhibition of DC migration from the skin can impair the initiation of a T_CD8⁺ response (2). After influenza infection in the lungs, there is a burst of DC migration, followed by a refractory period in which no DC migration occurs (19). The functional consequences of this refractory period of DC migration have not been explored.A number of viruses, particularly human papillomaviruses, infect the skin and are ignored by the immune response for extended periods of time (31). We sought to explore the possibility that, after a low-level peripheral virus infection of the skin, changes in DC migration may limit the availability of antigen in the draining lymph node and thus the induction of a T_CD8⁺ response. There are a number of confounding factors that make the study of DC migration in the initiation of an antiviral T_CD8⁺ response difficult. Virus particles may directly drain to the lymph node within seconds (11, 13, 25). In addition, many viruses will alter DC functions, including migration, after infection of the DC itself. This may occur via specific viral modulation of DC function (16) or via nonspecific shut down of host protein synthesis (26), both of which will affect migration. Thus, it is often not possible to distinguish between the effects of virus infection upon DC migration, drainage of virus directly to the lymph node, and the natural response that follows migration of DC responding to a peripheral virus infection.There is currently no mouse model of a peripheral virus infection that is confined to the skin, as no natural mouse papillomavirus has ever been isolated. Therefore, to address these issues, we have made use of another small DNA virus, namely, an adenovirus vector that is replication deficient (rAd). These vectors express influenza virus nucleoprotein (NP) under the control of a ubiquitous (cytomegalovirus [CMV] immediate-early) or tissue-targeted promoter (K14, targeted to keratinocytes, the site of papillomavirus replication). Antigen driven by the K14 promoter is expressed only in skin cells, so only uninfected DC can present antigen in this system, removing the need to account for modulation of the function of virus-infected DC.We demonstrate that when antigen is expressed in only keratinocytes in the skin, the efficiency of T_CD8⁺ induction is reduced and the time period for which antigen is available to prime effector cells is reduced dramatically. DC-mediated transport is required for antigen to reach the lymph node where a T_CD8⁺ response is initiated. The reduced time period of antigen presentation is the result of a transient blockade in DC migration from the site of infection. The blockade in DC migration reduced the delivery of viral antigen to the lymph node needed to induce a T_CD8⁺ response. The resulting T_CD8⁺ response to peripheral viral antigen is not capable of clearing all target cells presenting a viral peptide, thus allowing the persistence of peripheral virus-infected cells. These results provide a potential mechanism for the long-term evasion of the immune response by papillomaviruses following natural infection and also have important implications for tissue targeted gene therapy vectors.     

Protective HLA Class I Alleles That Restrict Acute-Phase CD8+ T-Cell Responses Are Associated with Viral Escape Mutations Located in Highly Conserved Regions of Human Immunodeficiency Virus Type 1

The control of human immunodeficiency virus type 1 (HIV-1) associated with particular HLA class I alleles suggests that some CD8⁺ T-cell responses may be more effective than others at containing HIV-1. Unfortunately, substantial diversities in the breadth, magnitude, and function of these responses have impaired our ability to identify responses most critical to this control. It has been proposed that CD8 responses targeting conserved regions of the virus may be particularly effective, since the development of cytotoxic T-lymphocyte (CTL) escape mutations in these regions may significantly impair viral replication. To address this hypothesis at the population level, we derived near-full-length viral genomes from 98 chronically infected individuals and identified a total of 76 HLA class I-associated mutations across the genome, reflective of CD8 responses capable of selecting for sequence evolution. The majority of HLA-associated mutations were found in p24 Gag, Pol, and Nef. Reversion of HLA-associated mutations in the absence of the selecting HLA allele was also commonly observed, suggesting an impact of most CTL escape mutations on viral replication. Although no correlations were observed between the number or location of HLA-associated mutations and protective HLA alleles, limiting the analysis to mutations selected by acute-phase immunodominant responses revealed a strong positive correlation between mutations at conserved residues and protective HLA alleles. These data suggest that control of HIV-1 may be associated with acute-phase CD8 responses capable of selecting for viral escape mutations in highly conserved regions of the virus, supporting the inclusion of these regions in the design of an effective vaccine.Despite substantial advances in antiretroviral therapies, development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine remains a critical goal (6, 39, 82). Unfortunately, current vaccine efforts have failed to reduce infection rates in humans (9, 75) and have only achieved modest decreases in viral loads in the simian immunodeficiency virus (SIV)/SHIV macaque model (21, 44, 81). A majority of these vaccine approaches have focused on inducing T-cell responses, utilizing large regions of the virus in an attempt to induce a broad array of immune responses (6, 34, 44, 81). While it is well established that CD8⁺ T-cell responses play a critical role in the containment of HIV-1 (45, 49, 67), supported in part by the strong association of particular HLA class I alleles with control of HIV (20, 33, 42, 61), it remains unclear which particular CD8⁺ T-cell responses are best able to control the virus and thus should be preferentially targeted by a vaccine. Studies comparing the magnitude, breadth, and function of CD8⁺ T-cell responses in subjects exhibiting either enhanced or poor control of HIV-1 have yielded few clues as to the specific factors associated with an effective CD8⁺ T-cell response (2, 28, 64, 67). Various differences in the functional capacity of T-cell responses have been observed in long-term nonprogressors (1, 26, 64), although it is possible that these differences may be reflective of an intact immune response, as opposed to having had directly enhanced immune control. As such, efforts are needed to identify factors or phenotypes associated with protective CD8⁺ T-cell responses in order to enable vaccines to induce the most effective responses.Recent studies have begun to suggest that the specificity of the CD8⁺ T-cell response, or the targeting of specific regions of the virus, may be associated with control of HIV-1. Preferential targeting of Gag, a structurally conserved viral protein responsible for multiple functions, has been associated with lower viral loads (25, 43, 56, 60, 77, 85). Furthermore, Kiepiela et al. (43) recently illustrated in a large cohort of 578 clade C-infected subjects that Gag-specific responses were associated with lowered viremia, in contrast to Env-specific responses, which were associated with higher viremia. These data are in line with previous observations that many of the major histocompatibility complex (MHC) class I alleles most strongly associated with control of HIV-1 and SIV, namely, HLA-B57, HLA-B27, and Mamu-A*01, restrict immunodominant CD8⁺ T-cell responses against the Gag protein (8, 10, 24, 63, 68, 83). However, other alleles associated with slower disease progression, such as HLA-B51 in humans and Mamu-B08 and B-17 in the rhesus macaque, do not immunodominantly target Gag, suggesting that targeting of some other regions of the virus may also be capable of eliciting control (8, 52-54). In addition, recent studies investigating the pattern of HIV-1-specific CD8⁺ T-cell responses during acute infection reveal that only a small subset of CD8⁺ T-cell responses restricted by any given HLA allele arise during acute infection and that there exist clear immunodominance patterns to these responses (8, 77, 85). Since control of HIV-1 is likely to be established or lost during the first few weeks of infection, these data suggest that potentially only a few key CD8⁺ T-cell responses may be needed to adequately establish early control of HIV-1.One of the major factors limiting the effectiveness of CD8⁺ T-cell responses is the propensity for HIV-1 to evade these responses through sequence evolution or viral escape (3, 13, 66). Even single point mutations within a targeted CD8 epitope can effectively abrogate recognition by either the HLA allele or the T-cell receptor. However, recent studies have begun to highlight that many sequence polymorphisms will revert to more common consensus residues upon transmission of HIV-1 to a new host, including many cytotoxic T-lymphocyte (CTL) escape mutations (4, 30, 33, 48, 50). Notably, the more rapidly reverting mutations have been observed to preferentially occur at conserved residues, indicating that structurally conserved regions of the virus may be particularly refractory to sequence changes (50). In support of these data, many CTL escape mutations have now been observed to directly impair viral replication (15, 23, 55, 74), in particular those known to either revert or require the presence of secondary compensatory mutations (15, 23, 73, 74). Taken together, these data suggest that, whereas CTL escape mutations provide a benefit to the virus to enable the evasion of host immune pressures, some of these mutations may come at a substantial cost to viral replication. These data may also imply that the association between Gag-specific responses and control of HIV-1 may be due to the targeting of highly conserved regions of the virus that are difficult to evade through sequence evolution.The propensity by which HIV-1 escapes CD8⁺ T-cell responses, and the reproducibility by which mutations arise at precise residues in targeted CD8 epitopes (3, 48), also enables the utilization of sequence data to predict which responses may be most capable of exerting immune selection pressure on the virus. Studies in HIV-1, SIV, and hepatitis C virus (16, 58, 65, 78) are now rapidly identifying immune-driven CTL escape mutations across these highly variable pathogens at the population level by correlating sequence polymorphisms in these viruses with the expression of particular HLA alleles. We provide here an analysis of HLA-associated mutations across the entire HIV-1 genome using a set of sequences derived from clade B chronically infected individuals. Through full-length viral genome coverage, these data provide an unbiased analysis of the location of these mutations and suggest that the control of HIV-1 by particular HLA alleles correlates with their ability to preferentially restrict early CD8⁺ T-cell responses capable of selecting for viral escape mutations at highly conserved residues of the virus. These data provide support for the inclusion of specific highly conserved regions of HIV-1 into vaccine antigens.