Similar Impact of CD8+ T Cell Responses on Early Virus Dynamics during SIV Infections of Rhesus Macaques and Sooty Mangabeys

Despite comparable levels of virus replication, simian immunodeficiency viruses (SIV) infection is non-pathogenic in natural hosts, such as sooty mangabeys (SM), whereas it is pathogenic in non-natural hosts, such as rhesus macaques (RM). Comparative studies of pathogenic and non-pathogenic SIV infection can thus shed light on the role of specific factors in SIV pathogenesis. Here, we determine the impact of target-cell limitation, CD8+ T cells, and Natural Killer (NK) cells on virus replication in the early SIV infection. To this end, we fit previously published data of experimental SIV infections in SMs and RMs with mathematical models incorporating these factors and assess to what extent the inclusion of individual factors determines the quality of the fits. We find that for both rhesus macaques and sooty mangabeys, target-cell limitation alone cannot explain the control of early virus replication, whereas including CD8+ T cells into the models significantly improves the fits. By contrast, including NK cells does only significantly improve the fits in SMs. These findings have important implications for our understanding of SIV pathogenesis as they suggest that the level of early CD8+ T cell responses is not the key difference between pathogenic and non-pathogenic SIV infection.     

Comparative Analysis of Cytotoxic T Lymphocytes in Lymph Nodes and Peripheral Blood of Simian Immunodeficiency Virus-Infected Rhesus Monkeys

Most studies of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) have been confined to the evaluation of these effector cells in the peripheral blood. What has not been clear is the extent to which CTL activity in the blood actually reflects this effector cell function in the lymph nodes, the major sites of HIV-1 replication. To determine the concordance between CTL activity in lymph nodes and peripheral blood lymphocytes (PBL), CTL specific for simian immunodeficiency virus of macaques (SIVmac) have been characterized in lymph nodes of infected, genetically selected rhesus monkeys by using both Gag peptide-specific functional CTL assays and tetrameric peptide-major histocompatibility complex (MHC) class I molecule complex staining techniques. In studies of six chronically SIVmac-infected rhesus monkeys, Gag epitope-specific functional lytic activity and specific tetrameric peptide-MHC class I staining were readily demonstrated in lymph node T lymphocytes. Although the numbers of tetramer-binding cells in some animals differed from those documented in their PBL, the numbers of tetramer-binding cells from these two different compartments were not statistically different. Phenotypic characterization of the tetramer-binding CD8⁺ lymph node T lymphocytes of the infected monkeys demonstrated a high level of expression of the activation-associated adhesion molecules CD11a and CD49d, the Fas molecule CD95, and MHC class II-DR. These studies documented a low expression of the naive T-cell marker CD45RA and the adhesion molecule CD62L. This phenotypic profile of the tetramer-binding lymph node CD8⁺ T cells was similar to that of tetramer-binding CD8⁺ T cells from PBL. These observations suggest that characterization of AIDS virus-specific CTL activity by sampling of cells in the peripheral blood should provide a reasonable estimation of CTL in an individual’s secondary lymphoid tissue.CD8⁺ cytotoxic T lymphocytes (CTL) are important in containing the spread of human immunodeficiency virus type 1 (HIV-1) in infected individuals. Studies have shown that virus-specific CD8⁺ CTL can inhibit AIDS virus replication in autologous CD4⁺ T lymphocytes in vitro, probably by release of chemokines and cytokines, as well as by lysis of infected cells (35, 36). In vivo the containment of HIV-1 replication that occurs during the period of primary infection coincides temporally with the generation of virus-specific CTL (8, 17, 29). Finally, a potent CTL response is correlated with low virus load and a stable clinical status in individuals chronically infected with HIV-1 (25, 27).HIV-1 replication occurs predominantly in the lymph nodes of the infected individual (30). However, most studies of HIV-1-specific CTL have been confined to the evaluation of these effector cells in the peripheral blood. It is not clear to what extent CTL activity in the blood actually reflects this effector cell function at the major sites of HIV-1 replication. An extensive evaluation of CTL in lymph nodes of HIV-1-infected humans has not been undertaken, at least in part because of the numerous surgical procedures that would be required for such a study. The use of such procedures in clinically stable individuals might be difficult to rationalize.The simian immunodeficiency virus (SIV)-infected macaque provides an ideal animal model in which to examine AIDS virus-specific CTL in lymph nodes. SIVmac-infected rhesus monkeys develop a disease with remarkable similarities to HIV-1-induced disease in humans (19, 20). SIVmac-specific CTL are readily detected in infected monkeys by functional killing assays (21, 38). We have made use of a dominant CTL response to the SIVmac Gag epitope p11C, C-M in rhesus monkeys expressing the major histocompatibility complex (MHC) class I molecule Mamu-A*01 to explore the role of CTL in the immunopathogenesis of AIDS (1, 22). In the present study, CTL specific for SIVmac have been characterized in lymph nodes of infected, Mamu-A*01⁺ rhesus monkeys using both Gag peptide-specific functional CTL assays and tetrameric peptide-MHC class I molecule complex staining techniques (2, 6, 12, 18, 24, 27).     

Treatment with IL-7 Prevents the Decline of Circulating CD4+ T Cells during the Acute Phase of SIV Infection in Rhesus Macaques

Although treatment with interleukin-7 (IL-7) was shown to transiently expand the naïve and memory T-cell pools in patients with chronic HIV-1 infection receiving antiretroviral therapy (ART), it is uncertain whether a full immunologic reconstitution can be achieved. Moreover, the effects of IL-7 have never been evaluated during acute HIV-1 (or SIV) infection, a critical phase of the disease in which the most dramatic depletion of CD4⁺ T cells is believed to occur. In the present study, recombinant, fully glycosylated simian IL-7 (50 µg/kg, s.c., once weekly for 7 weeks) was administered to 6 rhesus macaques throughout the acute phase of infection with a pathogenic SIV strain (mac251); 6 animals were infected at the same time and served as untreated controls. Treatment with IL-7 did not cause clinically detectable side effects and, despite the absence of concomitant ART, did not induce significant increases in the levels of SIV replication except at the earliest time point tested (day 4 post-infection). Strikingly, animals treated with IL-7 were protected from the dramatic decline of circulating naïve and memory CD4⁺ T cells that occurred in untreated animals. Treatment with IL-7 induced only transient T-cell proliferation, but it was associated with sustained increase in the expression of the anti-apoptotic protein Bcl-2 on both CD4⁺ and CD8⁺ T cells, persistent expansion of all circulating CD8⁺ T-cell subsets, and development of earlier and stronger SIV Tat-specific T-cell responses. However, the beneficial effects of IL-7 were not sustained after treatment interruption. These data demonstrate that IL-7 administration is effective in protecting the CD4⁺ T-cell pool during the acute phase of SIV infection in macaques, providing a rationale for the clinical evaluation of this cytokine in patients with acute HIV-1 infection.     

Reactivation of Latent Tuberculosis in Cynomolgus Macaques Infected with SIV Is Associated with Early Peripheral T Cell Depletion and Not Virus Load

HIV-infected individuals with latent Mycobacterium tuberculosis (Mtb) infection are at significantly greater risk of reactivation tuberculosis (TB) than HIV-negative individuals with latent TB, even while CD4 T cell numbers are well preserved. Factors underlying high rates of reactivation are poorly understood and investigative tools are limited. We used cynomolgus macaques with latent TB co-infected with SIVmac251 to develop the first animal model of reactivated TB in HIV-infected humans to better explore these factors. All latent animals developed reactivated TB following SIV infection, with a variable time to reactivation (up to 11 months post-SIV). Reactivation was independent of virus load but correlated with depletion of peripheral T cells during acute SIV infection. Animals experiencing reactivation early after SIV infection (<17 weeks) had fewer CD4 T cells in the periphery and airways than animals reactivating in later phases of SIV infection. Co-infected animals had fewer T cells in involved lungs than SIV-negative animals with active TB despite similar T cell numbers in draining lymph nodes. Granulomas from these animals demonstrated histopathologic characteristics consistent with a chronically active disease process. These results suggest initial T cell depletion may strongly influence outcomes of HIV-Mtb co-infection.     

Epitope-Specific Regulatory CD4 T Cells Reduce Virus-Induced Illness while Preserving CD8 T-Cell Effector Function at the Site of Infection

The role of epitope-specific regulatory CD4 T cells in modulating CD8 T-cell-mediated immunopathology during acute viral infection has not been well defined. In the murine model of respiratory syncytial virus (RSV) infection, CD8 T cells play an important role in both viral clearance and immunopathology. We have previously characterized two RSV epitope-specific CD4 T-cell responses with distinct phenotypic properties. One of them, the IA^bM₂₀₉-specific subset, constitutively expresses FoxP3 and modulates CD8 T-cell function in vitro. We show here that the IA^bM₂₀₉-specific CD4 T-cell response regulates CD8 T-cell function in vivo and is associated with diminished RSV-induced illness without affecting viral clearance at the site of infection. Achieving the optimal balance of regulatory and effector T-cell function is an important consideration for designing future vaccines.A subset of CD4 T cells with regulatory function (Treg) has been shown to play an important role in modulating adaptive immune responses. Natural Tregs are characterized by the expression of FoxP3 and participate in reducing the activation of CD8 T-cell responses in peripheral lymphoid organs (11, 20, 35). This modulation can diminish the ability of adaptive immune responses to control systemic infections (4). However, the presence of natural regulatory CD4 T cells can have a beneficial effect on immune-mediated pathology, particularly at the site of infection. Tregs have been shown to limit pulmonary inflammation and lung injury induced by pneumocystis infection (29) and to modulate herpes simplex virus-induced inflammatory lesions of the eye (46). Natural Tregs also reduce the symptoms of West Nile virus infections in both humans and mice; Treg-deficient mice were more likely to develop lethal infection (25). Viral infection can also induce antigen-specific CD4 T cells that express FoxP3 (27), and their role in protective immunity and immunopathology needs more detailed investigation.T lymphocytes are key components of adaptive immunity against respiratory syncytial virus (RSV) infection. Children with T-cell deficiencies have delayed virus clearance and are more susceptible to fatal RSV infection (10, 18). The absence of T cells infiltrating into lung is associated with fatal RSV infections in children without recognized underlying disease (49). In the murine model, CD8 T cells play a major role in RSV clearance, presumably through direct cytotoxicity to infected cells and the generation of immunocompetent molecules (2, 15, 43); depletion of CD8 T cells in mice results in delayed viral clearance (14). The CD8 T-cell response also induces immunopathology in primary infection of mice (15, 32, 48). Transferring high doses of CD8 T cells facilitates virus clearance but also causes hemorrhagic pneumonia and enhanced disease (6, 14). These studies demonstrate that while CD8 T cells are required for viral clearance, they are responsible for immunopathology. We have described the pattern of CD8 T-cell responses that occur in mice that are the F₁ hybrid (H-2^d/b) between BALB/c (H-2^d) and C57BL/6 (H-2^b) (39). These mice respond both to the well-characterized K^dM2₈₂ epitope (24) and to a more recently described D^bM₁₈₇ epitope (38). Both CD8 T-cell responses are dominant in the parent strains but assume a hierarchy (K^dM2₈₂ > D^bM₁₈₇) in the F₁ hybrid (39). This model infection allows the analysis of factors that determine T-cell response hierarchy and provides multiple endpoints for the assessment of factors that modify or regulate CD8 T-cell responses.We recently described epitope-specific CD4 T-cell responses distinguished by novel major histocompatibility complex (MHC) class II tetramers in RSV infection. The IA^bM₂₀₉-specific CD4 T cells have a high frequency of FoxP3 expression and suppress RSV-specific CD8 T-cell cytokine production in vitro (27). To investigate the regulatory role of IA^bM₂₀₉-specific CD4 T cells in vivo, we sought to determine how immunizing mice with the CD4 T-cell epitope peptide M₂₀₉ would affect the RSV-specific CD8 T-cell response. We show here that the IA^bM₂₀₉-specific CD4 T cells have a regulatory effect on the dominant CD8 T-cell response to RSV infection, reducing both the magnitude and effector cytokine production in peripheral lymphoid organs while allowing effector functions at the site of infection to clear virus with normal kinetics. Viral clearance was thus achieved with less illness.     

Evolution of CD8+ T Cell Responses after Acute PARV4 Infection

PARV4 is a small DNA human virus that is strongly associated with hepatitis C virus (HCV) and HIV infections. The immunologic control of acute PARV4 infection has not been previously described. We define the acute onset of PARV4 infection and the characteristics of the acute-phase and memory immune responses to PARV4 in a group of HCV- and HIV-negative, active intravenous drug users. Ninety-eight individuals at risk of blood-borne infections were tested for PARV4 IgG. Gamma interferon enzyme-linked immunosorbent spot assays, intracellular cytokine staining, and a tetrameric HLA-A2–peptide complex were used to define the T cell populations responding to PARV4 peptides in those individuals who acquired infection during the study. Thirty-five individuals were found to be PARV4 seropositive at the end of the study, eight of whose baseline samples were found to be seronegative. Persistent and functional T cell responses were detected in the acute infection phase. These responses had an active, mature, and cytotoxic phenotype and were maintained several years after infection. Thus, PARV4 infection is common in individuals exposed to blood-borne infections, independent of their HCV or HIV status. Since PARV4 elicits strong, broad, and persistent T cell responses, understanding of the processes responsible may prove useful for future vaccine design.     

Infection with Host-Range Mutant Adenovirus 5 Suppresses Innate Immunity and Induces Systemic CD4+ T Cell Activation in Rhesus Macaques

Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis and conjunctivitis, and seroconversion before adolescence is common in humans. To gain some insight into how Ad5 infection affects the immune system of rhesus macaques (RM) 18 RM were infected with a host-range mutant Ad5 (Ad5hr) by 3 mucosal inoculations. There was a delay of 2 to 6 weeks after the first inoculation before plasmacytoid dendritic cell (pDC) frequency and function increased in peripheral blood. Primary Ad5hr infection suppressed IFN-γ mRNA expression, but the second Ad5hr exposure induced a rapid increase in IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC). Primary Ad5hr infection suppressed CCL20, TNF and IL-1 mRNA expression in PBMC, and subsequent virus exposures further dampened expression of these pro-inflammatory cytokines. Primary, but not secondary, Ad5hr inoculation increased the frequency of CXCR3+ CD4+ T cells in blood, while secondary, but not primary, Ad5hr infection transiently increased the frequencies of Ki67+, HLADR+ and CD95+/CCR5+ CD4+ T cells in blood. Ad5hr infection induced polyfunctional CD4 and CD8+ T cells specific for the Ad5 hexon protein in all of the animals. Thus, infection with Ad5hr induced a complex pattern of innate and adaptive immunity in RM that included transient systemic CD4+ T cell activation and suppressed innate immunity on re-exposure to the virus. The complex effects of adenovirus infection on the immune system may help to explain the unexpected results of testing Ad5 vector expressing HIV antigens in Ad5 seropositive people.     

Hypoxia Is a Dominant Remodeler of the Effector T Cell Surface Proteome Relative to Activation and Regulatory T Cell Suppression

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture–based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8⁺ surface proteome (surfaceome). Surprisingly, coculturing primary CD8⁺ T cells with Tregs only modestly affected the CD8⁺ surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8⁺ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4⁺ T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function.     

Genome-Wide Analysis of T Cell Responses during Acute and Latent Simian Varicella Virus Infections in Rhesus Macaques

Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (HZ [shingles]). Clinical observations suggest that VZV-specific T cell immunity plays a more critical role than humoral immunity in the prevention of VZV reactivation and development of herpes zoster. Although numerous studies have characterized T cell responses directed against select VZV open reading frames (ORFs), a comprehensive analysis of the T cell response to the entire VZV genome has not yet been conducted. We have recently shown that intrabronchial inoculation of young rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we characterized the specificity of T cell responses during acute and latent SVV infection. Animals generated a robust and broad T cell response directed against both structural and nonstructural viral proteins during acute infection in bronchoalveolar lavage (BAL) fluid and peripheral blood. During latency, T cell responses were detected only in the BAL fluid and were lower and more restricted than those observed during acute infection. Interestingly, we identified a small set of ORFs that were immunogenic during both acute and latent infection in the BAL fluid. Given the close genome relatedness of SVV and VZV, our studies highlight immunogenic ORFs that may be further investigated as potential components of novel VZV vaccines that specifically boost T cell immunity.     

Concurrent Generation of Effector and Central Memory CD8 T Cells during Vaccinia Virus Infection

It is generally thought that during the contraction phase of an acute anti-viral T cell reponse, the effector T cells that escape activation-induced cell death eventually differentiate into central memory T cells over the next several weeks. Here we report that antigen-specific CD8T cells with the phenotype and function of central memory cells develop concomitantly with effector T cells during vaccinia virus (vv) infection. As soon as 5 days after an intraperitoneal infection with vv, we could identify a subset of CD44^hi and CD62L⁺ vv-specific CD8 T cells in the peritoneal exudate lymphocytes. This population constituted approximately 10% of all antigen-specific T cells and like central memory T cells, they also expressed high levels of CCR7 and IL-7R but expressed little granzyme B. Importantly, upon adoptive transfer into naïve congenic hosts, CD62L⁺, but not CD62L⁻ CD8 T cells were able to expand and mediate a rapid recall response to a new vv challenge initiated 6 weeks after transfer, confirming that the CD62L⁺ vv-specific CD8 T cells are bonafide memory cells. Our results are thus consistent with the branched differentiation model, where effector and memory cells develop simultaneously. These results are likely to have implications in the context of vaccine design, particularly those based on vaccinia virus recombinants.     

Acute SIV Infection in Sooty Mangabey Monkeys Is Characterized by Rapid Virus Clearance from Lymph Nodes and Absence of Productive Infection in Germinal Centers

Lymphoid tissue immunopathology is a characteristic feature of chronic HIV/SIV infection in AIDS-susceptible species, but is absent in SIV-infected natural hosts. To investigate factors contributing to this difference, we compared germinal center development and SIV RNA distribution in peripheral lymph nodes during primary SIV infection of the natural host sooty mangabey and the non-natural host pig-tailed macaque. Although SIV-infected cells were detected in the lymph node of both species at two weeks post infection, they were confined to the lymph node paracortex in immune-competent mangabeys but were seen in both the paracortex and the germinal center of SIV-infected macaques. By six weeks post infection, SIV-infected cells were no longer detected in the lymph node of sooty mangabeys. The difference in localization and rate of disappearance of SIV-infected cells between the two species was associated with trapping of cell-free virus on follicular dendritic cells and higher numbers of germinal center CD4⁺ T lymphocytes in macaques post SIV infection. Our data suggests that fundamental differences in the germinal center microenvironment prevent productive SIV infection within the lymph node germinal centers of natural hosts contributing to sustained immune competency.