Defective Human Immunodeficiency Virus-Specific CD8+ T-Cell Polyfunctionality,Proliferation, and Cytotoxicity Are Not Restored by Antiretroviral Therapy

Identifying the functions of human immunodeficiency virus (HIV)-specific CD8⁺ T cells that are not merely modulated by the level of virus but clearly distinguish patients with immune control from those without such control is of paramount importance. Features of the HIV-specific CD8⁺ T-cell response in antiretroviral-treated patients (designated Rx <50) and untreated patients (long-term nonprogressors [LTNP]) matched for very low HIV RNA levels were comprehensively examined. The proliferative capacity of HIV-specific CD8⁺ T cells was not restored in Rx <50 to the level observed in LTNP, even though HIV-specific CD4⁺ T-cell proliferation in the two patient groups was comparable. This diminished HIV-specific CD8⁺ T-cell proliferation in Rx <50 was primarily due to a smaller fraction of antigen-specific cells recruited to divide and not to the numbers of divisions that proliferating cells had undergone. Exogenous interleukin-2 (IL-2) induced proliferating cells to divide further but did not rescue the majority of antigen-specific cells with defective proliferation. In addition, differences in HIV-specific CD8⁺ T-cell proliferation could not be attributed to differences in cellular subsets bearing a memory phenotype, IL-2 production, or PD-1 expression. Although polyfunctionality of HIV-specific CD8⁺ T cells in Rx <50 was not restored to the levels observed in LTNP despite prolonged suppression of HIV RNA levels, per-cell cytotoxic capacity was the functional feature that most clearly distinguished the cells of LTNP from those of Rx <50. Taken together, these data suggest that there are selective qualitative abnormalities within the HIV-specific CD8⁺ T-cell compartment that persist under conditions of low levels of antigen.Understanding the features of an effective immune response to human immunodeficiency virus (HIV) is among the most important goals for the design of HIV vaccines and immunotherapies. Most HIV-infected patients develop persistent viremia and CD4⁺ T-cell decline in the absence of antiviral therapy. However, evidence that immunologic control of HIV is possible can be drawn from a small group of rare patients who maintain normal CD4⁺ T-cell counts and restrict HIV replication to below 50 copies/ml plasma for up to 25 years without antiretroviral therapy (ART) (4, 22, 31, 40). Historically, these unique individuals were included within heterogeneous cohorts referred to as long-term survivors or long-term nonprogressors (LTNP), categorized solely based on their disease-free survival exceeding 7 to 10 years and their stable CD4⁺ T-cell counts (21). Over time, it became apparent that only a small subset of individuals within these cohorts had truly nonprogressive infection, maintaining good health with nondeclining CD4⁺ T-cell counts, and these true nonprogressors tended to have HIV type 1 (HIV-1) RNA levels below the lower detection limits of the newly available assays (23, 31). Some investigators have adopted other designations more recently, including elite controllers, elite suppressors, or HIV controllers. These designations vary by institution and, in some cases, rely only upon viral load measurements without a requirement for stable CD4⁺ T-cell counts (4, 22, 40). However, for our designation of true LTNP, we employ the inclusion criteria of stable health, nondeclining CD4⁺ T-cell counts, and maintenance of plasma viral RNA levels below 50 copies/ml without ART (29-31).Several lines of evidence strongly suggest that CD8⁺ T cells mediate this control of HIV in LTNP. HLA B*5701 is highly overrepresented in these patients, and in B*5701⁺ patients, the HIV-specific CD8⁺ T-cell response is largely focused on peptides restricted by the B57 protein (15, 31). In addition, similar control of simian immunodeficiency virus replication has been described in rhesus macaques carrying the Mamu B*08 or B*17 allele (25, 49). In these macaques, CD8⁺ T-cell depletion studies have strongly suggested that control of viral replication is mediated by CD8⁺ T cells (14). Although these results support the idea that CD8⁺ T cells are responsible for immunologic control, the mechanism remains incompletely understood.Several lines of evidence suggest that immunologic control in LTNP is not simply due to differences in autologous virus recognition by CD8⁺ T cells. The frequencies of CD8⁺ T cells specific for HIV or individual HIV-encoded gene products in the peripheral blood are not different in LTNP and untreated progressors (reviewed in reference 32). Putative “escape” mutations are found in viruses of both HLAB*57⁺ LTNP and HLA-matched progressors (4, 6, 28, 33, 34). In addition, comparable frequencies of CD8⁺ T cells of LTNP and progressors recognize autologous CD4⁺ T cells infected with the autologous virus (12, 28). Similar observations have recently been made in the rhesus macaque model (26). Collectively, these observations strongly suggest that features of the CD8⁺ T-cell response associated with immunologic control are not due to quantitative differences in the numbers of HIV-specific cells or to differential abilities of the autologous virus gene products to be recognized between patient groups.Several qualitative features in the HIV-specific CD8⁺ T-cell response have been associated with immunologic control in LTNP. LTNP have been found to have higher frequencies of “polyfunctional” CD8⁺ T cells, named for their ability to degranulate and produce multiple cytokines, including interleukin-2 (IL-2) (2, 5, 51). However, these cells comprise an extremely small proportion of the HIV-specific CD8⁺ T-cell response. In addition, there is considerable overlap between patient groups, and many LTNP have few or no such cells. Compared to those of progressors, HIV-specific CD8⁺ T cells of LTNP have a dramatically higher proliferative capacity, a greater ability to upregulate granzyme B (GrB) and perforin production, and a greater cytolytic capacity against autologous HIV-infected CD4⁺ T cells (3, 17, 24, 29, 30). Increased HIV-specific CD8⁺ T-cell proliferative capacity in LTNP compared to progressors has also been associated with lower PD-1 expression or IL-2 production by HIV-specific CD4⁺ or CD8⁺ T cells (11, 24, 48, 51).Considerable controversy exists over the cause-and-effect relationships between these qualitative differences in the CD8⁺ T-cell response and HIV viremia between patient groups. High levels of antigen can have potent effects on diverse cell types in humans and in animal models. For HIV, lowering the level of viremia through ART has been observed to increase the function of CD4⁺ and CD8⁺ T cells, NK cells, monocytes, and plasmacytoid dendritic cells (16, 18, 20, 37, 41, 45-47, 50). However, the vast majority of treated progressors will not control HIV replication when ART is interrupted (7, 9, 35), suggesting that many of the qualitative differences in the CD4⁺ or CD8⁺ T-cell response between LTNP and untreated progressors are not the cause of control over HIV but rather are likely an effect of viremia. In some but not all studies, ART was sufficient to restore the proliferative capacity, phenotype, and cytokine production by CD4⁺ T cells to levels similar to responses to other viruses or to the HIV-specific response of LTNP (13, 16, 18, 20, 37, 46, 50). Because better IL-2 production or function of HIV-specific CD4⁺ T cells has been associated with increased CD8⁺ T-cell proliferative capacity (24), it has also been suggested that diminished proliferative capacity of progressor CD8⁺ T cells may be an effect of viremia during the chronic phase of infection. In some studies, ART is sufficient to increase the frequency of polyfunctional HIV-specific CD8⁺ T cells or to decrease PD-1 expression (30, 41). However, the interpretations of the observations within these studies have relied on extrapolations between studies based upon cohorts with differing levels and durations of viral suppression or on examination of a limited number of functions or subsets in either CD4⁺ or CD8⁺ T cells.In the present study, we extended our earlier work and comprehensively examined a broad array of functions of HIV-specific T cells derived from two large patient groups, LTNP and progressors on ART, who possess comparable levels of HIV viremia as determined by a sensitive single-copy assay. In response to autologous HIV-infected CD4⁺ T cells, HIV-specific CD8⁺ T-cell proliferative capacity, IL-2 responsiveness, surface phenotype, PD-1 expression, polyfunctionality, and cytotoxic capacity were measured in considerable detail. We observe that although ART results in restoration of many of these functions, HIV-specific CD8⁺ T-cell polyfunctionality and proliferative and killing capacities are not restored to levels observed in LTNP.     

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1

The role of CD4⁺ helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4⁺ T cells are needed for the generation of the protective HSV-1-specific CD8⁺-T-cell response. This study examined the contribution of CD4⁺ T cells in the generation of the primary CD8⁺-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8⁺-T-cell response generated in the draining lymph nodes of CD4⁺-T-cell-depleted C57BL/6 mice and B6-MHC-II^−/− mice is quantitatively and qualitatively distinct from the CD8⁺ T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8⁺ T cells express comparable levels of the activation marker CD44 in mice lacking CD4⁺ T cells and normal mice. In contrast, CD8⁺ T cells generated in the absence of CD4⁺ T cells express the interleukin 2 receptor α-chain (CD25) at lower levels. Importantly, the CD8⁺ T cells in the CD4⁺-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8⁺ T cells is diminished in the absence of CD4⁺-T-cell help. These results suggest that CD4⁺-T-cell help is essential for the generation of fully functional CD8⁺ T cells during the primary response to HSV-1 infection.Infection due to herpes simplex virus type 1 (HSV-1) results in a wide spectrum of clinical presentations depending on the host''s age, the host''s immune status, and the route of inoculation (47). HSV-1 typically causes mild and self-limited lesions on the orofacial areas or genital sites. However, the disease can be life-threatening, as in the case of neonatal and central nervous system infections (18). The host''s immune responses, particularly CD8⁺ T cells, play an important role in determining the outcome of HSV infections in both the natural human host (18, 19, 28) and experimental murine models (11, 43). Immunodepletion and adoptive transfer studies have demonstrated the role of CD8⁺ T cells in reducing viral replication, resolving cutaneous disease, and providing overall protection upon rechallenge (6, 25, 26). CD8⁺ T cells play a particularly important role in preventing infection of the peripheral nervous system (PNS) and the reactivation of latent virus from neurons in the sensory ganglia of infected mice (21, 24, 36). The mechanisms that CD8⁺ T cells employ include gamma interferon (IFN-γ) production and functions associated with cytolytic granule content at the sites of primary infection (23, 31, 38). In the PNS of infected mice, the mechanisms primarily involve IFN-γ secretion (16, 20, 29), particularly against infected neurons expressing surface Qa-1 (41). Histopathological evidence from HSV-1-infected human ganglion sections show a large CD8⁺-T-cell infiltrate and the presence of inflammatory cytokines, suggesting that the presence of activated, effector memory cells within the PNS is important for maintaining HSV-1 latency in the natural human host (10, 42).The generation of a robust CD8⁺-T-cell response is essential for the control of various infectious pathogens. Some studies suggest that a brief interaction with antigen-presenting cells (APCs) is sufficient for CD8⁺-T-cell activation and expansion into functional effectors (44). However, the magnitude and quality of the overall CD8⁺-T-cell response generated may be dependent on additional factors (49). Recent evidence suggests that CD4⁺ T cells facilitate the activation and development of CD8⁺-T-cell responses either directly through the provision of cytokines or indirectly by the conditioning of dendritic cells (DC) (8, 48, 51). Those studies suggested that the latter mechanism is the dominant pathway, wherein CD4⁺ T cells assist CD8⁺-T-cell priming via the engagement of CD40 ligand (CD154) on CD4⁺ T cells and CD40 expressed on DC (4, 30, 33). This interaction results in the activation and maturation of DC, making them competent to stimulate antigen-specific CD8⁺-T-cell responses (35, 37).The requirement for CD4⁺-T-cell help in the generation of primary and secondary CD8⁺-T-cell responses to antigen varies. Primary CD8⁺-T-cell responses to infectious pathogens, such as Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV), influenza virus, and vaccinia virus, can be mounted effectively independently of CD4⁺-T-cell help (3, 12, 22, 34). In contrast, primary CD8⁺-T-cell responses to nonmicrobial antigens display an absolute dependence on CD4⁺-T-cell help (4, 5, 30, 33, 46). This observed difference in the requirement for CD4⁺-T-cell help may ultimately be a product of the initial inflammatory stimulus generated following immunization (49). Microbial antigens trigger an inflammatory response that can lead to the direct activation and priming of APCs, such as DC, thereby bypassing the need for CD4⁺-T-cell help. Nonmicrobial antigens, however, trigger an attenuated inflammatory response that does not directly activate and prime DCs. In the absence of this inflammation, CD4⁺ T cells are thought to condition and license DC functions through CD154/CD40 interactions, which leads to the subsequent activation of antigen-specific CD8⁺-T-cell responses (5, 49). Even in the case of pathogens where primary CD8⁺-T-cell responses were independent of CD4⁺-T-cell help, the secondary responses to these pathogens were found to be defective in the absence of CD4⁺-T-cell help (3, 12, 34, 40).The requirement for CD4⁺-T-cell help in priming CD8⁺-T-cell responses against HSV-1 infection is not well defined. Earlier studies with HSV-1 suggested that CD4⁺ T cells play an important role in the generation of primary CD8⁺-T-cell responses, detected in vitro, to acute infection with HSV-1 (14), principally through the provision of interleukin 2 (IL-2) for optimal CD8⁺-T-cell differentiation and proliferation. Subsequent studies, utilizing an in vivo approach, indicated that CD4⁺ T cells were not required for CD8⁺-T-cell-mediated cytolytic function (23). CD4⁺ T cells are thought to provide help by conditioning DC in a cognate, antigen-specific manner, thereby making them competent to stimulate HSV-1-specific CD8⁺-T-cell responses (37). By contrast, findings from other studies show that CD4⁺-T-cell-depleted mice were able to fully recover from acute infection with HSV-1 (38). These studies imply that the absence of CD4⁺ T cells does not prevent priming of CD8⁺ T cells in vivo.Studies from this laboratory have identified two distinct HSV-1-specific CD8⁺-T-cell subpopulations generated during the primary response, based upon the ability to synthesize IFN-γ following antigenic stimulation in vitro (1). To better understand the need for CD4⁺-T-cell help, we examined the functional characteristics and phenotypes of these CD8⁺-T-cell populations generated during a primary response to acute infection with HSV-1 in mice lacking CD4⁺ T cells. Our findings show that primary CD8⁺-T-cell responses to HSV-1 are compromised in the absence of CD4⁺-T-cell help. Specifically, the HSV-1 gB-specific CD8⁺ T cells produced in the absence of CD4⁺ T cells were found to be active with regard to cytolysis in vivo but were functionally impaired in the production of IFN-γ and TNF-α compared with intact C57BL/6 mice. Virus-specific CD8⁺ T cells were also reduced in number in CD4-depleted mice and in B6 mice lacking major histocompatibility complex (MHC) class II expression (B6-MHC-II^−/−) compared to wild-type (WT) mice. In addition, our data showed higher virus burdens in the infectious tissues obtained from mice lacking CD4⁺ T cells than in those from intact mice. Collectively, these findings demonstrate that CD4⁺-T-cell help is essential for the generation of primary CD8⁺-T-cell responses following acute cutaneous infection with HSV-1.     

Simian Immunodeficiency Virus-Specific CD8+ T Cells Recognize Vpr- and Rev-Derived Epitopes Early after Infection

The kinetics of CD8⁺ T cell epitope presentation contribute to the antiviral efficacy of these cells yet remain poorly defined. Here, we demonstrate presentation of virion-derived Vpr peptide epitopes early after viral penetration and prior to presentation of Vif-derived epitopes, which required de novo Vif synthesis. Two Rev epitopes exhibited differential presentation kinetics, with one Rev epitope presented within 1 h of infection. We also demonstrate that cytolytic activity mirrors the recognition kinetics of infected cells. These studies show for the first time that Vpr- and Rev-specific CD8⁺ T cells recognize and kill simian immunodeficiency virus (SIV)-infected CD4⁺ T cells early after SIV infection.The antiviral activity of AIDS virus-specific CD8⁺ T cells is well documented in both in vivo (1, 4, 21) and in vitro (8, 24, 29) studies. Accordingly, human immunodeficiency virus (HIV) vaccine modalities that focus on engendering antiviral CD8⁺ T cells are being developed (13, 26, 28). Ideally, a CD8⁺ T cell-based vaccine would stimulate responses against epitopes that are presented by major histocompatibility complex class I (MHC-I) molecules early after infection of a target cell. However, successful selection of antigenic sequences for a CD8⁺ T cell-based vaccine has been frustrated in part by an incomplete understanding of the properties of effective CD8⁺ T cell responses (25).     

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.     

Proliferation Capacity and Cytotoxic Activity Are Mediated by Functionally and Phenotypically Distinct Virus-Specific CD8 T Cells Defined by Interleukin-7Rα (CD127) and Perforin Expression

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127⁺ perforin⁻/CD127⁻ perforin⁺, and CD127⁻/perforin⁻ CD8 T cells, respectively. CD127⁻/perforin⁻ and CD127⁻/perforin⁺ cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin⁺/IL-2⁻ or perforin⁻/IL-2⁺ cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127⁺/IL-2-secreting) and cytotoxic (perforin⁺) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.Cytotoxic CD8 T cells are a fundamental component of the immune response against viral infections and mediate an important role in immunosurveillance (7, 10, 55), and the induction of vigorous CD8 T-cell responses after vaccination is thought to be a key component of protective immunity (37, 41, 49, 50, 58, 60, 69). Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules containing perforin (pore-forming protein) and several granule-associated proteases, including granzymes (Grms) (5, 15, 20, 44). Several studies have recently advanced the characterization of the mechanism of granule-dependent cytotoxic activity and performed a comprehensive investigation of the content of cytotoxic granules in human virus-specific CD8 T cells (2, 19, 29, 44, 53).Heterogeneous profiles of cytotoxic granules have been identified in different virus-specific memory CD8 T cells and associated with distinct differentiation stages of memory CD8 T cells (2, 19, 29, 44). Furthermore, we have observed a hierarchy among the cytotoxic granules in setting the efficiency of cytotoxic activity and demonstrated that perforin (and to a lesser extent GrmB) but not GrmA or GrmK were associated with cytotoxic activity (29). Recently, a novel mechanism of perforin-dependent granule-independent CTL cytotoxicity has also been demonstrated (45).Major advances in the characterization of antigen (Ag)-specific CD4 and CD8 T cells have been made recently and have aimed at identifying functional profiles that may correlate with protective CD8 T-cell responses (1, 3, 4, 12, 13, 24, 28, 36-38, 40, 41, 49, 50, 56-58, 60, 64, 68). In particular, the functional characterization of antigen-specific T cells was mainly performed on the basis of (i) the pattern of cytokines secreted (i.e., gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], or macrophage inflammatory protein 1β [MIP-1β]), (ii) the proliferation capacity, and (iii) the cytotoxic capacity (13, 28, 59). Of note, degranulation activity (i.e., CD107a mobilization following specific stimulation) has been used as a surrogate marker of cytotoxic activity (11, 13).The term “polyfunctional” has been used to define T-cell immune responses that, in addition to typical effector functions such as secretion of IFN-γ, TNF-α, or MIP-1β and cytotoxic activity (measured by the degranulation capacity), comprise distinct T-cell populations able to secrete IL-2 and retain proliferation capacity (13, 28, 49, 50). Some evidence indicates that a hallmark of protective immune responses is the presence of polyfunctional T-cell responses (59). Furthermore, the ability to secrete IL-2 was shown to be linked to proliferation capacity, and both factors have been associated with protective antiviral immunity (13, 28, 49, 50). Although a lack of correlation between degranulation activity and GrmB expression was reported in mice (65), the relationship between degranulation activity and perforin expression has never been comprehensively investigated in mice and in humans.The private α chain of the IL-7 receptor (IL-7Rα, also called CD127) has been suggested to selectively identify CD8 T cells that will become long-lived memory cells (6, 34, 36). Moreover, it was shown in mice (34, 36) and humans (14, 48, 63) that the CD127^high memory-precursor CD8 T cells produced IL-2 in contrast to CD127^low effector CD8 T cells. Of interest, CD127 expression has also been shown to correlate with Ag-specific proliferation capacity in mice (34, 36). A similar correlation was observed in humans, although only for polyclonal stimulations (48). With the exception of studies performed in HIV-1 infection, where an association between CD127 expression and HIV-1 viremia has been shown (21, 22, 42, 48, 54), very limited information is available on the CD127 expression in human virus-specific CD8 T cells other that HIV-1.Although cytotoxic activity and proliferation capacity are key components of the antiviral cellular immune response, the relationship between these functions has been only investigated in nonprogressive HIV-1 infection (46), where these two functions were shown to be related. However, it still remains to be determined whether these functions are mediated by the same or by different T-cell populations.In the present study, we performed a comprehensive characterization of virus-specific CD8 T-cell responses against HIV-1, cytomegalovirus (CMV), Epstein Barr virus (EBV), and influenza virus (Flu) in order to (i) analyze the degree of concordance between degranulation activity and perforin/Grm expression; (ii) identify the relevance of CD127 in identifying virus-specific CD8 T cells endowed with proliferation capacity; (iii) delineate the relationship between proliferation capacity, cytotoxic activity, activation/differentiation stage, and level of exhaustion of CD8 T cells; and (iv) determine the influence of antigen exposure in shaping the functional composition of virus-specific CD8 T cells.Our data indicate that cytotoxic (as defined by perforin expression) and proliferating (as defined by CD127 expression or IL-2 secretion) virus-specific CD8 T cells are contained within distinct CD8 T-cell populations. Furthermore, the proportion of proliferating and cytotoxic T cells within a given virus-specific CD8 T-cell population appears to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferative capacity, differentiation stage, and Ag exposure of memory CD8 T cells.     

Enhancement of human immunodeficiency virus (HIV)-specific CD8+ T cells in cerebrospinal fluid compared to those in blood among antiretroviral therapy-naive HIV-positive subjects

During untreated human immunodeficiency virus type 1 (HIV-1) infection, virus-specific CD8⁺ T cells partially control HIV replication in peripheral lymphoid tissues, but host mechanisms of HIV control in the central nervous system (CNS) are incompletely understood. We characterized HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) and peripheral blood among seven HIV-positive antiretroviral therapy-naïve subjects. All had grossly normal brain magnetic resonance imaging and spectroscopy and normal neuropsychometric testing. Frequencies of epitope-specific CD8⁺ T cells by direct tetramer staining were on average 2.4-fold higher in CSF than in blood (P = 0.0004), while HIV RNA concentrations were lower. Cells from CSF were readily expanded ex vivo and responded to a broader range of HIV-specific human leukocyte antigen class I restricted optimal peptides than did expanded cells from blood. HIV-specific CD8⁺ T cells, in contrast to total CD8⁺ T cells, in CSF and blood were at comparable maturation states, as assessed by CD45RO and CCR7 staining. The strong relationship between higher T-cell frequencies and lower levels of viral antigen in CSF could be the result of increased migration to and/or preferential expansion of HIV-specific T cells within the CNS. This suggests an important role for HIV-specific CD8⁺ T cells in control of intrathecal viral replication.Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) early during primary infection (21, 30, 35), and proviral DNA persists in the brain throughout the course of HIV-1 disease (7, 25, 29, 47, 77, 83). Limited data from human and nonhuman primate studies suggest that little or no viral replication occurs in the brain during chronic, asymptomatic infection, based on the absence of demonstrable viral RNA or proteins (8, 85). In contrast, cognitive impairment affects approximately 40% of patients who progress to advanced AIDS without highly active antiretroviral therapy (21, 30, 35, 65). During HIV-associated dementia, there is active HIV-1 replication in the brain (23, 52, 61, 81), and viral sequence differences between cerebrospinal fluid (CSF) and peripheral tissues suggest distinct anatomic compartments of replication (18, 19, 22, 53, 75, 76, 78). Host mechanisms that control viral replication in the CNS during chronic, asymptomatic HIV-1 infection are incompletely understood.Anti-HIV CD8⁺ T cells are present in blood and peripheral tissues throughout the course of chronic HIV-1 infection (2, 14). Multiple lines of evidence support a critical role for these cells in controlling HIV-1 replication. During acute HIV-1 infection, the appearance of CD8⁺ T-cell responses correlates temporally with a decline in viremia (11, 43), and a greater proliferative capacity of peripheral blood HIV-specific CD8⁺ T cells correlates with better control of viremia (36, 54). In addition, the presence of certain major histocompatibility complex class I human leukocyte antigen (HLA) alleles, notably HLA-B*57, predicts slower progression to AIDS and death during chronic, untreated HIV-1 infection (55, 62). Finally, in the simian immunodeficiency virus (SIV) model, macaques depleted of CD8⁺ T cells experience increased viremia and rapid disease progression (39, 51, 67).Little is known regarding the role of intrathecal anti-HIV CD8⁺ T cells in HIV neuropathogenesis. Nonhuman primate studies have identified SIV-specific CD8⁺ T cells in the CNS early after infection (16, 80). Increased infiltration of SIV antigen-specific CD8⁺ T cells and cytotoxic T lymphocytes has been detected only in CSF of slow progressors without neurological symptoms (72). In chronically infected macaques with little or no SIV replication in the brain, the frequency of HIV-specific T cells was higher in CSF than in peripheral blood but did not correlate with the level of plasma viremia or CD4⁺ T-cell counts (56). Although intrathecal anti-HIV CD8⁺ T cells may help control viral replication, a detrimental role in the neuropathogenesis of HIV-1 has also been postulated (38). Immune responses contribute to neuropathogenesis in models of other infectious diseases, and during other viral infections cytotoxic T lymphocytes can worsen disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (3, 17, 31, 37, 42, 44, 71).We tested the hypothesis that quantitative and/or qualitative differences in HIV-specific CD8⁺ T-cell responses are present in CSF compared to blood during chronic, untreated HIV-1 infection. We characterized HIV-specific CD8⁺ T-cell responses in CSF among seven antiretroviral therapy-naïve adults with chronic HIV-1 infection, relatively high peripheral blood CD4⁺ T-cell counts, and low plasma HIV-1 RNA concentrations. We show that among these HIV-positive individuals with no neurological symptoms and with little or no HIV-1 RNA in CSF, frequencies of HIV-specific T cells are significantly higher in CSF than in blood. These CSF cells are at a state of differentiation similar to that of T cells in blood and are functionally competent for expansion and IFN-γ production. The higher frequency of functional HIV-specific CD8⁺ T cells in CSF, in the context of low or undetectable virus in CSF, suggests that these cells play a role in the control of intrathecal viral replication.     

The Receptor Complex Associated with Human T-Cell Lymphotropic Virus Type 3 (HTLV-3) Env-Mediated Binding and Entry Is Distinct from,but Overlaps with,the Receptor Complexes of HTLV-1 and HTLV-2

Little is known about the transmission or tropism of the newly discovered human retrovirus, human T-cell lymphotropic virus type 3 (HTLV-3). Here, we examine the entry requirements of HTLV-3 using independently expressed Env proteins. We observed that HTLV-3 surface glycoprotein (SU) binds efficiently to both activated CD4⁺ and CD8⁺ T cells. This contrasts with both HTLV-1 SU, which primarily binds to activated CD4⁺ T cells, and HTLV-2 SU, which primarily binds to activated CD8⁺ T cells. Binding studies with heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), two molecules important for HTLV-1 entry, revealed that these molecules also enhance HTLV-3 SU binding. However, unlike HTLV-1 SU, HTLV-3 SU can bind efficiently in the absence of both HSPGs and NRP-1. Studies of entry performed with HTLV-3 Env-pseudotyped viruses together with SU binding studies revealed that, for HTLV-1, glucose transporter 1 (GLUT-1) functions at a postbinding step during HTLV-3 Env-mediated entry. Further studies revealed that HTLV-3 SU binds efficiently to naïve CD4⁺ T cells, which do not bind either HTLV-1 or HTLV-2 SU and do not express detectable levels of HSPGs, NRP-1, and GLUT-1. These results indicate that the complex of receptor molecules used by HTLV-3 to bind to primary T lymphocytes differs from that of both HTLV-1 and HTLV-2.The primate T-cell lymphotropic virus (PTLV) group of deltaretroviruses consists of three types of human T-cell lymphotropic viruses (HTLVs) (HTLV-1, HTLV-2, HTLV-3), their closely related simian T-cell lymphotropic viruses (STLVs) (STLV-1, STLV-2, STLV-3), an HTLV (HTLV-4) for which a simian counterpart has not been yet identified, and an STLV (STLV-5) originally described as a divergent STLV-1 (5-7, 30, 35, 37, 38, 45, 51, 53). HTLV-1 and HTLV-2, which have a 70% nucleotide homology, differ in both their pathobiology and tropism (reviewed in reference 13). While HTLV-1 causes a neurological disorder (tropical spastic paraparesis/HTLV-1-associated myelopathy) and a hematological disease (adult T-cell leukemia/lymphoma) (15, 42, 55), HTLV-2 is only rarely associated with tropical spastic paraparesis/HTLV-1-associated myelopathy-like disease and is not definitively linked to any lymphoproliferative disease (12, 20). In vivo, both HTLV-1 and HTLV-2 infect T cells. Although HTLV-1 is primarily found in CD4⁺ T cells, other cell types in the peripheral blood of infected individuals have been found to contain HTLV-1, including CD8⁺ T cells, dendritic cells, and B cells (19, 29, 33, 36, 46).Binding and entry of retroviruses requires specific interactions between the Env glycoproteins on the virus and cell surface receptor complexes on target cells. For HTLV-1, three molecules have been identified as important for entry, as follows: heparan sulfate proteoglycans (HSPGs), neuropilin-1 (NRP-1), and glucose transporter 1 (GLUT-1) (16, 22, 26, 28, 29, 34, 39, 44). Recent studies support a model in which HSPG and NRP-1 function during the initial binding of HTLV-1 to target cells, and GLUT-1 functions at a postattachment stage, most likely to facilitate fusion (29, 34, 49). Efficient HTLV-2 binding and entry requires NRP-1 and GLUT-1 but not HSPGs (16, 26, 39, 49).This difference in the molecules required for binding to target cells reflects differences in the T-cell tropisms of these two viruses. Activated CD4⁺ T cells express much higher levels of HSPGs than CD8⁺ T cells (26). In infected individuals, HTLV-1 is primarily found in CD4⁺ T cells, while HTLV-2 is primarily found in CD8⁺ T cells (21, 43, 46). In vitro, HTLV-1 preferentially transforms CD4⁺ T cells while HTLV-2 preferentially transforms CD8⁺ T cells, and this difference has been mapped to the Env proteins (54).We and others have reported the discovery of HTLV-3 in two Cameroonese inhabitants (6, 7, 53). We recently uncovered the presence of a third HTLV-3 strain in a different population living several hundred kilometers away from the previously identified groups (5), suggesting that this virus may be common in central Africa. Since the HTLV-3 sequences were obtained by PCR amplification of DNA isolated from peripheral blood mononuclear cells (PBMCs) of infected individuals, little is known about its tropism and pathobiology in vivo. Based on the correlation between HSPG expression levels and viral tropisms of HTLV-1 and HTLV-2, we reasoned that knowledge about the HTLV-3 receptors might provide insight into the tropism of this virus. We therefore generated vectors expressing HTLV-3 Env proteins and used them to begin to characterize the receptor complex used by HTLV-3 to bind and enter cells.     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

Polyfunctional CD4+ T-Cell Induction in Neutralizing Antibody-Triggered Control of Simian Immunodeficiency Virus Infection

Rapid depletion of memory CD4⁺ T cells and delayed induction of neutralizing antibody (NAb) responses are characteristics of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. Although it was speculated that postinfection NAb induction could have only a limited suppressive effect on primary HIV replication, a recent study has shown that a single passive NAb immunization of rhesus macaques 1 week after SIV challenge can result in reduction of viral loads at the set point, indicating a possible contribution of postinfection NAb responses to virus control. However, the mechanism accounting for this NAb-triggered SIV control has remained unclear. Here, we report rapid induction of virus-specific polyfunctional T-cell responses after the passive NAb immunization postinfection. Analysis of SIV Gag-specific responses of gamma interferon, tumor necrosis factor alpha, interleukin-2, macrophage inflammatory protein 1β, and CD107a revealed that the polyfunctionality of Gag-specific CD4⁺ T cells, as defined by the multiplicity of these responses, was markedly elevated in the acute phase in NAb-immunized animals. In the chronic phase, despite the absence of detectable NAbs, virus control was maintained, accompanied by polyfunctional Gag-specific T-cell responses. These results implicate virus-specific polyfunctional CD4⁺ T-cell responses in this NAb-triggered virus control, suggesting possible synergism between NAbs and T cells for control of HIV/SIV replication.Virus-specific CD4⁺ and CD8⁺ T-cell responses are crucial for the control of pathogenic human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) infections (5, 6, 20, 23, 30, 39, 40). However, CD4⁺ T cells, especially CCR5⁺ memory CD4⁺ T cells, are themselves targets for these viruses, which may be an obstacle to potent virus-specific CD4⁺ T-cell induction (10, 47, 52). Indeed, HIV-1/SIV infection causes rapid, massive depletion of memory CD4⁺ T cells (26, 31), and host immune responses fail to contain viral replication and allow persistent chronic infection, although virus-specific CD8⁺ T-cell responses exert suppressive pressure on viral replication (15).Recently, the importance of T-cell quality in virus containment has been high-lighted, and T-cell polyfunctionality, which is defined by their multiplicity of antigen-specific cytokine production, has been analyzed as an indicator of T-cell quality (4, 8, 11, 41). However, there has been no evidence indicating an association of polyfunctional T-cell responses in the acute phase with HIV-1/SIV control. Even in the chronic phase, whether polyfunctional CD4⁺ T-cell responses may be associated with virus control has been unclear, although an inverse correlation between polyfunctional CD8⁺ T-cell responses and viral loads has been shown in HIV-1-infected individuals (4).Another characteristic of HIV-1/SIV infections is the absence of potent neutralizing antibody (NAb) induction during the acute phase (7). This is mainly due to the unusually neutralization-resistant nature of the virus, such as masking of target epitopes in viral envelope proteins (24). Whether this lack of effective NAb response contributes to the failure to control the virus, and whether NAb induction in the acute phase can contribute to virus control, remains unclear. Previous studies documenting virus escape from NAb recognition suggested that NAbs can also exert selective pressure on viral replication to a certain extent (38, 45, 49), but it was speculated that postinfection NAb induction could have only a limited suppressive effect on primary HIV-1/SIV replication (34, 37).By passive NAb immunization of rhesus macaques after SIV challenge, we recently provided evidence indicating that the presence of NAbs during the acute phase can result in SIV control (50). In that study, passive NAb immunization 1 week after SIVmac239 challenge resulted in transient detectable NAb responses followed by reduction in set point viral loads compared to unimmunized macaques. However, the mechanism of this virus control has remained unclear. In the present study, we found rapid appearance of polyfunctional Gag-specific CD4⁺ T-cell responses after such passive NAb immunization postinfection. These animals maintained virus control for more than 1 year in the absence of detectable plasma NAbs, which was accompanied by potent Gag-specific T-cell responses. These results implicate virus-specific polyfunctional CD4⁺ T-cell responses in this NAb-triggered primary and long-term SIV control.     

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.     

Impact of Cytotoxic-T-Lymphocyte Memory Induction without Virus-Specific CD4+ T-Cell Help on Control of a Simian Immunodeficiency Virus Challenge in Rhesus Macaques

Despite many efforts to develop AIDS vaccines eliciting virus-specific T-cell responses, whether induction of these memory T cells by vaccination before human immunodeficiency virus (HIV) exposure can actually contribute to effective T-cell responses postinfection remains unclear. In particular, induction of HIV-specific memory CD4⁺ T cells may increase the target cell pool for HIV infection because the virus preferentially infects HIV-specific CD4⁺ T cells. However, virus-specific CD4⁺ helper T-cell responses are thought to be important for functional CD8⁺ cytotoxic-T-lymphocyte (CTL) induction in HIV infection, and it has remained unknown whether HIV-specific memory CD8⁺ T cells induced by vaccination without HIV-specific CD4⁺ T-cell help can exert effective responses after virus exposure. Here we show the impact of CD8⁺ T-cell memory induction without virus-specific CD4⁺ T-cell help on the control of a simian immunodeficiency virus (SIV) challenge in rhesus macaques. We developed a prophylactic vaccine by using a Sendai virus (SeV) vector expressing a single SIV Gag_241-249 CTL epitope fused with enhanced green fluorescent protein (EGFP). Vaccination resulted in induction of SeV-EGFP-specific CD4⁺ T-cell and Gag_241-249-specific CD8⁺ T-cell responses. After a SIV challenge, the vaccinees showed dominant Gag_241-249-specific CD8⁺ T-cell responses with higher effector memory frequencies in the acute phase and exhibited significantly reduced viral loads. These results demonstrate that virus-specific memory CD8⁺ T cells induced by vaccination without virus-specific CD4⁺ T-cell help could indeed facilitate SIV control after virus exposure, indicating the benefit of prophylactic vaccination eliciting virus-specific CTL memory with non-virus-specific CD4⁺ T-cell responses for HIV control.Virus-specific T-cell responses are crucial for controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication (3, 4, 12, 20, 28, 36, 37). Therefore, a great deal of effort has been exerted to develop AIDS vaccines eliciting virus-specific T-cell responses (23, 27, 30, 47), but whether this approach actually results in HIV control remains unclear (1, 6). It is important to determine which T-cell responses need to be induced by prophylactic vaccination for HIV control after virus exposure.Because HIV preferentially infects HIV-specific CD4⁺ T cells (5), induction of HIV-specific memory CD4⁺ T cells by vaccination may increase the target cell pool for HIV infection and could enhance viral replication (42). However, CD4⁺ helper T-cell responses are important for functional CD8⁺ cytotoxic-T-lymphocyte (CTL) induction (11, 40, 43, 46), and it has remained unknown whether HIV-specific memory CD8⁺ T cells induced by vaccination with non-virus-specific CD4⁺ T-cell help (but without HIV-specific CD4⁺ T-cell help) can exert effective responses after virus exposure. Indeed, the real impact of prophylactic induction of CTL memory itself on HIV replication has not been well documented thus far.We previously developed a prophylactic AIDS vaccine consisting of DNA priming followed by boosting with a recombinant Sendai virus (SeV) vector expressing SIVmac239 Gag (26). Evaluation of this vaccine''s efficacy against a SIVmac239 challenge in Burmese rhesus macaques showed that some vaccinees contained SIV replication whereas unvaccinated animals developed AIDS (15, 27). In particular, vaccination consistently resulted in control of SIV replication in those animals possessing the major histocompatibility complex class I (MHC-I) haplotype 90-120-Ia. Gag_206-216 (IINEEAADWDL) and Gag_241-249 (SSVDEQIQW) epitope-specific CD8⁺ T-cell responses were shown to be involved in SIV control in these vaccinated macaques (14, 16).In the present study, focusing on CD8⁺ T-cell responses directed against one of these epitopes, we have evaluated the efficacy of a vaccine expressing the Gag_241-249 epitope fused with enhanced green fluorescent protein (EGFP) against a SIVmac239 challenge in 90-120-Ia-positive rhesus macaques. The animals exhibited this single-epitope-specific CD8⁺ T-cell response and SeV-EGFP-specific CD4⁺ T-cell responses after vaccination and showed rapid, dominant induction of potent secondary Gag_241-249-specific CD8⁺ T-cell responses after a SIV challenge. Plasma viral loads in these vaccinees were significantly reduced compared to those of naive controls. These results indicate that induction of CD8⁺ T-cell memory without virus-specific CD4⁺ T-cell help by prophylactic vaccination can result in effective CD8⁺ T-cell responses after virus exposure.     

Impact of HLA in Mother and Child on Disease Progression of Pediatric Human Immunodeficiency Virus Type 1 Infection

A broad Gag-specific CD8⁺ T-cell response is associated with effective control of adult human immunodeficiency virus (HIV) infection. The association of certain HLA class I molecules, such as HLA-B*57, -B*5801, and -B*8101, with immune control is linked to mutations within Gag epitopes presented by these alleles that allow HIV to evade the immune response but that also reduce viral replicative capacity. Transmission of such viruses containing mutations within Gag epitopes results in lower viral loads in adult recipients. In this study of pediatric infection, we tested the hypothesis that children may tend to progress relatively slowly if either they themselves possess one of the protective HLA-B alleles or the mother possesses one of these alleles, thereby transmitting a low-fitness virus to the child. We analyzed HLA type, CD8⁺ T-cell responses, and viral sequence changes for 61 mother-child pairs from Durban, South Africa, who were monitored from birth. Slow progression was significantly associated with the mother or child possessing one of the protective HLA-B alleles, and more significantly so when the protective allele was not shared by mother and child (P = 0.007). Slow progressors tended to make CD8⁺ T-cell responses to Gag epitopes presented by the protective HLA-B alleles, in contrast to progressors expressing the same alleles (P = 0.07; Fisher''s exact test). Mothers expressing the protective alleles were significantly more likely to transmit escape variants within the Gag epitopes presented by those alleles than mothers not expressing those alleles (75% versus 21%; P = 0.001). Reversion of transmitted escape mutations was observed in all slow-progressing children whose mothers possessed protective HLA-B alleles. These data show that HLA class I alleles influence disease progression in pediatric as well as adult infection, both as a result of the CD8⁺ T-cell responses generated in the child and through the transmission of low-fitness viruses by the mother.Human immunodeficiency virus (HIV)-specific CD8⁺ T cells play a central role in controlling viral replication (12). It is the specificity of the CD8⁺ T-cell response, particularly the response to Gag, that is associated with low viral loads in HIV infection (7, 17, 34). Although immune control is undermined by the selection of viral mutations that prevent recognition by the CD8⁺ T cells, evasion of Gag-specific responses mediated by protective class I HLA-B alleles typically brings a reduction in viral replicative capacity, facilitating subsequent immune control of HIV (2, 20, 21). The same principle has been demonstrated in studies of simian immunodeficiency virus infection (18, 22).Recent studies showed that the class I HLA-B alleles that protect against disease progression present more Gag-specific CD8⁺ T-cell epitopes and drive the selection of more Gag-specific escape mutations than those alleles that are associated with high viral loads (23). These protective HLA-B alleles not only are beneficial to infected individuals expressing those alleles but also benefit a recipient following transmission, since the transmitted virus carrying multiple Gag escape mutations may have substantially reduced fitness (3, 4, 8). However, there is no benefit to the recipient if he or she shares the same protective allele as the donor because the transmitted virus carries escape mutations in the Gag epitopes that would otherwise be expected to mediate successful immune control in the recipient (8, 11).The sharing of HLA alleles between donor and recipient occurs frequently in mother-to-child transmission (MTCT). The risk of MTCT is related to viral load in the mother, and a high viral load is associated with nonprotective alleles, such as HLA-B*18 and -B*5802. This may contribute in two distinct ways to the more rapid progression observed in pediatric HIV infection (24, 26, 27). First, because infected children share 50% or more of their HLA alleles with the transmitting mother, they are less likely than adults to carry protective HLA alleles (16). Thus, infected children as a group carry fewer protective HLA alleles and more nonprotective HLA alleles. Second, even when the child has a protective allele, such as HLA-B*27, this allele does not offer protection if the maternally transmitted virus carries escape mutations within the key Gag epitopes that are presented by the protective allele (11, 19).However, it is clear that infected children who possess protective alleles, such as HLA-B*27 or HLA-B*57, can achieve durable immune control of HIV infection if the virus transmitted from the mother is not preadapted to those alleles (6, 10). HIV-specific CD8⁺ T-cell responses are detectable from birth in infected infants (32). Furthermore, as in adult infection (3, 8), HIV-infected children have the potential to benefit from transmission of low-fitness viruses in the situation where the mother possesses protective HLA alleles and the child does not share those protective alleles. MTCT of low-fitness viruses carrying CD8⁺ T-cell escape mutations was recently documented (28; J. Prado et al., unpublished data).In this study, undertaken in Durban, South Africa, we set out to test the hypothesis that HIV-infected children are less likely to progress rapidly to disease if either the infected child or the transmitting mother possesses a protective HLA allele that is not shared. The HLA alleles most strongly associated with low viral loads and high CD4 counts in a cohort of >1,200 HIV-infected adults in Durban are HLA-B*57 (-B*5702 and -B*5703), HLA-B*5801, and HLA-B*8101 (16; A. Leslie et al., unpublished data). These four alleles all present Gag-specific CD8⁺ T-cell epitopes, and in each case the escape mutations selected in these epitopes reduce viral replicative capacity (2-4, 8, 21, 23).Analyzing a previously described cohort of 61 HIV-infected children in Durban (24, 26, 32), South Africa, who were all monitored from birth, we first addressed the question of whether possession of any of these four alleles by either mother or child is associated with slower disease progression in the child and then determined whether sharing of protective alleles by mother and child affects the ability of the child to make the Gag-specific CD8⁺ T-cell responses restricted by the shared allele.     

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.     

Route of Adenovirus-Based HIV-1 Vaccine Delivery Impacts the Phenotype and Trafficking of Vaccine-Elicited CD8+ T Lymphocytes

Candidate HIV-1 vaccine regimens utilizing intramuscularly (i.m.) administered recombinant adenovirus (rAd)-based vectors can induce potent mucosal cellular immunity. However, the degree to which mucosal rAd vaccine routing might alter the quality and anatomic distribution of vaccine-elicited CD8⁺ T lymphocytes remains unclear. We show that the route of vaccination critically impacts not only the magnitude but also the phenotype and trafficking of antigen-specific CD8⁺ T lymphocytes in mice. I.m. rAd immunization induced robust local transgene expression and elicited high-frequency, polyfunctional CD8⁺ T lymphocytes that trafficked broadly to both systemic and mucosal compartments. In contrast, intranasal (i.n.) rAd immunization led to similarly robust local transgene expression but generated low-frequency, monofunctional CD8⁺ T lymphocytes with restricted anatomic trafficking patterns. Respiratory rAd immunization elicited systemic and mucosal CD8⁺ T lymphocytes with phenotypes and trafficking properties distinct from those elicited by i.m. or i.n. rAd immunization. Our findings indicate that the anatomic microenvironment of antigen expression critically impacts the phenotype and trafficking of antigen-specific CD8⁺ T lymphocytes.Acute human immunodeficiency virus type 1 (HIV-1) infection is accompanied by a massive, irreversible destruction of memory CD4⁺ T lymphocytes, particularly within the intestinal mucosa (11, 26, 30, 42), as a result of the high proportion of effector/memory target cells within the intestinal lamina propria. Chronic HIV-1 infection is characterized by inflammation within the intestinal mucosa, breakdown of epithelial-barrier integrity, and translocation of gut microflora from the intestinal lumen (10, 24). These processes may drive systemic inflammation and contribute to HIV-1 disease progression. Therefore, vaccination strategies that enhance mucosal cellular immunity and attenuate the mucosal immunopathology of HIV-1 infection would be desirable.Recombinant adenovirus (rAd) vectors are potent inducers of cellular immunity (3, 12, 25), and we have recently demonstrated that intramuscular (i.m.) rAd immunization transiently activates peripheral antigen-specific CD8⁺ T lymphocytes and allows them to migrate to mucosal surfaces and establish potent, durable mucosal cellular immunity (22). Moreover, we have shown that an i.m. delivered heterologous rAd prime-boost regimen prevented the destruction of CD4⁺ T lymphocytes within the intestinal mucosa and attenuated disease progression following simian immunodeficiency virus (SIV) challenge (29). Notably, this vaccine regimen did not contain the SIV Env protein, indicating that cellular mucosal immunity likely played a critical role in abrogating mucosal CD4⁺ T-lymphocyte destruction.While our laboratory and others have observed potent mucosal CD8⁺ T-lymphocyte responses after i.m. immunization with rAd vectors (2, 21, 28, 41) and other vaccine modalities (40-41), other studies have suggested that mucosal routing of vaccine vectors may optimize mucosal cellular immunity (4-7, 16, 33, 36, 46). We therefore assessed the phenotype and anatomic trafficking patterns of antigen-specific CD8⁺ T-lymphocyte responses following i.m. and mucosal rAd immunization in mice. We found that the immunization route dramatically impacted the phenotype of vaccine-elicited systemic and mucosal CD8⁺ T lymphocytes. In particular, while both i.m. and intranasal (i.n.) rAd immunization resulted in efficient local transgene expression, only i.m. immunization induced potent, polyfunctional cellular immune memory in both systemic and mucosal anatomic compartments, while i.n. immunization elicited lower-frequency cellular immune responses that were restricted to mucosal surfaces and characterized by monofunctional gamma interferon (IFN-γ) secretion. Our data highlight the critical impact of the route of antigen delivery and the anatomic microenvironment of transgene expression on the quality and distribution of vaccine-elicited CD8⁺ T-lymphocyte responses.