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.     

CD4 T-Cell Help Programs a Change in CD8 T-Cell Function Enabling Effective Long-Term Control of Murine Gammaherpesvirus 68: Role of PD-1-PD-L1 Interactions

We previously showed that agonistic antibodies to CD40 could substitute for CD4 T-cell help and prevent reactivation of murine gammaherpesvirus 68 (MHV-68) in the lungs of major histocompatibility complex (MHC) class II^−/− (CII^−/−) mice, which are CD4 T cell deficient. Although CD8 T cells were required for this effect, no change in their activity was detected in vitro. A key question was whether anti-CD40 treatment (or CD4 T-cell help) changed the function of CD8 T cells or another cell type in vivo. To address this question, in the present study, we showed that adoptive transfer of CD8 T cells from virus-infected wild-type mice or anti-CD40-treated CII^−/− mice caused a significant reduction in lung viral titers, in contrast to those from control CII^−/− mice. Anti-CD40 treatment also greatly prolonged survival of infected CII^−/− mice. This confirms that costimulatory signals cause a change in CD8 T cells enabling them to maintain effective long-term control of MHV-68. We investigated the nature of this change and found that expression of the inhibitory receptor PD-1 was significantly increased on CD8 T cells in the lungs of MHV-68-infected CII^−/−, CD40^−/−, or CD80/86^−/− mice, compared with that in wild-type or CD28/CTLA4^−/− mice, correlating with the level of viral reactivation. Furthermore, blocking PD-1-PD-L1 interactions significantly reduced viral reactivation in CD4 T-cell-deficient mice. In contrast, the absence of another inhibitory receptor, NKG2A, had no effect. These data suggest that CD4 T-cell help programs a change in CD8 T-cell function mediated by altered PD-1 expression, which enables effective long-term control of MHV-68.Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen which is closely related to Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) (17, 64). Intranasal administration of MHV-68 to mice results in acute productive infection of lung epithelial cells and a latent infection in various cell types, including B lymphocytes, dendritic cells, epithelial cells, and macrophages (18, 19, 52, 53, 61, 65). The virus induces an inflammatory infiltrate in the lungs, lymph node enlargement, splenomegaly, and mononucleosis comprising increased numbers of activated CD8 T cells in the blood (53, 58). It has also been reported to induce lymphoproliferative disease/lymphoma in immunocompromised mice (30, 55, 60). Thus, the pathogenesis resembles that of EBV in humans, although structurally, the virus is more closely related to KSHV.Infectious MHV-68 is cleared from the lungs by a T-cell-dependent mechanism 10 to 15 days after infection (18, 53, 56). In wild-type mice, the lungs remain clear of replicating virus thereafter. Although CD4 T cells are not essential for primary clearance of replicating virus, they are required for effective long-term control (11). Thus, major histocompatibility complex (MHC) class II^−/− mice that lack CD4 T cells or mice rendered CD4 deficient by antibody treatment initially clear infectious virus from the lungs. However, infectious virus reactivates in the lungs 10 to 15 days later and gradually increases in titer (11, 43). The infected CD4-deficient mice eventually die, apparently from long-term lung damage due to continuing lytic viral replication (11). MHC class II^−/− mice do not produce antibody to T-dependent antigens (10). Cytotoxic T-lymphocyte (CTL) epitopes have been identified in open reading frame (ORF) 6 (p56, H-2D^b-restricted), and ORF 61 (p79, H-2K^b-restricted) gene products, which appear to encode early lytic-phase proteins (32, 49). The epitopes are presented during two distinct phases during MHV-68 infection, which changes the pattern of CTL dominance (32, 51). However, there is no significant difference in the numbers of CD8 T cells specific for each epitope in wild-type mice and CD4 T-cell-deficient mice (4, 50). In addition, CTL activity measured in vitro does not differ substantially in the lungs of wild-type mice or CD4 T-cell-deficient mice (4, 11, 50). Furthermore, postexposure vaccination with the p56 epitope failed to prevent viral reactivation in class II^−/− mice, despite dramatically expanding the number of CD8 T cells specific for the peptide (5). In contrast, vaccination of wild-type mice against these epitopes reduced lytic viral titers in the lung dramatically on subsequent challenge with MHV-68. B-cell-deficient mice clear MHV-68 with the kinetics of wild-type mice and do not show viral reactivation in the lungs (13, 61), suggesting that antibody is not essential for control of the virus. Depletion of CD4 T cells during the latent phase of infection in B-cell-deficient mice does not induce viral reactivation, whereas depletion of both CD4 and CD8 T-cell subsets provokes viral reactivation in the lungs (52). Short-term depletion of both CD4 and CD8 T-cell subsets during the latent phase of infection in wild-type mice does not lead to viral reactivation probably due to the presence of neutralizing antibody (11). Taken together, these results suggest that CD4 and CD8 T cells and B cells play overlapping roles in preventing or controlling reactivation of MHV-68 during the latent phase of infection. However, the B-cell- and CD8 T-cell-mediated control mechanisms do not develop in the absence of CD4 T cells.We, and others, have previously shown that the costimulatory molecule CD28 is not required for long-term control of MHV-68 (28, 29). However, interestingly, mice lacking both of the ligands for CD28, CD80 and CD86, show viral reactivation in the lung (21, 35). Our previously published data showed that agonistic antibodies to CD40 could substitute for CD4 T-cell function in the long-term control of MHV-68 (46). CD8 T-cell receptor-positive (TCR+) cells were required for this effect, while antibody production was not restored (45, 46). MHV-68-infected CD40L^−/− mice (7) and CD40^−/− mice (29) also showed viral reactivation in the lungs. However, no change in CD8 CTL activity was detected in in vitro assays following anti-CD40 treatment (46). A key question was whether anti-CD40 treatment (or CD4 T-cell help) caused a direct change in CD8 T-cell function or whether both CD8 T cells and an independent anti-CD40-sensitive step were required for viral control. To address this question, we used adoptive transfer of CD8 T cells from MHV-68-infected wild-type mice, anti-CD40-treated mice, or control MHC class II^−/− mice to MHV-68-infected class II^−/− recipients. We also investigated whether anti-CD40 treatment prolonged survival in addition to reducing lung viral titers. The heterodimeric molecule CD94/NKG2A has been implicated in negatively regulating the CD8 T-cell response to polyomavirus (38) and herpes simplex virus (HSV) (54), while the inhibitory receptor PD-1 (programmed death 1) has been implicated in T-cell exhaustion following infection with several other persistent viruses (2, 15, 20, 22, 26, 36, 39-41, 57, 67). In the present study, we investigated the effect of signaling via various costimulatory molecules on the expression of NKG2A and PD-1 and how these molecules influenced viral control.     

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.     

Effective Downregulation of HLA-A*2 and HLA-B*57 by Primary Human Immunodeficiency Virus Type 1 Isolates Cultured from Elite Suppressors

Elite controllers or suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who control viral replication to <50 copies/ml without antiretroviral therapy. Downregulation of HLA class I molecules is an important mechanism used by HIV-1 to evade the immune system. In this study, we showed that primary isolates from ES are as effective as isolates obtained from patients with progressive HIV-1 disease at downregulating HLA-A*2 and HLA-B*57 molecules on primary CD4⁺ T cells. Thus, a diminished ability of viral isolates from ES to evade HIV-specific immune responses probably does not contribute to the control of viral replication in these patients.Long-term nonprogressors (LTNP) are human immunodeficiency virus type 1 (HIV-1)-infected individuals who maintain normal CD4⁺ T-cell counts and remain asymptomatic for longer than 10 years without therapy (7). Although many LTNP have detectable levels of HIV-1 RNA in their plasma, patients known as elite suppressors (ES) have viral loads of <50 RNA copies/ml. Understanding the factors involved in the maintenance of LTNP and ES statuses may be critical for the development of effective vaccines and immunotherapeutic treatments. One such factor under investigation is the role of cytotoxic-T-lymphocyte (CTL) responses. Several studies have shown that the HLA-B*27 and -B*57 alleles are overrepresented in cohorts of ES (13, 16, 19, 28, 29, 34). These findings suggest important roles for major histocompatibility complex class I (MHC-I) restriction and CD8⁺ T-cell responses in the control of viremia. Indeed, multiple studies have documented qualitatively superior CD8⁺ T cell function in ES compared to that in chronic HIV progressors (CP) (2, 5, 12, 27, 28, 37, 47).Other studies suggest that some ES and LTNP are infected with attenuated viruses. One illustrative example comes from studies done on the Sydney Blood Bank Cohort, in which an LTNP donor transmitted an HIV-1 isolate with a large deletion in nef and the U3 region of the long terminal repeat to multiple recipients, all of whom became LTNP (11, 21). As in the Sydney Blood Bank Cohort studies, several other investigators have detected viruses with defective nef genes in LTNP and ES (1, 8, 18, 23, 25, 35, 36, 38, 43). In contrast, other studies showed that CD4⁺ T cells from ES could produce Gag when they were stimulated in vitro (20, 26), and full-length sequence analyses of plasma and proviral genomes revealed no evidence of significant deletions (30). Recent studies have suggested that plasma isolates (31) and replication-competent viruses (32) from HLA-B*57/B58*01 ES and LTNP, respectively, are less fit than isolates from B*57/B*5801 CP, but the difference in fitness observed is unlikely to fully explain the control of viral replication in these patients. Furthermore, we recently performed detailed genotypic and phenotypic analyses of replication-competent viruses isolated from ES and showed that these viruses were fully replication competent (6) Although nef is not required for viral replication in vitro, it has been strongly associated with pathogenesis in vivo (reviewed in reference 14). It is thus possible that some ES isolates are replication competent but have mutations in nef that result in diminished pathogenesis.nef has been shown to be involved in the downregulation of both CD4 (15) and MHC-I (41). Several studies have shown that nef-induced MHC-I downregulation has a major impact on CTL function. In a seminal study, a dramatic reduction in HLA-A*2 expression by CD4⁺ T cells infected with wild-type virus but not by those infected with a virus carrying a defective nef gene was demonstrated. This downregulation resulted in diminished killing of HIV-1-infected cells by CTL clones specific for an HLA-A*2-restricted HIV-1 Gag epitope (10). Similarly, nef-mediated MHC-I downregulation was shown to impair the ability of HIV-1-specific CTL clones to suppress viral replication (42, 44). While these findings strongly suggest that HIV-1 partially evades the immune response by inducing MHC-I downregulation, other studies have demonstrated that primary CD8⁺ T cells from some ES and CP could effectively respond to autologous viral replication in autologous CD4⁺ T cells (26).We tested the hypothesis that ES are infected with HIV-1 isolates that are less capable of downregulating MHC-I molecules. This could potentially cause the isolates to be more susceptible to CD8⁺ T-cell suppression of replication and may explain the superior CD8⁺ T-cell responses reported in prior ES studies (2, 5, 12, 27, 28, 37, 47). To date, fully characterized replication-competent isolates have been reported from just six ES subjects (1, 3, 6). We compared the MHC-I downregulation capacity of isolates from five of these ES to that of isolates obtained from resting CD4⁺ T cells of eight patients with progressive disease (viral load, >10,000 copies/ml). In order to develop a physiological model for HIV-1-induced MHC-I downregulation, we enriched primary CD4⁺ T cells from peripheral blood mononuclear cells (PBMC) from donors who were HLA-A*2 and/or HLA-B*57 positive by CD8⁺ T cell depletion with magnetic beads (Dynal), followed by activation in vitro with phytohemagglutinin for 3 days. For evaluation of HLA-A*2 downregulation, CD4⁺ T cells were obtained from HIV-seronegative donors. CD4⁺ T cells from ES were used for the evaluation of HLA-B*57 downregulation. This allele was as effectively downregulated in these ES as it was in multiple HLA-B*57 CP (data not shown). Following activation, the cells were infected with primary HIV-1 isolates from ES or CP by spinoculation (33). The primary isolates were obtained as previously described from latently infected CD4⁺ T cells (9). The median peak viral load and CD4⁺ T-cell nadir of the CP from whom viral isolates were obtained was 81,000 copies/ml and 279 cells/μl, respectively, and thus these isolates should be effective at HLA downregulation (22).At different time points, the cells were harvested and stained with either fluorescein isothiocyanate (FITC)-conjugated anti-HLA-A*2 (Becton Dickinson) and tricolor-conjugated anti-CD4 antibodies (Caltag) or biotinylated anti-HLA-B*57 antibody (One Lambda) followed by FITC-conjugated streptavidin, peridinin chlorophyll protein-Cy5.5-conjugated anti-CD4 antibody (Becton Dickenson), and allophycocyanin-conjugated anti-CD3 antibody. The cells were fixed and permeabilized with Cytofix/Cytoperm solution (Becton Dickenson). Intracellular staining was then performed with the phycoerythrin-conjugated Gag-specific monoclonal antibody Kc57 or an immunoglobulin G1 mouse isotype control (Beckman Coulter). A total of 100,000 to 500,000 events were analyzed for each sample. HLA typing of ES was performed as previously described (4). The HLA-specific antibodies were tested on cells from a panel of ES with known HLA types to confirm specificity.MHC-I downregulation was measured by comparing the mean fluorescence intensities (MFI) of HLA-A*2 and HLA-B*57 on HIV-1-infected versus noninfected CD4⁺ T cells. Infected cells were defined as cells that stained positive for intracellular Gag and had downregulated CD4 (Fig. ​(Fig.1).1). Uninfected CD4⁺ T cells were defined as cells that expressed high levels of CD4 and were negative for intracellular Gag protein. In order to standardize values, we determined relative MFI by dividing the MFI of the infected population by that of the CD4-positive, uninfected population. The Wilcoxon Mann-Whitney test was used to analyze the data.Open in a separate windowFIG. 1.Analysis of HLA-B*57 downregulation on HIV-1-infected cells. (A) CD8⁺ T-cell-depleted PBMC were stained with anti-HLA-B*57 and anti-CD4 monoclonal antibodies 3 days after infection with primary isolates from an ES (ES8) or a CP (CP2). Cells in quadrant 1 are uninfected CD4⁺ T cells, and cells in quadrant 4 (Gag-positive, low levels of CD4) are infected cells that have downregulated CD4. (PE, phycoerythrin; IgG, immunoglobulin G.) (B) The MFI of HLA-B*57 were compared for uninfected (quadrant 1) and infected (quadrant 4) cells from each sample.To determine if there was a difference in the ability of HIV-1 isolates cultured from ES versus CP to downregulate HLA-A*2, we measured the MFI of this molecule on infected CD4⁺ T cells that had downregulated CD4. On average, primary CD4⁺ T cells infected by ES viruses had levels of MHC-I downregulation of about two- to threefold, with relative MFI of 0.51, 0.37, and 0.30 on days 2, 3, and 4, respectively. Similarly, cells infected by isolates cultured from CP had relative MFI of 0.46, 0.36, and 0.33 on days 2, 3, and 4, respectively (Fig. ​(Fig.2B).2B). These differences were not significantly different at any time point.Open in a separate windowFIG. 2.(A) Relative MFIs of HLA-A*02 on cells infected with isolates from five ES (triangles) and eight CP (squares) on days 2 to 4 postinfection. The relative MFI is defined as the MFI of the infected cells divided by the MFI of the uninfected CD4⁺ T cells in each sample. The horizontal bars represent the median for each group. (B) Average relative MFI of HLA-A*02 for cells infected with isolates from ES and CP on each day. (C) Average relative MFI of HLA-A*02 for cells infected with the wild-type NL4-3-green fluorescent protein virus (diamonds) or the Nef⁻ Vpr⁻ mutant virus (circles).In order to rule out nonspecific downregulation of MHC-I on infected cells, we determined the MFI of HLA-DR and CD45 RO on cells infected with isolates from two subjects. The average relative MFI of the two proteins were 1.28 and 1.48, respectively, indicating that the MHC-I was in fact specifically downregulated. Since mutations in Nef have been shown to abrograte HLA downregulation, we also compared HLA-A2 downregulation by the HIV-1-based reporter construct NL4-3-green fluorescent protein and a Nef⁻ Vpr⁻ mutant vector (45, 46). As shown in Fig. ​Fig.2C,2C, no downregulation of HLA-A2 was seen at any point after infection with the Nef⁻ Vpr⁻ mutant virus, whereas infection with wild-type virus caused a degree of downregulation that was similar to that seen with primary isolates from ES and CP. Finally, we also looked at CD3 downregulation, as this molecule has been shown to be downregulated by Nef from HIV-2 and many simian immunodeficiency virus (SIV) isolates but not from HIV-1 (39). Furthermore, since SIVsmm nef isolated from sooty mangabeys with preserved CD4⁺ T-cell counts causes significantly more downregulation than SIVsmm nef from sooty mangabeys with CD4⁺ T-cell depletion (40), we determined whether isolates from ES also selectively downregulated this molecule. As shown in Fig. ​Fig.3A,3A, there was no significant downmodulation of CD3 after infection of cells with isolates from ES or CP.Open in a separate windowFIG. 3.(A) Relative MFI of CD3 on cells infected with isolates from five ES (triangles) and five CP (squares) on day 3 postinfection. The horizontal bars represent the median for each group. (B) Relative MFI of HLA-B*57 on cells infected with isolates from ES and CP.Epidemiologic studies have suggested that HLA-B alleles play a larger role than HLA-A alleles in determining the outcome of infection (17). Furthermore, while HLA-B*57 is the most overrepresented allele seen in ES, there have not been any studies looking at downregulation of this MHC-I protein. Activated CD4⁺ T cells from an HLA-B*5703-positive ES were infected with isolates from five ES and five CP, and the degree of HLA-B*57 downregulation was measured on day 3. As shown in Fig. ​Fig.3B,3B, the average relative MFI of cells infected with isolates from five ES was 0.53, which was not significantly different from the average relative MFI of 0.64 that was seen in cells infected with isolates from five progressors.While it appeared that there was generally more downregulation of HLA-A*2 than HLA-B*57, the studies were performed in cells from different donors, and this precluded a direct comparison of the MFI of the two MHC-I alleles. Two ES in our cohort were positive for both HLA alleles, and the degrees of downregulation of these proteins could thus be compared. CD4⁺ T cells from ES8 were infected with autologous virus (6), and cells from ES9 were infected with a primary isolate from the CP who transmitted virus to her (3). For patient ES8, HLA-A2 showed a greater degree of downregulation than HLA-B57 at day 3 (a relative MFI of 0.36 versus 0.62) (Fig. ​(Fig.4).4). In contrast, in ES9 the degrees of downregulation of the two proteins were nearly identical (a relative MFI of 0.35 for HLA-A2 versus 0.31 for HLA-B*57).Open in a separate windowFIG. 4.Comparison of the relative MFI of HLA-A*02 and HLA-B*57 on CD8⁺ T-cell-depleted PBMC from ES8 and ES9 that were infected with autologous virus (ES8) or with the primary isolate from the CP who transmitted the virus to ES9. The MFI of HLA-A*2 or HLA-B*57 on uninfected CD4⁺ T cells (top panels) and infected cells that had downregulated CD4 (bottom panels) are shown.This is the first study to look at downregulation of MHC-I proteins on CD4⁺ T cells infected with HIV-1 isolates cultured from ES CD4⁺ T cells. We used a physiological model where primary CD4⁺ T cells were infected with primary HIV-1 isolates. One advantage of this approach is that it accounts for HLA downregulation mediated by viral proteins such as Tat (24), as well as Nef. Similar amounts of MHC-I downregulation were seen for cells infected with replication-competent isolates cultured from ES and progressors. These results demonstrate that most ES are not infected by HIV-1 virions that are deficient in downregulating MHC-I compared to those of CP. Thus, it is likely that other factors enable ES to control viremia. The identification of these factors will have implications for the design of HIV-1 vaccines.     

Influence of Disulfide-Stabilized Structure on the Specificity of Helper T-Cell and Antibody Responses to HIV Envelope Glycoprotein gp120

CD4⁺ helper T cells specific for human immunodeficiency virus type 1 (HIV-1) are associated with control of viremia. Nevertheless, vaccines have had limited effectiveness thus far, in part because sequence variability and other structural features of the HIV envelope glycoprotein deflect the immune response. Previous studies indicated that CD4⁺ T-cell epitope dominance is controlled by antigen three-dimensional structure through its influence on antigen processing and presentation. In this work, three disulfide bonds in the outer domain of gp120 were individually deleted in order to destabilize the local three-dimensional structure and enhance the presentation of nearby weakly immunogenic epitopes. However, upon immunization of groups of BALB/c mice, the CD4⁺ T-cell response was broadly reduced for all three variants, and distinct epitope profiles emerged. For one variant, antibody titers were sharply increased, and the antibody exhibited significant CD4-blocking activity.The development of an effective vaccine against HIV has been hampered by an incomplete understanding of the correlates of protection against the virus. It is generally accepted that a robust antibody response and cytotoxic T-lymphocyte (CTL) response are required to control the disease and to prevent progression to AIDS (2, 17, 19, 20, 36, 38-42). Both of these arms of the immune system require help from CD4⁺ helper T cells (1, 27, 48). However, several important aspects of the CD4⁺ helper T-cell response remain poorly defined; these include the factors that determine epitope immunodominance in the CD4⁺ T-cell response, the relationship of specificity in the CD4⁺ T-cell response to specificity in the antibody and CD8⁺ responses, and the investment made by HIV (or any pathogen) to control the CD4⁺ T-cell response.Previous studies of mice showed that antigen structure modulates antigen processing and presentation of CD4⁺ helper T-cell epitopes (3-6, 9, 10, 23, 24, 43). Immunodominant CD4⁺ helper T-cell epitopes raised in response to immunization with the HIV envelope glycoprotein gp120 were found adjacent to flexible loops between elements of secondary structure (10). This was rationalized by the fact that flexible loops more readily conform to protease active sites and therefore are preferentially cleaved by proteases during antigen processing (10, 14, 15). Helper T-cell epitopes of gp120 in humans infected with HIV were also found flanking flexible loops (30). Dominant epitopes were located in the outer domain, an average of 12 residues C-terminal to flexible loops. In the less immunogenic inner domain, epitopes were found an average of five residues N-terminal to conserved regions of the protein, once again placing the epitopes C-terminal to flexible loops (30). These results suggested that antigen structure plays a significant role in the shaping of the helper T-cell response against HIV gp120 in both mice and humans.In reviewing previous studies mapping the helper T-cell response to gp120, we noted a marked absence of CD4⁺ T-cell responses to regions of the outer domain that coincided with the locations of highly conserved disulfide bonds (Fig. ​(Fig.1).1). Disulfide bonds have previously been shown to interfere with presentation of nearby helper T-cell epitopes (13, 26). Thus, we hypothesized that disulfide bonds stabilized these regions of the protein, protecting them from proteolysis. This resulted in the exclusion of these regions from presentation to helper T cells. We further hypothesized that the deletion of these disulfide bonds would result in the production of new helper T-cell epitopes by creating localized regions of flexibility that could now be processed and presented to T cells. The creation of new helper T-cell epitopes could also potentially lead to changes in the antibody response.Open in a separate windowFIG. 1.Gaps in helper T-cell epitope frequency in the outer domain of HIV gp120 coincide with the locations of disulfide bonds. The graph illustrates the frequencies of responses by residue for the combined profiles from immunized BALB/c and CBA mice (gray area) and for a group of seven HIV-infected human subjects (black line) (10, 30).For the present work, we constructed three disulfide-bond variants of gp120 by replacing paired cysteines in the outer domain with alanines. Characterization of the variants revealed that the proteins were structurally distinct from one another and from wild-type gp120. Groups of 10 BALB/c mice immunized with these proteins produced patterns of helper T-cell responses that were very different from each other and from that of a group of 10 BALB/c mice immunized with wild-type gp120. In general, the T-cell response was reduced in mice immunized with the variant proteins. For one of the variants, anti-gp120 antibody titers were increased and exhibited CD4-blocking activity.     

Pseudotyping Incompatibility between HIV-1 and Gibbon Ape Leukemia Virus Env Is Modulated by Vpu

The Env protein from gibbon ape leukemia virus (GaLV) has been shown to be incompatible with human immunodeficiency virus type 1 (HIV-1) in the production of infectious pseudotyped particles. This incompatibility has been mapped to the C-terminal cytoplasmic tail of GaLV Env. Surprisingly, we found that the HIV-1 accessory protein Vpu modulates this incompatibility. The infectivity of HIV-1 pseudotyped with murine leukemia virus (MLV) Env was not affected by Vpu. However, the infectivity of HIV-1 pseudotyped with an MLV Env with the cytoplasmic tail from GaLV Env (MLV/GaLV Env) was restricted 50- to 100-fold by Vpu. A Vpu mutant containing a scrambled membrane-spanning domain, Vpu_RD, was still able to restrict MLV/GaLV Env, but mutation of the serine residues at positions 52 and 56 completely alleviated the restriction. Loss of infectivity appeared to be caused by reduced MLV/GaLV Env incorporation into viral particles. The mechanism of this downmodulation appears to be distinct from Vpu-mediated CD4 downmodulation because Vpu-expressing cells that failed to produce infectious HIV-1 particles nonetheless continued to display robust surface MLV/GaLV Env expression. In addition, if MLV and HIV-1 were simultaneously introduced into the same cells, only the HIV-1 particle infectivity was restricted by Vpu. Collectively, these data suggest that Vpu modulates the cellular distribution of MLV/GaLV Env, preventing its recruitment to HIV-1 budding sites.The gammaretrovirus gibbon ape leukemia virus (GaLV) has been widely used for gene therapy because of its wide host cell tropism and nonpathogenicity (1, 6, 10, 12, 13, 20). The host cell receptor for GaLV Env has been cloned and identified as a sodium-dependent phosphate transporter protein (25, 26). Like other retroviruses, GaLV encodes a single transmembrane surface glycoprotein (GaLV Env), which is cleaved into surface (SU) and transmembrane (TM) subunits (Fig. ​(Fig.1).1). The TM domain of GaLV Env contains a short 30-amino-acid C-terminal cytoplasmic tail. Although GaLV Env functions well when coupled (pseudotyped) with murine leukemia virus (MLV)-based retroviral vectors, it has been shown to be completely incompatible with HIV-1 (4, 35). When GaLV Env is expressed with HIV-1, essentially no infectious HIV-1 particles are produced (4, 35). The mechanism for this infectivity downmodulation is unknown, but the component of GaLV Env responsible for the restriction has been mapped to the cytoplasmic tail. Replacing the cytoplasmic tail of GaLV Env with the equivalent sequence from MLV Env ameliorates the restriction. Likewise, replacing the cytoplasmic tail of MLV Env with that from GaLV Env confers the restriction (4).Open in a separate windowFIG. 1.Schematic of MLV Env protein. Sequences are the C-terminal cytoplasmic tails of MLV Env, GaLV Env, and human CD4. GaLV sequences in boldface are residues that have been shown to modulate the HIV-1 incompatibility (4). Underlined sequences in CD4 are amino acids required for Vpu-mediated downmodulation (2, 15). Arrows denote the location of MLV/GaLV tail substitution. SU, surface domain; TM, transmembrane domain.Vpu is an 81-amino-acid HIV-1 accessory protein produced from the same mRNA as the HIV-1 Env gene. The N terminus of Vpu contains a membrane-spanning domain, followed by a 50-amino-acid cytoplasmic domain. Vpu is unique to HIV-1 and a few closely related SIV strains. The best-characterized roles for Vpu in the HIV-1 life cycle are modulation of host proteins CD4 and tetherin (also known as BST-2, CD317, and HM1.24) (24, 38, 39). Vpu promotes the degradation of CD4 in the endoplasmic reticulum through a proteasome-dependent mechanism (29). The cytoplasmic tail of Vpu physically interacts with the cytoplasmic tail of CD4 and recruits the human β-transducing repeat-containing protein (β-TrCP) and E3 ubiquitin ligase components to polyubiquitinate and ultimately trigger the degradation of CD4 (18). Two serine residues at positions 52 and 56 of Vpu are phosphorylated by casein kinase-2 and are required for CD4 degradation (31, 32). The membrane-spanning domain of Vpu is not specifically required for CD4 degradation. A mutant protein containing a scrambled membrane-spanning sequence, Vpu_RD, is still able to trigger the degradation of CD4 (32). The region of CD4 that is targeted by Vpu is approximately 17 to 13 amino acids from the C terminus in the cytoplasmic tail (Fig. ​(Fig.1)1) (2, 15).In addition to degrading CD4, Vpu has also long been known to result in enhanced viral release (EVR) in certain cell lines (14, 36). Recently, the type I interferon-induced host protein tetherin was identified as being responsible for this Vpu-modulated restriction (24, 38). In the absence of Vpu, tetherin causes particles to remain tethered (hence the name) to the host cell postfission. Although Vpu counteracts the function of tetherin, the exact mechanism has not been fully elucidated. However, the mechanism for tetherin antagonism appears to be distinct from that for modulating CD4. Mutation of the serines 52 and 56 of Vpu abolish CD4 degradation, but only reduce EVR activity (5, 17, 21, 32). Some EVR activity remains even when much of the Vpu cytoplasmic tail is deleted (30). In addition, many mutations in the membrane-spanning domain, such as Vpu_RD, do not affect CD4 degradation and yet completely abolish EVR activity (27, 30, 37). The critical residues in tetherin for recognition by Vpu appear to be in the membrane-spanning domain and not the cytoplasmic tail (9, 19, 28). Although β-TrCP is required for complete EVR activity, there is no consensus whether the degradation of tetherin is proteasome or lysosome mediated (5, 7, 21) or whether degradation is required at all. In some cases there can be some EVR activity in the absence of tetherin degradation (17, 22).We demonstrate here that Vpu is responsible for the incompatibility between HIV-1 and GaLV Env. Glycoproteins containing the cytoplasmic tail from GaLV Env are prevented from being incorporated into HIV-1 particles by Vpu, effectively reducing infectious particle production by 50- to 100-fold. The serines at positions 52 and 56 are required for this restriction, but the membrane-spanning domain is not. Although the mechanism for this restriction appears similar to CD4 degradation, there are apparent differences. Vpu does not prevent surface expression, and it does not prevent its incorporation into MLV particles. Therefore, the mechanism of restriction appears to involve a system that does not rely directly on global protein degradation.     

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.     

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.     

Therapeutic Memory T Cells Require Costimulation for Effective Clearance of a Persistent Viral Infection

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

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.     

Adult AIDS-Like Disease in a Novel Inducible Human Immunodeficiency Virus Type 1 Nef Transgenic Mouse Model: CD4+ T-Cell Activation Is Nef Dependent and Can Occur in the Absence of Lymphophenia

CD4C/HIV^nef transgenic (Tg) mice express Nef in CD4⁺ T cells and in the cells of the macrophage/monocyte/dendritic lineage, and they develop an AIDS-like disease similar to human AIDS. In these mice, Nef is constitutively expressed throughout life. To rule out the contribution of any developmental defects caused by early expression of Nef, we generated inducible human immunodeficiency virus type 1 (HIV-1) Nef Tg mice by using the tetracycline-inducible system. Faithful expression of the Nef transgene was induced in (CD4C/rtTA × TRE/HIV^Nef) or (CD4C/rtTA2^S-M2 × TRE/HIV^Nef) double-Tg mice upon doxycycline (DOX) treatment in drinking water. Long-term treatment of these mice with DOX also led to loss, apoptosis, and activation of CD4⁺ T cells, this latter phenotype being observed even with low levels of Nef. These phenotypes could be transferred by bone marrow (BM) transplantation, indicating a hematopoietic cell autonomous effect. In addition, in mixed Tg:non-Tg BM chimeras, only Tg and not non-Tg CD4⁺ T cells exhibited an effector/memory phenotype in the absence of lymphopenia. Finally, the DOX-induced double-Tg mice developed nonlymphoid organ diseases similar to those of CD4C/HIV^Nef Tg mice and of humans infected with HIV-1. These results show for the first time that adult mice are susceptible to the detrimental action of Nef and that Nef-mediated T-cell activation can be independent of lymphopenia. These Tg mice represent a unique model which is likely to be instrumental for understanding the cellular and molecular pathways of Nef action as well as the main characteristics of immune reconstitution following DOX withdrawal.Small animal models able to express the entire human immunodeficiency virus (HIV) genome or selected HIV genes have provided useful information on the pathogenesis of AIDS and still represent important research tools toward this goal. Among these models, transgenic (Tg) mice containing intact copies of HIV DNA, defective provirus with the gag and pol genes deleted, or individual HIV-1 genes have been reported to develop various pathologies, some of which resemble those found in human AIDS (2, 3, 8, 9, 16, 17, 18, 24, 27, 29, 30, 38, 44, 45, 46, 49, 51, 52). The cell type context in which the HIV-1 transgene is expressed in these Tg mice appears to play an important role in determining the type of pathological lesions. Tg mice generated in our laboratory and expressing the entire coding sequence of HIV-1 (CD4C/HIV^WT) or HIV-1 Nef alone (CD4C/HIV^Nef) in the relevant target cells of HIV-1, namely, CD4⁺ T cells, macrophages, and dendritic cells, develop pathologies very similar to those in human AIDS (17, 18). The AIDS-like disease of CD4C/HIV^Nef Tg mice is characterized by immunodeficiency, loss of CD4⁺ T cells, thymic atrophy, activation of T cells and pathologies in heart, lungs, and kidneys (18, 53). Similarly, expression of simian immunodeficiency virus (SIV) Nef in Tg mice under the control of the same promoter sequences (CD4C) results in an AIDS-like disease (42). These studies demonstrated that Nef plays an important role in the development of the AIDS-like disease induced by HIV-1 or SIV in Tg mice.Among the AIDS-like phenotypes of these models, the T-cell activation observed by a number of groups in Tg mice expressing Nef (3, 33, 44, 53) may be of special interest for its resemblance to that of humans or macaques infected with HIV-1 or SIV, respectively. HIV infection results in a state of chronic immune activation which correlates very closely with disease progression in humans (11, 14, 23). Similarly, SIV-infected macaques which develop AIDS show aberrant immune activation (35), while SIV-infected sooty mangabey monkeys, natural hosts of SIV, do not develop immunopathologies and do not show immune activation either (41). Various factors may contribute to this immune activation, including increased plasma lipopolysaccharide levels due to microbial translocation from the gut (4), impaired regulatory T cell function (32), or the action of the HIV-1 gene products themselves, such as Env gp120 and Nef (10, 12, 43). Consistent with this latter scenario, we reported that in CD4C/HIV^Nef Tg mice the extent of T-cell activation correlates with levels of Nef expression in CD4⁺ T cells, thus suggesting a direct involvement of Nef in this activation (53). In contrast, Koenen and coworkers reported that T-cell activation in CD2/Nef Tg mice is induced indirectly by lymphophenia (26). In that study, chimeric mice, which were generated from a mixture of non-Tg and Nef Tg bone marrow (BM) cells, were not lymphopenic, and the donor-derived Nef-expressing Tg T cells did not show an activated phenotype. However, the donor Nef Tg T cells constituted only 1 to 2% of peripheral T cells of these chimeric mice (26). Clearly, alternative experimental approaches are needed to study this phenotype in a more physiological context.In the previously described CD4C/HIV^Nef Tg mice (18), Nef expression begins early in life and is constitutively expressed throughout the life of the animal. The AIDS-like disease caused by this early expression of Nef best represents a model for pediatric AIDS. However, in these Tg mice, Nef may interfere with normal developmental processes and these latter defects may contribute to some of the phenotypes observed. To assess the effects of Nef in fully mature adult animals, and thus develop a model of adult AIDS, temporal regulation of Nef expression in adult mice using an inducible system is required.In the present study, we chose the tet-On (rtTA and rtTA2^S-M2) system (13, 15, 25, 48) to induce expression of HIV-1 Nef in CD4⁺ T cells and cells of the macrophage/dendritic lineage of mice using the CD4C tissue-specific regulatory elements. These CD4C sequences were previously used to generate the constitutively Nef-expressing CD4C/HIV^Nef Tg mice (18). These inducible adult (TRE/HIV^Nef × CD4C/rtTA) and (TRE/HIV^Nef × CD4C/rtTA2^S-M2) double-Tg (DTg) mice express Nef when treated with doxycycline (DOX) and develop an AIDS-like disease very similar to that seen in constitutively Nef-expressing CD4C/HIV^Nef Tg mice. We took advantage of this novel biological system to reassess the role of Nef in T-cell activation. Using a mixed chimera made with BM cells from these inducible Nef Tg mice and from non-Tg mice, we could document CD4⁺ T-cell activation only in donor-derived Nef-expressing Tg cells, but not in non-Tg cells, in the absence of lymphopenia. This result strongly suggests that this CD4⁺ T-cell activation phenotype is most likely driven by expression of Nef in these cells.     

Effect of B-Cell Depletion on Viral Replication and Clinical Outcome of Simian Immunodeficiency Virus Infection in a Natural Host

Simian immunodeficiency virus (SIV)-infected African nonhuman primates do not progress to AIDS in spite of high and persistent viral loads (VLs). Some authors consider the high viral replication observed in chronic natural SIV infections to be due to lower anti-SIV antibody titers than those in rhesus macaques, suggesting a role of antibodies in controlling viral replication. We therefore investigated the impact of antibody responses on the outcome of acute and chronic SIVagm replication in African green monkeys (AGMs). Nine AGMs were infected with SIVagm.sab. Four AGMs were infused with 50 mg/kg of body weight anti-CD20 (rituximab; a gift from Genentech) every 21 days, starting from day −7 postinfection up to 184 days. The remaining AGMs were used as controls and received SIVagm only. Rituximab-treated AGMs were successfully depleted of CD20 cells in peripheral blood, lymph nodes (LNs), and intestine, as shown by the dynamics of CD20⁺ and CD79a⁺ cells. There was no significant difference in VLs between CD20-depleted AGMs and control monkeys: peak VLs ranged from 10⁷ to 10⁸ copies/ml; set-point values were 10⁴ to 10⁵ SIV RNA copies/ml. Levels of acute mucosal CD4⁺ T-cell depletion were similar for treated and nontreated animals. SIVagm seroconversion was delayed for the CD20-depleted AGMs compared to results for the controls. There was a significant difference in both the timing and magnitude of neutralizing antibody responses for CD20-depleted AGMs compared to results for controls. CD20 depletion significantly altered the histological structure of the germinal centers in the LNs and Peyer''s patches. Our results, although obtained with a limited number of animals, suggest that humoral immune responses play only a minor role in the control of SIV viral replication during acute and chronic SIV infection in natural hosts.In marked contrast to pathogenic human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections of humans and macaques, which are characterized by the constant progression to AIDS in a variable time frame (26), African monkey species naturally infected with SIV are generally spared from any signs of disease (reviewed in references 53 and 71).There are currently three animal models of SIV infection in natural hosts: SIVagm infection of African green monkeys (AGMs), SIVsmm infection of sooty mangabeys, and SIVmnd-1 and SIVmnd-2 infection of mandrills (53, 71). SIV infection in natural hosts is characterized by the following: (i) active viral replication, with set-point viral loads (VLs) similar to or even higher than those found in pathogenic infections (44-46, 49, 50, 52, 61-63); (ii) transient depletion of peripheral CD4⁺ T cells during primary infection, which rebound to preinfection levels during chronic infection (12, 30, 44-46, 49, 62); (iii) significant CD4⁺ T-cell depletion in the intestine, which can be partially restored during chronic infection in spite of significant viral replication (21, 48); (iv) low levels of CD4⁺ CCR5⁺ cells in blood and tissues (47); (v) transient and moderate increases in immune activation and T-cell proliferation during acute infection, with a return to baseline levels during the chronic phase (44-46, 49, 50, 52, 61-63), as a result of an anti-inflammatory milieu which is rapidly established after infection (14, 30); and (vi) no significant increase in CD4⁺ T-cell apoptosis during either acute or chronic infection (37, 48), thus avoiding enteropathy and microbial translocation, which control excessive immune activation and prevent disease progression by allowing CD4⁺ T-cell recovery in the presence of high VLs (21, 48). Hence, the current view is that the main reason behind the lack of disease progression in natural African hosts lies in a better adaptation of the host in response to the highly replicating virus. A better understanding of the mechanisms underlying the lack of disease in natural hosts for SIV infection may provide important clues for understanding the pathogenesis of HIV infection (53, 71).To date, it is still unknown whether or not immune responses are responsible for the lack of disease progression in natural hosts, since data are scarce. Studies of cellular immune responses are significantly more limited than is the case with pathogenic infection, and although not always in agreement (3, 13, 28, 29, 73, 76), their convergence point is that cellular immune responses are not essentially superior to those observed in pathogenic infections (3, 13, 28, 29, 73, 76). This observation is not surprising in the context of the high viral replication in natural hosts. Data are even scarcer on the role of humoral immune responses in the control of disease progression in natural hosts. However, several studies reported that anti-SIV antibody titers are lower in SIV infections of natural hosts, with a lack of anti-Gag responses being characteristic of natural SIV infections in African nonhuman primates (1, 6, 24, 25, 42, 43, 71). Because the viral replication in SIVagm-infected AGMs is of the same magnitude or higher than that in pathogenic infections of rhesus macaques (RMs), it has been hypothesized that these high VLs may be a consequence of the lower antibody titers. Moreover, a recent study has also shown that B cells in lymph nodes (LNs) of AGMs are activated at an earlier time point than is the case for SIVmac251-infected RMs, which implies that humoral immune responses may be important in controlling SIV replication in the natural hosts (9). Conversely, it has been shown that passively transferring immunoglobulins from animals naturally infected with SIVagm prior to infection with a low dose of SIVagm did not prevent infection in AGMs (42, 60), which is in striking contrast to results in studies of pathogenic infections, which convincingly demonstrated with animal models that intravenously administered or topically applied antibodies can protect macaques against intravenous or mucosal simian-human immunodeficiency virus challenge (34-36, 54, 72).Previous CD20⁺ B-cell-depletion studies during pathogenic RM infections have indicated that humoral immune responses may be important for controlling both the postpeak VL and disease progression (38, 57). However, these studies used strains that are highly resistant to neutralization (SIVmac251 and SIVmac239), making it difficult to assess the role that antibodies have in controlling SIV replication and disease progression. Moreover, our recent results suggested a limited impact of humoral immune responses in controlling replication of a neutralization-sensitive SIVsmm strain in rhesus macaques (18).To investigate the effect that CD20⁺ B cells and antibodies have on SIV replication in natural hosts, we have depleted CD20⁺ B cells in vivo in AGMs infected with SIVagm.sab92018. We assessed the impact of humoral immune responses on the control of viral replication and other immunological parameters, and we report that ablating humoral immune responses in SIVagm-infected AGMs does not significantly alter the course of virus replication or disease 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.