Antiviral CD8+ T Cells Restricted by Human Leukocyte Antigen Class II Exist during Natural HIV Infection and Exhibit Clonal Expansion.

CD8+ T cell recognition of virus-infected cells is characteristically restricted by major histocompatibility complex (MHC) class I, although rare examples of MHC class II restriction have been reported in Cd4-deficient mice and a macaque SIV vaccine trial using a recombinant cytomegalovirus vector. Here, we demonstrate the presence of human leukocyte antigen (HLA) class II-restricted CD8+ T cell responses with antiviral properties in a small subset of HIV-infected individuals. In these individuals, T cell receptor ß (TCRß) analysis revealed that class II-restricted CD8+ T cells underwent clonal expansion and mediated killing of HIV-infected cells. In one case, these cells comprised 12% of circulating CD8+ T cells, and TCRα analysis revealed two distinct co-expressed TCRα chains, with only one contributing to binding of the class II HLA-peptide complex. These data indicate that class II-restricted CD8+ T cell responses can exist in a chronic human viral infection, and may contribute to immune control.

Dysfunctional HIV-specific CD8+ T cell proliferation is associated with increased caspase-8 activity and mediated by necroptosis.

Decreased HIV-specific CD8(+) T cell proliferation is a hallmark of chronic infection, but the mechanisms of decline are unclear. We analyzed gene expression profiles from antigen-stimulated HIV-specific CD8(+) T cells from patients with controlled and uncontrolled infection and identified caspase-8 as a correlate of dysfunctional CD8(+) T cell proliferation. Caspase-8 activity was upregulated in HIV-specific CD8(+) T cells from progressors and correlated positively with disease progression and programmed cell death-1 (PD-1) expression, but negatively with proliferation. In addition, progressor cells displayed a decreased ability to upregulate membrane-associated caspase-8 activity and increased necrotic cell death following antigenic stimulation, implicating the programmed cell death pathway necroptosis. In vitro necroptosis blockade rescued HIV-specific CD8(+) T cell proliferation in progressors, as did silencing of necroptosis mediator RIPK3. Thus, chronic stimulation leading to upregulated caspase-8 activity contributes to dysfunctional HIV-specific CD8(+) T cell proliferation through activation of necroptosis and increased cell death.

HIV-1 Antibody Neutralization Breadth Is Associated with Enhanced HIV-Specific CD4+ T Cell Responses.

UNLABELLED: Antigen-specific CD4(+) T helper cell responses have long been recognized to be a critical component of effective vaccine immunity. CD4(+) T cells are necessary to generate and maintain humoral immune responses by providing help to antigen-specific B cells for the production of antibodies. In HIV infection, CD4(+) T cells are thought to be necessary for the induction of Env-specific broadly neutralizing antibodies. However, few studies have investigated the role of HIV-specific CD4(+) T cells in association with HIV neutralizing antibody activity in vaccination or natural infection settings. Here, we conducted a comprehensive analysis of HIV-specific CD4(+) T cell responses in a cohort of 34 untreated HIV-infected controllers matched for viral load, with and without neutralizing antibody breadth to a panel of viral strains. Our results show that the breadth and magnitude of Gag-specific CD4(+) T cell responses were significantly higher in individuals with neutralizing antibodies than in those without neutralizing antibodies. The breadth of Gag-specific CD4(+) T cell responses was positively correlated with the breadth of neutralizing antibody activity. Furthermore, the breadth and magnitude of gp41-specific, but not gp120-specific, CD4(+) T cell responses were significantly elevated in individuals with neutralizing antibodies. Together, these data suggest that robust Gag-specific CD4(+) T cells and, to a lesser extent, gp41-specific CD4(+) T cells may provide important intermolecular help to Env-specific B cells that promote the generation or maintenance of Env-specific neutralizing antibodies. IMPORTANCE: One of the earliest discoveries related to CD4(+) T cell function was their provision of help to B cells in the development of antibody responses. Yet little is known about the role of CD4(+) T helper responses in the setting of HIV infection, and no studies to date have evaluated the impact of HIV-specific CD4(+) T cells on the generation of antibodies that can neutralize multiple different strains of HIV. Here, we addressed this question by analyzing HIV-specific CD4(+) T cell responses in untreated HIV-infected persons with and without neutralizing antibodies. Our results indicate that HIV-infected persons with neutralizing antibodies have significantly more robust CD4(+) T cell responses targeting Gag and gp41 proteins than individuals who lack neutralizing antibodies. These associations suggest that Gag- and gp41-specific CD4(+) T cell responses may provide robust help to B cells for the generation or maintenance of neutralizing antibodies in natural HIV-infection.

Cooperativity of HIV-Specific Cytolytic CD4 T Cells and CD8 T Cells in Control of HIV Viremia.

UNLABELLED: CD4+ T cells play a pivotal role in the control of chronic viral infections. Recently, nontraditional CD4+ T cell functions beyond helper effects have been described, and a role for cytolytic CD4+ T cells in the control of HIV infection has been suggested. We define here the transcriptional, phenotypic, and functional profiles of HIV-specific cytolytic CD4+ T cells. Fluidigm BioMark and multiparameter flow cytometric analysis of HIV-specific cytolytic CD4+ T cells revealed a distinct transcriptional signature compared to Th1 CD4+ cells but shared similar features with HIV-specific cytolytic CD8+ T cells. Furthermore, HIV-specific cytolytic CD4+ T cells showed comparable killing activity relative to HIV-specific CD8+ T cells and worked cooperatively in the elimination of virally infected cells. Interestingly, we found that cytolytic CD4+ T cells emerge early during acute HIV infection and tightly follow acute viral load trajectory. This emergence was associated to the early viral set point, suggesting an involvement in early control, in spite of CD4 T cell susceptibility to HIV infection. Our data suggest cytolytic CD4+ T cells as an independent subset distinct from Th1 cells that show combined activity with CD8+ T cells in the long-term control of HIV infection. IMPORTANCE: The ability of the immune system to control chronic HIV infection is of critical interest to both vaccine design and therapeutic approaches. Much research has focused on the effect of the ability of CD8+ T cells to control the virus, while CD4+ T cells have been overlooked as effectors in HIV control due to the fact that they are preferentially infected. We show here that a subset of HIV-specific CD4+ T cells cooperate in the cytolytic control of HIV replication. Moreover, these cells represent a distinct subset of CD4+ T cells showing significant transcriptional and phenotypic differences compared to HIV-specific Th1 cells but with similarities to CD8+ T cells. These findings are important for our understanding of HIV immunopathology.

Induction of Gag-specific CD4 T cell responses during acute HIV infection is associated with improved viral control.

UNLABELLED: Effector CD4 T cell responses have been shown to be critically involved in the containment and clearance of viral pathogens. However, their involvement in the pathogenesis of HIV infection is less clear, given their additional role as preferred viral targets. We previously demonstrated that the presence of HIV-specific CD4 T cell responses is somewhat associated with HIV control and that specific CD4 T cell functions, such as direct cytolytic activity, can contribute to control of HIV viremia. However, little is known about how the induction of HIV-specific CD4 T cell responses during acute HIV infection influences disease progression and whether responses induced during the early phase of infection are preferentially depleted. We therefore longitudinally assessed, in a cohort of 55 acutely HIV-infected individuals, HIV-specific CD4 T cell responses from acute to chronic infection. Interestingly, we found that the breadth, magnitude, and protein dominance of HIV-specific CD4 T cell responses remained remarkably stable over time. Moreover, we found that the epitopes targeted at a high frequency in acute HIV infection were recognized at the same frequency by HIV-specific CD4 T cells in chronic HIV infection. Interestingly the induction of Gag-specific CD4 T cell responses in acute HIV infection was significantly inversely correlated with viral set point in chronic HIV infection (R = -0.5; P = 0.03), while the cumulative contribution of Env-specific CD4 T cell responses showed the reverse effect. Moreover, individuals with HIV-specific CD4 T cell responses dominantly targeting Gag over Env in acute HIV infection remained off antiretroviral therapy significantly longer (P = 0.03; log rank). Thus, our data suggest that the induction of HIV-specific CD4 T cell responses during acute HIV infection is beneficial overall and does not fuel disease progression. IMPORTANCE: CD4 T cells are critical for the clearance and control of viral infections. However, HIV preferentially infects HIV-specific CD4 T cells. Thus, their contribution to the control of HIV viremia is uncertain. Here, we study HIV-specific CD4 T cell responses from acute to chronic HIV infection and show that the generation of certain CD4 responses is associated with control rather than disease progression.

HIV control is mediated in part by CD8+ T-cell targeting of specific epitopes.

UNLABELLED: We investigated the hypothesis that the correlation between the class I HLA types of an individual and whether that individual spontaneously controls HIV-1 is mediated by the targeting of specific epitopes by CD8(+) T cells. By measuring gamma interferon enzyme-linked immunosorbent spot (ELISPOT) assay responses to a panel of 257 optimally defined epitopes in 341 untreated HIV-infected persons, including persons who spontaneously control viremia, we found that the correlation between HLA types and control is mediated by the targeting of specific epitopes. Moreover, we performed a graphical model-based analysis that suggested that the targeting of specific epitopes is a cause of such control--that is, some epitopes are protective rather than merely associated with control--and identified eight epitopes that are significantly protective. In addition, we use an in silico analysis to identify protein regions where mutations are likely to affect the stability of a protein, and we found that the protective epitopes identified by the ELISPOT analysis correspond almost perfectly to such regions. This in silico analysis thus suggests a possible mechanism for control and could be used to identify protective epitopes that are not often targeted in natural infection but that may be potentially useful in a vaccine. Our analyses thus argue for the inclusion (and exclusion) of specific epitopes in an HIV vaccine. IMPORTANCE: Some individuals naturally control HIV replication in the absence of antiretroviral therapy, and this ability to control is strongly correlated with the HLA class I alleles that they express. Here, in a large-scale experimental study, we provide evidence that this correlation is mediated largely by the targeting of specific CD8(+) T-cell epitopes, and we identify eight epitopes that are likely to cause control. In addition, we provide an in silico analysis indicating that control occurs because mutations within these epitopes change the stability of the protein structures. This in silico analysis also identified additional epitopes that are not typically targeted in natural infection but may lead to control when included in a vaccine, provided that other epitopes that would otherwise distract the immune system from targeting them are excluded from the vaccine.

HIV-specific CD4 T cell responses to different viral proteins have discordant associations with viral load and clinical outcome.

A successful prophylactic vaccine is characterized by long-lived immunity, which is critically dependent on CD4 T cell-mediated helper signals. Indeed, most licensed vaccines induce antigen-specific CD4 T cell responses, in addition to high-affinity antibodies. However, despite the important role of CD4 T cells in vaccine design and natural infection, few studies have characterized HIV-specific CD4 T cells due to their preferential susceptibility to HIV infection. To establish at the population level the impact of HIV-specific CD4 T cells on viral control and define the specificity of HIV-specific CD4 T cell peptide targeting, we conducted a comprehensive analysis of these responses to the entire HIV proteome in 93 subjects at different stages of HIV infection. We show that HIV-specific CD4 T cell responses were detectable in 92% of individuals and that the breadth of these responses showed a significant inverse correlation with the viral load (P = 0.009, R = -0.31). In particular, CD4 T cell responses targeting Gag were robustly associated with lower levels of viremia (P = 0.0002, R = -0.45). Importantly, differences in the immunodominance profile of HIV-specific CD4 T cell responses distinguished HIV controllers from progressors. Furthermore, Gag/Env ratios were a potent marker of viral control, with a high frequency and magnitude of Gag responses and low proportion of Env responses associated with effective immune control. At the epitope level, targeting of three distinct Gag peptides was linked to spontaneous HIV control (P = 0.60 to 0.85). Inclusion of these immunogenic proteins and peptides in future HIV vaccines may act as a critical cornerstone for enhancing protective T cell responses.

HIV-1-specific interleukin-21+ CD4+ T cell responses contribute to durable viral control through the modulation of HIV-specific CD8+ T cell function.

Functional defects in cytotoxic CD8(+) T cell responses arise in chronic human viral infections, but the mechanisms involved are not well understood. In mice, CD4 cell-mediated interleukin-21 (IL-21) production is necessary for the maintenance of CD8(+) T cell function and control of persistent viral infections. To investigate the potential role of IL-21 in a chronic human viral infection, we studied the rare subset of HIV-1 controllers, who are able to spontaneously control HIV-1 replication without treatment. HIV-specific triggering of IL-21 by CD4(+) T cells was significantly enriched in these persons (P = 0.0007), while isolated loss of IL-21-secreting CD4(+) T cells was characteristic for subjects with persistent viremia and progressive disease. IL-21 responses were mediated by recognition of discrete epitopes largely in the Gag protein, and expansion of IL-21(+) CD4(+) T cells in acute infection resulted in lower viral set points (P = 0.002). Moreover, IL-21 production by CD4(+) T cells of HIV controllers enhanced perforin production by HIV-1-specific CD8(+) T cells from chronic progressors even in late stages of disease, and HIV-1-specific effector CD8(+) T cells showed an enhanced ability to efficiently inhibit viral replication in vitro after IL-21 binding. These data suggest that HIV-1-specific IL-21(+) CD4(+) T cell responses might contribute to the control of viral replication in humans and are likely to be of great importance for vaccine design.

Dissection of multiple sclerosis genetics identifies B and CD4+ T cells as driver cell subsets.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune condition of the central nervous system with a well-characterized genetic background. Prior analyses of MS genetics have identified broad enrichments across peripheral immune cells, yet the driver immune subsets are unclear. RESULTS: We utilize chromatin accessibility data across hematopoietic cells to identify cell type-specific enrichments of MS genetic signals. We find that CD4 T and B cells are independently enriched for MS genetics and further refine the driver subsets to Th17 and memory B cells, respectively. We replicate our findings in data from untreated and treated MS patients and find that immunomodulatory treatments suppress chromatin accessibility at driver cell types. Integration of statistical fine-mapping and chromatin interactions nominate numerous putative causal genes, illustrating complex interplay between shared and cell-specific genes. CONCLUSIONS: Overall, our study finds that open chromatin regions in CD4 T cells and B cells independently drive MS genetic signals. Our study highlights how careful integration of genetics and epigenetics can provide fine-scale insights into causal cell types and nominate new genes and pathways for disease.

High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry.

Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single-cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to address a variety of questions in heterogeneous cell populations. We demonstrate antigen-specific upregulation of IFNγ and IL-2 mRNAs in HIV- and CMV-specific T cells. We show simultaneous detection of cytokine mRNA and corresponding protein in single cells. We apply this method to detect mRNAs for which flow antibodies against the corresponding proteins are poor or are not available. We use this technique to show modulation of a microRNA critical for T-cell function, miR-155. We adapt this assay for simultaneous detection of mRNA and proteins by ImageStream technology.

Association of HLA-DRB1-restricted CD4⁺ T cell responses with HIV immune control.

The contribution of HLA class II-restricted CD4(+) T cell responses to HIV immune control is poorly defined. Here, we delineated previously uncharacterized peptide-DRB1 restrictions in functional assays and analyzed the host genetic effects of HLA-DRB1 alleles on HIV viremia in a large cohort of HIV controllers and progressors. We found distinct stratifications in the effect of HLA-DRB1 alleles on HIV viremia, with HLA-DRB1*15:02 significantly associated with low viremia and HLA-DRB1*03:01 significantly associated with high viremia. Notably, a subgroup of HLA-DRB1 variants linked with low viremia showed the ability to promiscuously present a larger breadth of peptides with lower functional avidity when compared to HLA-DRB1 variants linked with high viremia. Our data provide systematic evidence that HLA-DRB1 variant expression has a considerable impact on the control of HIV replication, an effect that seems to be mediated primarily by the protein specificity of CD4(+) T cell responses to HIV Gag and Nef.

HIV-specific cytolytic CD4 T cell responses during acute HIV infection predict disease outcome.

Early immunological events during acute HIV infection are thought to fundamentally influence long-term disease outcome. Whereas the contribution of HIV-specific CD8 T cell responses to early viral control is well established, the role of HIV-specific CD4 T cell responses in the control of viral replication after acute infection is unknown. A growing body of evidence suggests that CD4 T cells-besides their helper function-have the capacity to directly recognize and kill virally infected cells. In a longitudinal study of a cohort of individuals acutely infected with HIV, we observed that subjects able to spontaneously control HIV replication in the absence of antiretroviral therapy showed a significant expansion of HIV-specific CD4 T cell responses-but not CD8 T cell responses-compared to subjects who progressed to a high viral set point (P = 0.038). Markedly, this expansion occurred before differences in viral load or CD4 T cell count and was characterized by robust cytolytic activity and expression of a distinct profile of perforin and granzymes at the earliest time point. Kaplan-Meier analysis revealed that the emergence of granzyme A(+) HIV-specific CD4 T cell responses at baseline was highly predictive of slower disease progression and clinical outcome (average days to CD4 T cell count <350/µl was 575 versus 306, P = 0.001). These data demonstrate that HIV-specific CD4 T cell responses can be used during the earliest phase of HIV infection as an immunological predictor of subsequent viral set point and disease outcome. Moreover, these data suggest that expansion of granzyme A(+) HIV-specific cytolytic CD4 T cell responses early during acute HIV infection contributes substantially to the control of viral replication.

Expansion of HIV-specific T follicular helper cells in chronic HIV infection.

HIV targets CD4 T cells, which are required for the induction of high-affinity antibody responses and the formation of long-lived B cell memory. The depletion of antigen-specific CD4 T cells during HIV infection is therefore believed to impede the development of protective B cell immunity. Although several different HIV-related B cell dysfunctions have been described, the role of CD4 T follicular helper (TFH) cells in HIV infection remains unknown. Here, we assessed HIV-specific TFH responses in the lymph nodes of treatment-naive and antiretroviral-treated HIV-infected individuals. Strikingly, both the bulk TFH and HIV-specific TFH cell populations were significantly expanded in chronic HIV infection and were highly associated with viremia. In particular, GAG-specific TFH cells were detected at significantly higher levels in the lymph nodes compared with those of GP120-specific TFH cells and showed preferential secretion of the helper cytokine IL-21. In addition, TFH cell expansion was associated with an increase of germinal center B cells and plasma cells as well as IgG1 hypersecretion. Thus, our study suggests that high levels of HIV viremia drive the expansion of TFH cells, which in turn leads to perturbations of B cell differentiation, resulting in dysregulated antibody production.

Cytolytic CD4(+) T cells in viral immunity.

It is generally believed that the role of CD4(+) T cells is to coordinate the different arms of the adaptive immune system to shape an effective response against a pathogen and regulate nonessential or deleterious activities. However, a growing body of evidence suggests that effector CD4(+) T cells can directly display potent antiviral activity themselves. The presence of cytolytic CD4(+) T cells has been demonstrated in the immune response to numerous viral infections in both humans and in animal models and it is likely that they play a critical role in the control of viral replication in vivo. This article describes the current research on virus-specific cytolytic CD4(+) T cells, with a focus on HIV-1 infection and the implications that this immune response has for vaccine design.