CD4 T follicular helper (TFH) cells interact with and stimulate the generation of antigen-specific B cells. TFH cell interaction with B cells correlates with production of SIV-specific immunoglobulins. However, the fate of TFH cells and their participation in SIV-induced antibody production is not well understood. We investigated the phenotype, function, location, and molecular signature of TFH cells in rhesus macaques. Similar to their human counterparts, TFH cells in rhesus macaques represented a heterogeneous population with respect to cytokine function. In a highly differentiated subpopulation of TFH cells, characterized by CD150^lo expression, production of Th1 cytokines was compromised while IL-4 production was augmented, and cells exhibited decreased survival, cycling, and trafficking capacity. TFH cells exhibited a distinct gene profile that was markedly altered by SIV infection. TFH cells were infected by SIV; yet, in some animals, these cells actually accumulated during chronic SIV infection. Generalized immune activation and increased IL-6 production helped drive TFH differentiation during SIV infection. Accumulation of TFH cells was associated with increased frequency of activated germinal center B cells and SIV-specific antibodies. Therefore, chronic SIV does not disturb the ability of TFH cells to help B cell maturation and production of SIV-specific immunoglobulins.

CD8+ T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8+ T cell differentiation, T-bet and Eomesodermin (Eomes), have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4), functional characteristics and memory differentiation of CD8+ T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8+ T cells was elevated in chronically infected individuals and highly associated with a T-bet^dimEomes^hi expressional profile. Interestingly, both resting and activated HIV-specific CD8+ T cells in chronic infection were almost exclusively T-bet^dimEomes^hi cells, while CMV-specific CD8+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-bet^dimEomes^hi virus-specific CD8+ T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector) characteristics. The transitional and exhausted phenotype of HIV-specific CD8+ T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART) initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8+ T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8+ T cells to control the viral replication post-ART cessation. CD8+ T cells display numerous traits of severe dysfunction in both treated and untreated HIV infection. Previous studies have demonstrated that HIV-specific CD8+ T cells in most individuals possess poor polyfunctionality, and an immature/skewed maturation phenotype. However, it remains unclear which transcriptional programming governs the regulation of CD8+ T cell differentiation and exhaustion in HIV infection. T-bet and Eomes represent two key transcription factors for CD8+ T cell differentiation and function, but surprisingly little is known about their influence of effector immunity following chronic viral infections in humans. In this study, we demonstrate that HIV-specific CD8+ T cells possess highly elevated levels of Eomes, but low T-bet expression. This differential relationship is linked to the up-regulation of several inhibitory receptors, impaired functional characteristics and a transitional memory differentiation phenotype for virus-specific CD8+ T cells. Importantly, these characteristics of HIV-specific CD8+ T cells remained stable despite suppressive ART for many years. These results implicate that reinvigoration of these cells might fail to elicit efficient responses to eradicate the viral reservoir.

Human immunodeficiency virus-1 (HIV-1) has infected more than 60 million people and caused nearly 30 million deaths worldwide, ultimately the consequence of cytolytic infection of CD4(+) T cells. In humans and in macaque models, most of these cells contain viral DNA and are rapidly eliminated at the peak of viraemia, yet the mechanism by which HIV-1 induces helper T-cell death has not been defined. Here we show that virus-induced cell killing is triggered by viral integration. Infection by wild-type HIV-1, but not an integrase-deficient mutant, induced the death of activated primary CD4 lymphocytes. Similarly, raltegravir, a pharmacologic integrase inhibitor, abolished HIV-1-induced cell killing both in cell culture and in CD4(+) T cells from acutely infected subjects. The mechanism of killing during viral integration involved the activation of DNA-dependent protein kinase (DNA-PK), a central integrator of the DNA damage response, which caused phosphorylation of p53 and histone H2AX. Pharmacological inhibition of DNA-PK abolished cell death during HIV-1 infection in vitro, suggesting that processes which reduce DNA-PK activation in CD4 cells could facilitate the formation of latently infected cells that give rise to reservoirs in vivo. We propose that activation of DNA-PK during viral integration has a central role in CD4(+) T-cell depletion, raising the possibility that integrase inhibitors and interventions directed towards DNA-PK may improve T-cell survival and immune function in infected individuals.

The interaction between follicular T helper cells (T_FH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral T_FH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral T_FH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7^highCXCR5^highCCR6^highPD-1^high CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral T_FH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral T_FH population, the expression level of T_FH-associated genes more closely resembles a memory, non-T_FH population, as opposed to a T_FH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory T_FH cells. Follicular T helper cells (T_FH) interact with B cells within germinal centers of lymphoid tissue to promote the survival, isotype switching and generation of high affinity memory B cells and plasma cells. Recently, a population of circulating CD4 T cells that shares phenotypic and functional characteristics with T_FH cells, named peripheral T_FH cells, has been identified. The relationship between peripheral T_FH cells in the blood and T_FH cells within the lymphoid tissue remains unclear, and whether or not peripheral T_FH cells can provide insight into T cell and B cell dynamics within lymphoid tissue during infection or vaccination is not understood. Here we characterize peripheral T_FH cells and show that unlike T_FH cells, peripheral T_FH cells secrete a diverse array of cytokines and decrease, rather than increase, during chronic HIV infection. Furthermore, we did not observe a relationship between peripheral T_FH cells and memory B cells, or with the production of neutralizing antibodies to HIV. Overall, our data indicate that while peripheral T_FH cells share some characteristics with T_FH cells, they may not represent a good surrogate to study T cell and B cell dynamics within lymphoid tissue.

A highly complex network of coinhibitory and costimulatory receptors regulates the outcome of virus-specific CD8+ T-cell responses. Here, we report on the expression patterns of multiple inhibitory receptors on HIV-specific, cytomegalovirus-specific, and bulk CD8+ T-cell memory populations. In contrast to cytomegalovirus-specific CD8+ T cells, the majority of HIV-specific CD8+ T cells exhibited an immature phenotype and expressed Programmed Death-1, CD160 and 2B4 but not lymphocyte activation gene-3. Notably, before antiretroviral therapy, simultaneous expression of these negative regulators correlated strongly with both HIV load and impaired cytokine production. Suppression of HIV replication by antiretroviral therapy was associated with reduced surface expression of inhibitory molecules on HIV-specific CD8+ T cells. Furthermore, in vitro manipulation of Programmed Death-1 and 2B4 inhibitory pathways increased the proliferative capacity of HIV-specific CD8+ T cells. Thus, multiple coinhibitory receptors can affect the development of HIV-specific CD8+ T-cell responses and, by extension, represent potential targets for new immune-based interventions in HIV-infected persons.