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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-betdimEomeshi expressional profile. Interestingly, both resting and activated HIV-specific CD8+ T cells in chronic infection were almost exclusively T-betdimEomeshi cells, while CMV-specific CD8+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-betdimEomeshi 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.

SUMMARY Epstein-Barr virus (EBV) represents a major global health problem. Though it is associated with infectious mononucleosis and over 200,000 cancers annually worldwide, a vaccine is not available. The major target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a symmetric array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine improved vaccine-induced protection in a mouse model by targeting a functionally conserved site of vulnerability with 10- to 100-fold increased neutralization compared to soluble gp350. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by focusing on a conserved viral entry domain, a strategy that can be applied to other viruses.

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.

The interaction between follicular T helper cells (TFH) 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 TFH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral TFH 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 CCR7highCXCR5highCCR6highPD-1high 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 TFH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral TFH population, the expression level of TFH-associated genes more closely resembles a memory, non-TFH population, as opposed to a TFH 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 TFH cells.

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Broadly neutralizing antibodies (bNAbs) protect against HIV-1 infection, yet how they are generated during chronic infection remains unclear. It is known that T follicular helper (TFH) cells are needed to promote affinity maturation of B cells during an immune response; however, the role of TFH during HIV-1 infection is undefined within lymph node germinal centers (GCs). We use nonhuman primates to investigate the relationship in the early stage of chronic SHIVAD8 (simian-human immunodeficiency virus AD8) infection between envelope (Env)-specific TFH cells, Env-specific B cells, virus, and the generation of bNAbs during later infection. We found that both the frequency and quality of Env-specific TFH cells were associated with an expansion of Env-specific immunoglobulin G-positive GC B cells and broader neutralization across HIV clades. We also found a correlation between breadth of neutralization and the degree of somatic hypermutation in Env-specific memory B cells. Finally, we observed high viral loads and greater diversity of Env sequences in rhesus macaques that developed cross-reactive neutralization as compared to those that did not. These studies highlight the importance of boosting high-quality TFH populations as part of a robust vaccine regimen aimed at eliciting bNabs.

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