Safety and practicality of an excisional lymph node study driving HIV cure research in South Africa.

Introduction: Studying diseased human tissues offers better insights into the intricate interactions between pathogens and the human host. In conditions such as HIV and cancers, where diseases primarily manifest in tissues, peripheral blood studies are limited in providing a thorough understanding of disease processes and localized immune responses. Methods: We describe a study designed to obtain excisional lymph nodes from volunteers for HIV reservoir studies. Since study commencement in 2015, 181 lymph node excisions have been performed, resulting in collection of 138 lymph node tissues. Lymph nodes were surgically excised from study volunteers using a minimally invasive procedure, performed in a minor theater under local anesthesia. Results: The surgery takes less than 30 minutes to complete, minimizing risk and stress on the volunteer. The small incision made during the procedure typically heals within a week. The associated discomfort is generally manageable, and participants are often able to resume their regular activities within a day. Only 5.5% of the study participants experienced minor adverse events, such as swelling and prolonged wound healing, recovering within 2 weeks with no serious adverse events reported. Discussion: Our study demonstrates that when done with outmost care, obtaining excised lymph nodes for research is relatively safe and practical.

The impact of HIV infection on the frequencies, function, spatial localization and heterogeneity of T follicular regulatory cells (TFRs) within human lymph nodes.

BACKGROUND: HIV eradication efforts have been unsuccessful partly due to virus persistence in immune sanctuary sites such as germinal centres within lymph node (LN) tissues. Recent evidence suggests that LNs harbour a novel subset of regulatory T cells, termed follicular regulatory T cells (TFRs), but their role in HIV pathogenesis is not fully elucidated. RESULTS: Paired excisional LN and peripheral blood samples obtained from 20 HIV-uninfected and 31 HIV-infected treated and 7 chronic untreated, were used to determine if and how HIV infection modulate frequencies, function and spatial localization of TFRs within LN tissues. Imaging studies showed that most TFRs are localized in extra-follicular regions. Co-culture assays showed TFRs suppression of TFH help to B cells. Importantly, epigenetic and transcriptional studies identified DPP4 and FCRL3 as novel phenotypic markers that define four functionally distinct TFR subpopulations in human LNs regardless of HIV status. Imaging studies confirmed the regulatory phenotype of DPP4+TFRs. CONCLUSION: Together these studies describe TFRs dynamic changes during HIV infection and reveal previously underappreciated TFR heterogeneity within human LNs.

Differential localization and limited cytotoxic potential of duodenal CD8+ T cells.

The duodenum is a major site of HIV persistence during suppressive antiretroviral therapy despite harboring abundant tissue-resident memory (Trm) CD8+ T cells. The role of duodenal Trm CD8+ T cells in viral control is still not well defined. We examined the spatial localization, phenotype, and function of CD8+ T cells in the human duodenal tissue from people living with HIV (PLHIV) and healthy controls. We found that Trm (CD69+CD103hi) cells were the predominant CD8+ T cell population in the duodenum. Immunofluorescence imaging of the duodenal tissue revealed that CD103+CD8+ T cells were localized in the intraepithelial region, while CD103-CD8+ T cells and CD4+ T cells were mostly localized in the lamina propria (LP). Furthermore, HIV-specific CD8+ T cells were enriched in the CD69+CD103-/lo population. However, the duodenal HIV-specific CD8+ Trm cells rarely expressed canonical molecules for potent cytolytic function (perforin and granzyme B) but were more polyfunctional than those from peripheral blood. Taken together, our results show that duodenal CD8+ Trm cells possess limited perforin-mediated cytolytic potential and are spatially separated from HIV-susceptible LP CD4+ T cells. This could contribute to HIV persistence in the duodenum and provides critical information for the design of cure therapies.

Hypermethylation at the CXCR5 gene locus limits trafficking potential of CD8+ T cells into B-cell follicles during HIV-1 infection.

CD8+ T cells play an important role in HIV control. However, in human lymph nodes (LNs), only a small subset of CD8+ T cells express CXCR5, the chemokine receptor required for cell migration into B-cell follicles, which are major sanctuaries for HIV persistence in individuals on therapy. Here, we investigate the impact of HIV infection on follicular CD8+ T cell (fCD8) frequencies, trafficking patterns, and CXCR5 regulation. We show that, although HIV infection results in a marginal increase in fCD8s in LNs, the majority of HIV-specific CD8+ T cells are CXCR5- (non-fCD8s) (P < .003). Mechanistic investigations using Assay for Transposase-Accessible Chromatin using sequencing showed that non-fCD8s have closed chromatin at the CXCR5 transcriptional start site (TSS). DNA bisulfite sequencing identified DNA hypermethylation at the CXCR5 TSS as the most probable cause of closed chromatin. Transcriptional factor footprint analysis revealed enrichment of transforming growth factors (TGFs) at the TSS of fCD8s. In vitro stimulation of non-fCD8s with recombinant TGF-ß resulted in a significant increase in CXCR5 expression (fCD8s). Thus, this study identifies TGF-ß signaling as a viable strategy for increasing fCD8 frequencies in follicular areas of the LN where they are needed to eliminate HIV-infected cells, with implications for HIV cure strategies.

Major Scientific Hurdles in HIV Vaccine Development: Historical Perspective and Future Directions.

Following the discovery of HIV as a causative agent of AIDS, the expectation was to rapidly develop a vaccine; but thirty years later, we still do not have a licensed vaccine. Progress has been hindered by the extensive genetic variability of HIV and our limited understanding of immune responses required to protect against HIV acquisition. Nonetheless, valuable knowledge accrued from numerous basic and translational science research studies and vaccine trials has provided insight into the structural biology of the virus, immunogen design and novel vaccine delivery systems that will likely constitute an effective vaccine. Furthermore, stakeholders now appreciate the daunting scientific challenges of developing an effective HIV vaccine, hence the increased advocacy for collaborative efforts among academic research scientists, governments, pharmaceutical industry, philanthropy, and regulatory entities. In this review, we highlight the history of HIV vaccine development efforts, highlighting major challenges and future directions.

Magnitude and Kinetics of CD8+ T Cell Activation during Hyperacute HIV Infection Impact Viral Set Point.

CD8(+) T cells contribute to the control of HIV, but it is not clear whether initial immune responses modulate the viral set point. We screened high-risk uninfected women twice a week for plasma HIV RNA and identified 12 hyperacute infections. Onset of viremia elicited a massive HIV-specific CD8(+) T cell response, with limited bystander activation of non-HIV memory CD8(+) T cells. HIV-specific CD8(+) T cells secreted little interferon-γ, underwent rapid apoptosis, and failed to upregulate the interleukin-7 receptor, known to be important for T cell survival. The rapidity to peak CD8(+) T cell activation and the absolute magnitude of activation induced by the exponential rise in viremia were inversely correlated with set point viremia. These data indicate that rapid, high magnitude HIV-induced CD8(+) T cell responses are crucial for subsequent immune control of acute infection, which has important implications for HIV vaccine design.

The Breadth of Expandable Memory CD8+ T Cells Inversely Correlates with Residual Viral Loads in HIV Elite Controllers.

UNLABELLED: Previous studies have shown that elite controllers with minimal effector T cell responses harbor a low-frequency, readily expandable, highly functional, and broadly directed memory population. Here, we interrogated the in vivo relevance of this cell population by investigating whether the breadth of expandable memory responses is associated with the magnitude of residual viremia in individuals achieving durable suppression of HIV infection. HIV-specific memory CD8(+) T cells were expanded by using autologous epitopic and variant peptides. Viral load was measured by an ultrasensitive single-copy PCR assay. Following expansion, controllers showed a greater increase in the overall breadth of Gag responses than did untreated progressors (P = 0.01) as well as treated progressors (P = 0.0003). Nef- and Env-specific memory cells expanded poorly for all groups, and their expanded breadths were indistinguishable among groups (P = 0.9 for Nef as determined by a Kruskal-Wallis test; P = 0.6 for Env as determined by a Kruskal-Wallis test). More importantly, we show that the breadth of expandable, previously undetectable Gag-specific responses was inversely correlated with residual viral load (r = -0.6; P = 0.009). Together, these data reveal a direct link between the abundance of Gag-specific expandable memory responses and prolonged maintenance of low-level viremia. Our studies highlight a CD8(+) T cell feature that would be desirable in a vaccine-induced T cell response. IMPORTANCE: Many studies have shown that the rare ability of some individuals to control HIV infection in the absence of antiretroviral therapy appears to be heavily dependent upon special HIV-specific killer T lymphocytes that are able to inhibit viral replication. The identification of key features of these immune cells has the potential to inform rational HIV vaccine design. This study shows that a special subset of killer lymphocytes, known as central memory CD8(+) T lymphocytes, is at least partially involved in the durable control of HIV replication. HIV controllers maintain a large proportion of Gag-specific expandable memory CD8(+) T cells involved in ongoing viral suppression. These data suggest that induction of this cell subset by future HIV vaccines may be important for narrowing possible routes of rapid escape from vaccine-induced CD8(+) T cell responses.

Mosaic HIV-1 Gag antigens can be processed and presented to human HIV-specific CD8+ T cells.

Polyvalent mosaic HIV immunogens offer a potential solution for generating vaccines that can elicit immune responses against genetically diverse viruses. However, it is unclear whether key T cell epitopes can be processed and presented from these synthetic Ags and recognized by epitope-specific human T cells. In this study, we tested the ability of mosaic HIV immunogens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors to process and present major HIV clade B and clade C CD8 T cell epitopes in human cells. A bivalent mosaic vaccine expressing HIV Gag sequences was used to transduce PBMCs from 12 HIV-1-infected individuals from the United States and 10 HIV-1-infected individuals from South Africa; intracellular cytokine staining, together with tetramer staining, was used to assess the ability of mosaic Gag Ags to stimulate pre-existing memory responses compared with natural clade B and C vectors. Mosaic Gag Ags expressed all eight clade B epitopes tested in 12 United States subjects and all 5 clade C epitopes tested in 10 South African subjects. Overall, the magnitude of cytokine production induced by stimulation with mosaic Ags was comparable to clade B and clade C Ags tested, but the mosaic Ags elicited greater cross-clade recognition. Additionally, mosaic Ags induced HIV-specific CD4 T cell responses. Our studies demonstrate that mosaic Ags express major clade B and clade C viral T cell epitopes in human cells, as well as support the evaluation of mosaic HIV-1 vaccines in humans.

Dynamic regulation of functionally distinct virus-specific T cells.

The functional capacities of CD8(+) T cells important for virus clearance are influenced by interactions with antigen presenting cells (APCs) and CD4(+) T cells during initial selection, subsequent expansion, and development of memory. Recently, investigators have shown that polyfunctional T cells correlate best with long-term protection, however, it is still unknown how to stimulate T cells to achieve these responses. To study this, we examined the phenotypes and functions of CD8(+) T cells specific for two different virus antigens stimulated ex vivo using either autologous monocyte-derived dendritic cells (moDCs) or HLA-A2-Ig-based artificial APCs (aAPCs). Although similar numbers of influenza virus and measles virus tetramer-positive cells were generated by stimulation with peptide-loaded moDCs and aAPCs, T cell function, assessed by expression of IL-2, IFN-gamma, TNF-alpha, MIP1beta, and CD107a, showed that aAPC-generated CD8(+) T cells were multifunctional, whereas moDC-generated cells were mostly monofunctional. aAPC-generated cells also produced more of each cytokine per cell than CD8(+) T cells generated with moDCs. These phenotypes were not fixed, as changing the culture conditions of expanding T cells from aAPCs to moDCs, and moDCs to aAPCs, reversed the phenotypes. We conclude that CD8(+) T cells are heterogeneous in their functionality and that this is dependent, in a dynamic way, on the stimulating APC. These studies will lead to understanding the factors that influence induction of optimal CD8(+) T cell function.