HIV persistence in tissue sites despite ART is a major barrier to HIV cure. Detailed studies of HIV-infected cells and immune responses in native lymph node tissue environment is critical for gaining insight into immune mechanisms impacting HIV persistence and clearance in tissue sanctuary sites. We compared HIV persistence and HIV-specific T cell responses in lymph node biopsies obtained from 14 individuals who initiated therapy in Fiebig stages I/II, 5 persons treated in Fiebig stages III-V and 17 late treated individuals who initiated ART in Fiebig VI and beyond. Using multicolor immunofluorescence staining and in situ hybridization, we detect HIV RNA and/or protein in 12 of 14 Fiebig I/II treated persons on suppressive therapy for 1 to 55 months, and in late treated persons with persistent antigens. CXCR3+ T follicular helper cells harbor the greatest amounts of gag mRNA transcripts. Notably, HIV-specific CD8+ T cells responses are associated with lower HIV antigen burden, suggesting that these responses may contribute to HIV suppression in lymph nodes during therapy. These results reveal HIV persistence despite the initiation of ART in hyperacute infection and highlight the contribution of virus-specific responses to HIV suppression in tissue sanctuaries during suppressive ART.

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.

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 + CD103 hi ) 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.

CD8 + T cells play an important role in viral and tumour control. However, in human lymph nodes (LNs), only a small subset of CD8 + T cells called follicular CD8 + T cells (fCD8s) expresses CXCR5, the chemokine receptor required for cell migration into B cell follicles, thought to promote immune evasion. Here we obtained LNs from HIV infected persons to investigate regulation of CXCR5 expression in lymphoid CD8 + T cells, and compared this to the more abundant CXCR5 expressing T follicular CD4 + helper cells (GCTfh). Our results show that DNA hypermethylation and closed chromatin at the transcriptional start site (TSS) prevent CXCR5 expression in non-fCD8s. We also found that greater nucleosomal density at the CXCR5 TSS could be responsible for reduced CXCR5 expression in fCD8s relative to GCTfh. Together, these data provide critical insights into both the underlying molecular mechanisms that repress CXCR5 expression in non-fCD8s and the plausible mechanism responsible for the low CXCR5 expression in fCD8s, with implications for HIV cure strategies.

HIV persistence in tissue sites despite ART is a major barrier to HIV cure. Detailed studies of HIV infected cells and immune responses in native lymph node (LN) tissue environment is critical for gaining insight into immune mechanisms impacting HIV persistence and clearance in tissue sanctuary sites. We compared HIV persistence and HIV-specific T cell responses in LN biopsies obtained from 14 individuals who initiated therapy in Fiebig stages I/II, 5 persons treated (Tx) in Fiebig stages III-V and 17 late Tx individuals who initiated ART in Fiebig VI and beyond. Using multicolor immunofluorescence staining and in situ hybridization, HIV RNA and/or protein was detected in 12 of 14 Fiebig I/II Tx persons who were on suppressive therapy for 1 to 55 months, while all late Tx persons had persistent antigens. CXCR3+T follicular helper T cells harbored the greatest amounts of gag mRNA transcripts. Notably, HIV-specific CD8+ T cells responses associated with lower HIV antigen burden in LNs, suggesting that these responses may contribute to HIV suppression in LNs during therapy. These results reveal HIV persistence despite the initiation of ART in hyperacute infection and highlight the contribution of virus-specific responses to HIV suppression in tissue sanctuaries during suppressive ART.