Since the 1990s, HIV-replicating cells have been described in the liver causing diseases and comorbidities. Understanding liver alterations related to CD4+ T cell infection is poorly addressed so far.

Herein, rhesus macaques (RMs) were infected with SIVmac251 (20 Animal Infectious Dose 50, AID50). On the day of euthanasia, liver cells were mechanically isolated from both infected and non-infected RMs. Phenotypic analyses were performed by flow cytometry. The levels of viral DNA were quantified by qPCR. Gene profile of sorted CD4+ T cells was assessed by RNA-sequencing and bioinformatic analyses.

We found that hepatic CD4+ T cells from healthy RMs express higher levels of CCR5 compared to those in the blood. Furthermore, consistent with CD4+ T cell depletion characterizing Aids, hepatic memory CD4+ T cells were also severely depleted in SIV-infected RMs. Interestingly, by analyzing the expression of CCR5 and programmed cell death 1 (PD-1), a marker of exhaustion, PD-1+CCR5- and PD-1+CCR5+ expressing CD4+ T cells were significantly higher in SIV-infected RMs compared to healthy RMs. Consistent with viral detection, transcriptomic analyses revealed inflammatory and interferon (IFN) gene signatures. Most importantly, our study revealed mitochondrial gene alteration supporting defective oxidative phosphorylation (OXPHOS). Whereas glycolytic gene machinery was not overexpressed in CD4+ T cells, our results highlighted a lipid metabolism signature that is increased in SIV-infected RMs.

Given the role of PD-1 in reprogramming T cell metabolism, our results provided new insights related to hepatic CD4+ T cell gene metabolism profiles during viral infection.

CD4+ T cells are the main cellular targets of both HIV and SIV. However, the cellular subsets in which SIV actively replicates in blood and tissues from non-human primates (NHPs) remain largely unidentified. We developed a novel flow-based approach, using two different antibodies specific for the p27 SIV protein (2F12 and 4B7), to enumerate and phenotype productively infected cells during untreated SIV infection. The assay (SIV-Flow) can detect a single infected cell in a million CD4+ T cells and has a large dynamic range (1 to 100,000 infected cells/106 CD4+ T cells).
We measured the SIV reservoirs in isolated CD4+ T cells from multiple tissues (blood, spleen and different lymph nodes) from 4 SIVmac239-infected ART-naïve rhesus macaques using SIV-Flow antibodies in an 18 parameters cytometry panel, as well as total SIV DNA and cell-associated (ca) SIV RNA quantifications.
We measured higher frequencies of p27+ cells in the spleen (mean 1,887 cells/106 CD4+ T cells) compared to blood and lymph nodes (means 842 and 848 cells/106 CD4+ T cells, respectively). Frequencies of p27+ cells measured by SIV-Flow correlated with total SIV DNA and ca-SIV RNA levels in blood (p=0.02; p=0.0014; respectively) and partially in the inguinal (p=0.051; p=0.02; respectively) and mesenteric lymph nodes (p=0.04; p=0.07; respectively). Analyzing all tissues together, when comparing with the general population, the effector memory cell subset was highly enriched in p27+ cells (enrichment 35.7 fold), and in stem-cell memory cells (enrichment 7.5 fold). Overall, compared to their uninfected counterparts, p27+ cells expressed higher levels of several cellular markers including PD-1 (enrichment 4.1 fold), CD69 (enrichment 2.9 fold), Ki67 (enrichment 5.6 fold) and CTLA-4 (enrichment 2.6 fold).
Our results suggest that activated CD4+ T cells in the spleen are important contributors to SIV replication in chronically infected NHPs.

Background: The foreskin is a primary site of HIV acquisition in heterosexual men. However, the biological basis of HIV susceptibility in foreskin tissue remains incompletely understood. Current techniques to analyze immune cell populations in the foreskin rely on processing tissue immediately after circumcision, which can be logistically difficult. We sought to determine whether viable cells could be recovered from cryopreserved foreskin tissues, and to characterize the effect of cryopreservation on the relative proportions of CD4+ T cell subsets.

Methods: Foreskin tissues from HIV-negative males aged >18 were collected immediately after voluntary circumcision (n=10). For each sample, tissues were sectioned into 0.25cm2 pieces, and half were cryopreserved in 10% DMSO to be thawed and digested later (“frozen”), while the remaining half was immediately digested into a cell suspension (“fresh”). In both conditions, cell suspensions were created through mechanical and enzymatic digestion, stained with antibodies against CD3, CD4, HLA-DR, CD38, CD25, CD127, CCR4, CXCR3, CCR6, CCR5, and CCR10; and analyzed by flow cytometry.

Results: We observed significant increases in the proportion of CD4+ T cells expressing CCR5 after cryopreservation (medians fresh vs. frozen: 55.1%, 67.7%, p=0.0012), CD38 (2.7%, 4.7%, p=0.0146), and CXCR3 (3.6%, 8.2%, p=0.0086) after cryopreservation. However, the relative rankings of matched participants' fresh and cryopreserved samples was maintained. Moreover, there were no significant differences between the proportion of CD25 (12.5%, 14.1%), CCR4 (54.0%, 54.9%), CCR6 (44.3%, 40.6%), HLA-DR (15.3%, 17.9%), CCR10 (6.0%, 5.7%), and CD127 (72.5%, 72.3%) between fresh and cryopreserved foreskin samples.

Significance:
Digestion and analysis of T cell subsets from foreskin tissue is feasible after cryopreservation. Despite some variation, the ranking of participants' matched samples was well maintained for these markers; thus, inter-individual comparisons remain valid. For HIV researchers lacking access to fresh foreskin tissue, these data suggest that cryopreservation and delayed immune analysis is a viable alternative.

The cervical mucosa is the primary barrier of the female reproductive tract where tissue-resident memory T (TRM) cells are crucial for optimal localized protection against sexually transmitted infections (STIs) and regulation of the local mucosal milieu. TRM subsets are conventionally characterized by surface expression of the C-type lectin CD69 and/or the integrin CD103, while their functional profiles in human cervical mucosa remain unclear. Our preliminary flow analysis showed that CD45+ immune cells and CD69+CD103+ cells were more frequently observed in the ectocervix.

Here, we used single cell RNA sequencing (scRNAseq) to construct transcriptomic profiles of 9,000 individual cells on the mucosal wall from the ectocervical biopsy and endocervical cytobrush samples. We identified 11 cell types; macrophage and epithelial cells were more frequently observed in the ectocervix, whereas lymphatic endothelial cells, MAST, and pDC were dominant in the endocervix. T and B cells were distributed evenly across the cervical mucosa. By further profiling subclusters of 2,855 T cells expressing CD3D gene, we confirm high expression of the CD69 gene markers and a high proportion of cell expressing T follicular helper cell-related genes. CD4+ Treg cells were concentrated in the ectocervix. Of four distinct CD8+ TRM subpopulations, the subset with GZMB+IFNG+ phenotypes were preferentially localized in the ectocervix, with upregulated energy metabolism-associated gene pathways, while the other three subsets were more confined to the endocervix.

Taken together, this work provides a valuable resource for deciphering the heterogeneity of the immune cells and TRM subsets in the cervical mucosa and a better understanding of their existence and functions is important for optimizing effective vaccines as well as new therapeutic agents against STIs.

A persistent latent reservoir in long-lived CD4+ T cells is the main obstacle to eradicating HIV-1 infection. Chronic HIV-1 infection functionally alters CD8+ T cells through upregulation of immune checkpoint (IC) receptors such as TIGIT (T cell immunoreceptor with Ig and ITIM domains). Expression of IC receptors can result in impaired cytolytic activity and failure to suppress viral replication. Therefore, blocking IC receptors could be an adjunct therapeutic approach targeting the HIV reservoir in eradication strategies.

This study employed TIGIT engagement and blockade on CD8+ T cells from people living with HIV (PLWH) to test how TIGIT expression affects T cell function. We tested TIGIT engagement on CD8+ T cells from PLWH in non-specific redirected cytotoxicity assays and tested the impact of TIGIT blockade on HIV antigen-specific CD8+ T cells.

For PLWH with circulating CD8+ T cell cytotoxicity >10% in redirected killing assays, TIGIT engagement reduced cytotoxicity in 8/14 cases, showing that TIGIT engagement impairs killing by CD8+ T cells from some PLWH. About 20% of subjects tested had strong interferon-gamma (IFN)-γ responses against HIV Gag and/or Nef peptides (>1000 spot-forming units/10⁶ peripheral blood mononuclear cells). Stimulation of HIV-specific CD8+ T cells with peptides in the presence of TIGIT-blocking monoclonal antibody increased CD8+ T cell degranulation and IFN-γ production in certain individuals. Thus, generalized and HIV-specific effector functions of CD8+ T cells from a subset of PLWH are inhibited by TIGIT expression.

These data show that TIGIT blockade can improve antiviral effector cell function in certain PLWH. Identifying features of the subset of responsive CD8+ T cells will help direct blockade therapy to those PLWH most likely to benefit. Supported by CIHR.

HIV-1 induced immune ‘exhaustion’ is marked by sustained and upregulated expression of various inhibitory receptors which play a role in dampening protective immune responses. Therefore, identification and blockade of inhibitory molecules that mediate T cell exhaustion during chronic HIV-1 infection, is a viable strategy to enhancing immune-directed clearance. Recently, Carcinoembryonic Antigen related Cell Adhesion Molecule 1 (CEACAM1), was identified as a negative regulator of T-cell responses in murine models of cancer and inflammatory disease. During chronic HIV-1 infection, CD4 and CD8 T-cells co-expressing CEACAM1 and its ligand TIM-3 exhibited the most dysfunctional responses, suggesting a role for CEACAM1 in HIV-1 pathogenesis. Therefore, we hypothesized that interactions between CEACAM1 and its ligands on HIV-specific T-cells, inhibits their responses and allows viral persistence. To investigate our hypothesis, we characterized CEACAM1 expression on CD4 and CD8 T-cells from HIV-1+ donors with various degrees of viral control and treatment status. Furthermore, we quantified and compared HIV-1 pro-viral DNA between CEACAM1+/- CD4 T-cells from treated and untreated chronically infected donors. Finally, we measured HIV-Gag specific CD4 and CD8 T-cell proliferation and IFNγ production, in the presence of CEACAM1 binding antibody, 18/20. We found that frequency of CEACAM1+CD4 T-cells was elevated during untreated HIV-1 infection (HIV-: 7.045%, acute: 25.66%, chronic: 22.51%) but reduced after treatment (chronic/treated: 12.31%). Differential enrichment of HIV DNA in CEACAM1-CD4 T-cells (76.23%) from untreated donors and in CEACAM1+CD4 T cells (90.04%) in treated donors was evident. Finally, CEACAM1 binding resulted in ~50% reduction in HIV-specific T-cell proliferation and IFNγ production. Together, our data suggests a complex but tuneable function for CEACAM1 during HIV-1 infection. Future work will elucidate the mechanism of 18/20 antibody function, and role of observed changes in killing of HIV-1 infected cells. Validation of this approach would suggest that targeting CEACAM1 may have immunotherapeutic potential during HIV-1 infection.

Abstract The intestinal environment facilitates HIV-1 replication/viral reservoir (VR) persistence via mechanisms involving the gut-homing elixir retinoic acid (RA), which transcriptionally reprograms CD4+ T-cells for increased HIV-1 infection. Consistently, colon-infiltrating CD4+ T-cells carry viral reservoirs (VRs) in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Intriguingly, integrative infection in colon macrophages (MΦ), a pool constantly replenished by circulating monocytes, represents a rare event in ART-treated PLWH, thus raising new questions on HIV-1 permissiveness/persistence in gut-residing MΦ during ART. We hypothesized that the rarity of VRs in colon-infiltrating MDM of ART-treated PLWH is likely not the consequence of their resistance to infection, but rather due to their rapid turn-over in vivo. Thus, we aimed i) to explore the effect of RA on HIV-1 replication and ii) to identify molecular mechanisms by which RA modulates HIV-1 replication in monocyte-derived MΦ (MDM) in vitro.

MDM generated in the presence/absence of RA in vitro were analyzed by FACS for CD4, CCR5 and CXCR4 expression, and exposed to replication-competent and single-round VSV-G-pseudotyped HIV constructs. HIV replication was measured by ELISA and FACS, as well as nested real-time PCR using specific primers for early/late reverse transcription and integrated HIV-DNA. RNA-Sequencing was performed using the Illumina technology. Validations were performed by RT-PCR or ELISA.

Results demonstrated that RA promotes HIV-1 replication in MDM by facilitating CD4/CCR5-mediated HIV entry and post-entry mechanisms. Gene set variation analysis and HIV interactor NCBI database interrogation revealed profound RA-mediated transcriptional reprogramming associated with effector functions, metabolic/signaling status, and HIV-1 dependency/restriction factors. Functional validations and pharmacological antagonism demonstrated the contribution of RA-modulated mTOR/S6K and Wnt/-catenin/TCF4 pathways.

These results support a model in which macrophages in RA-rich tissues represent important HIV-1 targets via mTOR/S6K and Wnt/β-catenin/TCF4-dependent mechanisms that can be therapeutically targeted.

Th17 cells are rapidly depleted during HIV infection and contribute to HIV persistence, whereas their frequencies and functions are not fully restored by antiretroviral therapy (ART). However, molecular mechanisms underlying these alterations are understudied. RNA-sequencing (Illumina Technology) was performed in freshly FACS-sorted Th17 cells (CD4+CD25-CD45RA-CCR6+) from aviremic ART-Treated (ST, n=6), elite controllers (EC, n=3) and uninfected controls (HD, n=5). Th17 cells from STs vs. HDs showed alterations in the TGF-β1/smad2-3 pathway, indicative of impairments in their generation and stability. Other pathways related to Th17 differentiation were downregulated in STs vs. HDs, including p38, PTEN, RUNX1, NFAT, and Notch. Consistently, the RORC2 repressor NR1D1 was upregulated, while Semaphorin 4D, a RORC2 inducer, decreased in STs vs. HDs. Migration markers CCR9, α4β1, CXCR5 and CCR7 decreased in STs vs. HDs, indicating diminished migration potential towards the gut and inflammatory tissues. Metabolic alterations were observed in STs vs. HDs, comprising decreased expression of mitochondria genes, energy metabolism, and PI3K-AKT-mTOR pathway. Furthermore, in STs, sphingolipids synthesis and amino acid transport were downregulated. In STs vs. HDs, decreased expression of transcription factors RUNX1, NFAT, and CREB, along with lower CDK6 expression and increased expression of EED and TP53BP1, are associated with increased HIV persistence/latency. Similarly to the ST group, Th17 cells in ECs showed decreased expression of Semaphorin 4D and downregulated PI3K-AKT-mTOR pathway. Decreased synthesis of sphingolipids and amino acid transport and higher expression of NR1D1 were also observed in Th17 cells from EC and ST vs. HD donors. Furthermore, compared to HD donors, Th17 cells in ECs showed a unique metabolic profile related to an increase in the TCA cycle, fatty acid β-oxidation, cellular response to hypoxia, and decreased inositol phosphate metabolism. The Th17 transcriptional profiling revealed impaired differentiation, stability, migration, and metabolic transcriptomic signature linked to chronic HIV infection despite viral-suppressive ART.