Death and survival of gut CD4 T cells following HIV-1 infection ex vivo

Abstract

The gastrointestinal tract is ground zero for the massive and sustained CD4 T cell depletion during acute HIV-1 infection. To date, the molecular mechanisms governing this fundamental pathogenic process remain unclear. HIV-1 infection in the gastrointestinal tract is associated with chronic inflammation due to a disrupted epithelial barrier that results in microbial translocation. Here, we utilized the lamina propria aggregate culture model to demonstrate that the profound induction of granzyme B by bacteria in primary gut CD4 T cells ex vivo significantly contributes to HIV-1-mediated CD4 T cell death. Counterintuitively, a substantial fraction of gut granzyme B+ CD4 T cells harboring high levels of HIV-1 infection survive via a pathway linked to CD120b/TNFR2. Our findings underscore previously undescribed mechanisms governing the death and survival of gut CD4 T cells during HIV-1 infection that could inform strategies to counter HIV-1 pathogenesis and persistence in this critical tissue compartment.

Keywords: HIV-1; TNF response; cytotoxic CD4+ T lymphocytes; granzyme B; gut immunology.

Fig. 1.

HIV-1 infection and survival of GZB+ CD4 T cells in the LPAC model. A) Infection time course. At 0 dpi, LPMCs are spinoculated with TF HIV-1 for 2 h and then immediately cultured with commensal E. coli lysate. At 2 and 4 dpi, HIV-1 infection and viable CD4 T cell counts are measured. B) Gating strategy for GZB+ and GZB^neg cells. Note that CD4 T cells were determined from CD3+ CD8− cells. C) Up-regulation of (Left) HIV-1-infected CD4 T cells (n = 9 donors; paired t test) and (Right) GZB (n = 8 donors for mock, n = 9 donors for HIV-1-infected; mixed-effects model) following E. coli stimulation. D) HIV-1 infection in GZB+ versus GZB− CD4 T cells, based on the gating strategy in Fig. 1B (n = 9 donors; two-tailed paired t test). E) Survival of GZB+ versus GZB− CD4 T cells following HIV-1 infection (n = 9 donors; one-way ANOVA with Sidak's multiple comparisons test) in the presence of E.coli lysate. Δ corresponds to the average CD4 T cell depletion relative to mock. CD4 T cell counts were normalized to mock-infected cultures as 100% for each donor. For each of C to E, each shape corresponds to a distinct LPMC donor. Bars represent mean (± SEM); *P < 0.05, ***P < 0.001. ns, not significant.

Fig. 2.

HIV-1 infection and GZB expression are associated with apoptosis. LPMCs were spinoculated with TF HIV-1 or mock infected and cultured with commensal E. coli lysate for 2 or 4 days. A) Gating strategy. Early apoptotic cells are defined as viable (Aqua Viability dye negative) and Annexin V positive. B) Percentages of early apoptotic GZB+ or GZB− CD3+ CD8− T cells CD3+ CD8− T cells at 2 dpi (n = 6) and 4 dpi (n = 5). C) Gating strategy for early apoptotic HIV-1-infected (p24+) or uninfected (p24−) CD4 T cells. D) Percentages of early apoptotic infected or bystander CD3+ CD8− T cells at 2 dpi (n = 6) and 4 dpi (n = 5). Statistical analyses for B and D were performed using two-tailed paired t test. **P < 0.01.

Fig. 3.

Effect of CD4 T cell-intrinsic GZB on HIV-1-mediated CD4 T cell death. A), B) LPMCs were spinoculated with TF HIV-1 or mock and cultured with or without commensal E. coli lysate in the presence/absence of the GZB inhibitor Z-AAD-CMK (250 μM) for 4 days. A) Intracellular GZB activity in cell lysates (n = 6 donors; two-tailed paired t test) was determined using a colorimetric assay. GZB cleavage of Ac-IEPD-pNA releases pNA, which can be detected at 405 nm. B). Total CD4 T cell survival normalized to donor-matched mock infection (n = 6 donors; Repeated Measures (RM) one-way ANOVA with Sidak's multiple comparisons test). C) LPMCs depleted of CD8 cells (–CD8) using magnetic beads were confirmed by flow cytometry (n = 4 donors, two-tailed paired t test). D) to F) Total or CD8-depleted LPMCs were spinoculated with TF HIV-1 or mock then cultured with E. coli lysate for 4 days. D) HIV-1 infection levels in LPMC versus CD8-depleted LPMCs (n = 4 donors, two-tailed paired t test). E) Intracellular GZB activity in cell lysates measured as in A. F) Total CD4 T cell survival normalized to matched mock control (n = 4 donors, RM one-way ANOVA with Sidak's multiple comparisons test). For A to F, bars correspond to mean values; P < 0.05, NS, not significant; P-values < 0.1 were noted. G) Model of HIV-1-mediated CD4 T cell death ± GZB activity (Biorender.com). Microbial translocation up-regulates GZB in gut CD4 T cells. When GZB activity is present, more HIV-1-mediated CD4 T cell death occurs for both infected and bystander CD4 T cells (blebbing cells). When GZB activity is blocked, less HIV-1-mediated CD4 T cell death occurs, leaving a larger surviving population of CD4 T cells.

Fig. 4.

A subset of GZB+ CD4 T cells express markers associated with cell survival. A) to C) Viable CD4 T cells from LPMC (n = 6 donors) cultured with E. coli lysate for 2 days were sorted prior to generation of V3 Chromium Single Cell 3′ Transcriptome libraries. Of 48,934 CD4 T cells captured, 15,317 (31%) were GZB+. A) Uniform Manifold Approximation and Projection (UMAP) graph of scRNAseq data; each dot represents a single cell; darker dots correspond to GZB+ CD4 T cells; lighter dots depict GZB− CD4 T cells. B) DICE analysis of CD4 T cell subsets in GZB+ and GZB− CD4 T cells. C) Select transcripts up-regulated (blue) and down-regulated (red) in GZB+ versus GZB− CD4 T cells. Arrows indicate TNFRSF genes. The full list of altered 58 genes is given in Table S1. D), F) GZB expression at 2 days (n = 4) and 4 days (n = 5) ± E. coli lysate stimulation. E), G) TNFRSF protein expression measured by flow cytometry at 2 days (n = 4) and 4 days (n = 5). For D) to G), differences between groups were evaluated by two-tailed paired t test. Bars represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Fig. 5.

CD120b marks infected GZB+ CD4 T cells in the LPAC model. LPMCs were spinoculated with TF HIV-1 and then cultured for 4 days (A, B) or 6 days (C). HIV-1 infection (in CD3+ CD8− cells) was measured using intracellular p24 flow cytometry. Two-tailed paired t test, n = 5 donors (A, B), n = 6 donors (C). A), C) HIV-1 infection in CD120b ± GZB+ CD4 T cells 4 and 6 dpi. B) HIV-1 infection in GITR ± GZB+ CD4 T cells. D) to F). Single-cell transcriptome analysis (n = 6 donors; described in Fig. 4A) of 15,317 GZB+ CD4 T cells from LPMC cultured with E. coli lysate for 2 days. D) UMAP graph of scRNAseq data; each dot represents a single cell; darker dots correspond to CD120b+ GZB+ CD4 T cells; lighter dots depict CD120b− GZB+ CD4 T cells. E) DICE analysis of CD4 T cell subsets found in CD120b+ versus CD120b− GZB+ CD4 T cells. F) Select transcripts up-regulated in CD120b+ versus CD120b− GZB+ CD4 T cells. The full list of genes, fold-changes, and adjusted P-values are given in Table S2. Bars represent mean ± SEM. For A to D, *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.

Fig. 6.

CD120b signaling is associated with GZB+ CD4 T cell survival in the LPAC model. LPMCs were spinoculated with TF HIV-1 and then cultured with E. coli lysate for 6 days. A) CD120b expression in gut CD4 T cells in HIV-1- versus mock-infected cultures with E. coli (n = 6 donors). B) Difference in absolute CD4 T cell counts between HIV-1-infected and mock (n = 6). C) Percent survival of GZB+ versus GZB− CD4 T cells (n = 6). D) Percent survival of CD120b+ versus CD120b− GZB+ CD4 T cells (n = 6 donors). E), F) Effect of CD120b blockade on CD4 T cell survival following HIV-1 infection. LPMCs were incubated with anti-CD120b (αCD120b) or a matched isotype control at 0 dpi. E) Percent survival of CD120b ± GZB+ CD4 T cells. F) Absolute number of infected (p24+) CD120b+ GZB+ CD4 T cells with or without αCD120 treatment. For C, D, and F, the percent survival was calculated relative to mock (100%) for each donor; Δ corresponds to the mean CD4 T cell depletion relative to mock. Group differences in A and B were evaluated by two-tailed paired t test; those in C to E were evaluated using RM one-way ANOVA with Sidak's multiple comparisons test. Bars represent mean (± SEM); *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant. G) Model of HIV-1 persistence in CD120b+ GZB+ CD4 T cells (Biorender.com). Cells with a receptor tag represent CD120b+ GZB+ CD4 T cells which have a survival advantage over CD120b− cells. Moreover, CD120b+ cells are more likely to be infected (integrated provirus).