BACH2-driven tissue resident memory programs promote HIV-1 persistence

Abstract

Transcription repressor BACH2 redirects short-lived terminally differentiated effector into long-lived memory cells. We postulate that BACH2-mediated long-lived memory programs promote HIV-1 persistence in gut CD4+ T cells. We coupled single-cell DOGMA-seq and TREK-seq to capture chromatin accessibility, transcriptome, surface proteins, T cell receptor, HIV-1 DNA and HIV-1 RNA in 100,744 gut T cells from ten aviremic HIV-1+ individuals and five HIV-1- donors. BACH2 was the leading transcription factor that shaped gut tissue resident memory T cells (TRMs) into long-lived memory with restrained interferon-induced effector function. We found that HIV-1-infected cells were enriched in TRMs (80.8%). HIV-1-infected cells had increased BACH2 transcription factor accessibility, TRM (CD49a, CD69, CD103) and survival (IL7R) gene expression, and Th17 polarization (RORC, CCR6). In vitro gut CD4+ T cell infection revealed preferential infection and persistence of HIV-1 in CCR6+ TRMs. Overall, we found BACH2-driven TRM program promotes HIV-1 persistence and BACH2 as a new therapeutic target.

Keywords: BACH2; HIV-1 persistence; HIV-1 reservoir; T cell clonal expansion; tissue resident memory T cell.

Figure 1.. BACH2 drives tissue resident memory CD4+ T cells in human gut.

(A) WNN UMAP plot of distinct cell subsets in human gut (n = 17,642 in PLWH and 22,422 in HIV− individuals). (B) Proportions of gut cell subsets in PLWH and HIV− individuals. (C) Proportions of gut cells subsets per participant. (D) WNN UMAP plot of distinct CD4+ T cell subsets in human gut (n = 43,113 in PLWH and 6,092 in HIV− individuals), including CD4+ T cells from gut cell aliquots and CD3+ enriched aliquots. (E) Proportions of gut CD4+ T cell subsets PLWH and HIV− individuals. (F) Mean regulation scores (signed, −log10 scale) across domains of regulatory chromatin (DORC)(n = 639) per transcription factor (TF)(n = 95) for all significant TF-DORC interactions determined in PLWH. Top TF activators (left) and top TF repressors (right) are highlighted. (G) WNN UMAP plot of BACH2 transcription factor accessibility in gut CD4+ T cells, measured by chromVAR bias-corrected deviations. (H) WNN UMAP plot of per cell RNA module scores in gut CD4+ T cells from PLWH. The module score was built on DORC genes that had upregulated RNA expression in PLWH and were predicted to be activated by BACH2 (BACH2 regulation scores > 0). (I) Heatmap indicating differential transcription factors binding motifs accessibility (measured by chromVAR bias-corrected deviations) between CD4+ TRM cells and non-TRM CD4+ cells. FDR-adjusted P < 0.05, average Z-score difference > 0.2. (J) Heatmap indicating differentially expressed genes (normalized and scaled) between CD4+ TRM cells and non-TRM CD4+ cells. FDR-adjusted P < 0.05, min.pct. > 0.25, log2FC > 0.6. (K) Heatmap indicating differentially expressed surface proteins (normalized and scaled) between CD4+ TRM cells and non-TRM CD4+ cells. FDR-adjusted P < 0.05, min.pct. > 0.15, log2FC > 0.2. The mean expression of all protein features shown were tested to be greater than the mean expression of their specific isotype controls (Z > 2; two-sample Z test) in CD4+ T cells. (L) To identify enriched pathways in CD4+ TRM T cells, all 36,601 genes were ranked by log2-fold change in normalized gene expression between CD4+ TRM cells and non-TRM CD4+ cells. Significantly enriched TNF signaling pathway was identified using GSEA with leading-edge genes shown. See also Figure S1 – S3.

Figure 2.. BACH2 drives tissue resident memory program of CD8+ T cells in human gut while interferon regulatory factors (IRF) drive effector programs.

(A) WNN UMAP plot of distinct CD4+ T cell subsets in human gut (n = 45,475 in PLWH and 6,064 in HIV− individuals), including CD4+ T cells from gut cell aliquots and CD3+ enriched aliquots. (B) Proportions of CD8+ T cell subsets in PLWH and HIV− individuals. (C) Mean regulation scores (signed, −log10 scale) across DORCs (n = 671) per TF (n = 105) for significant TF-DORC interactions determined in PLWH. (D) BACH2 transcription factor accessibility in CD8+ T cells, measured by chromVAR bias-corrected deviations. (E) Differences in BACH2 transcription factor accessibility in CD8+ T cells between TRM and non-TRM cells in PLWH and HIV− individuals. (F) WNN UMAP plot of per cell module scores. The module score was built on DORC genes that had upregulated RNA expression in PLWH and were predicted to be activated by BACH2 (BACH2 regulation scores > 0) in PLWH CD8+ T cells. (G) Differences in module score of DORC genes (upregulated in PLWH and predicted to be activated by BACH2) in CD8+ T cells and CD8+ TRM cells between PLWH and HIV− individuals. (H,I) To identify enriched pathway in CD8+ T cell, all 36,601 genes were ranked by log2-fold change in normalized gene expression between CD8+ TRM cell and CD8+ TEM cells in PLWH and in HIV− individuals. (H) TNF signaling via NF-κB was enriched in TRM. (I) Leukocyte mediated cytotoxicity was enriched in TEM. Significantly enriched pathways were identified using GSEA with leading-edge genes shown. (J) Heatmap showing differential transcription factor binding motif accessibility (measured by chromVAR bias-corrected deviations) between CD8+ TRM cells and CD8+ TEM cells. FDR-adjusted P < 0.05, average Z-score difference > 0.15. Heatmap indicating differentially expressed genes (normalized and scaled) between CD8+ TRM cells and CD8+ TEM cells (middle). FDR-adjusted p < 0.05, min.pct > 0.25, log2FC > 0.6. (K) Heatmap indicating differentially expressed surface proteins (normalized and scaled) between CD8+ TRM cells and CD8+ TEM cells. FDR-adjusted P < 0.05, min.pct > 0.15, log2FC > 0.25. (L) The mean expression of all protein features shown were tested to be greater than the mean expression of their specific isotype controls (Z > 2; two-sample Z test) in CD8+ T cells. *p < 0.05, ** p < 0.01, ***p < 0.001, Wilcoxon rank-sum test. See also Figure S4.

Figure 3.. BACH2 restrains terminal effector programs and drives increased long-lived memory cytokine receptor expression in tissue resident memory CD4+ and CD8+ T cells.

(A) WNN UMAP plot highlighting CD4 TRM Th1_1 and CD4 TRM Th1_2 cells (left) and BACH2 transcription factor accessibility in CD4+ T cells, measured by chromVAR bias-corrected deviations. (B) WNN UMAP plot highlighting CD8 TRM_1 cells (left) and BACH2 transcription factor accessibility in CD8+ T cells, measured by chromVAR bias-corrected deviations. (C – F) Diffusion heatmap indicating dynamic changes in CD4+ TRM Th1 cells and CD8+ TRM cells ordered from low to high BACH2 accessibility (by chromVAR deviations). Cells were binned into 10 groups by BACH2 accessibility and enriched features were determined in each bin. (C) Changes in global chromatin accessibility of transcription factors (measured by chromVAR bias-corrected deviations) from low BACH2 accessible CD4+ TRM Th1 cells (TRM Th1_2) to high BACH2 accessible CD4+ TRM Th1 cells (TRM Th1_1). FDR-adjusted P < 0.05, average Z-score difference > 0.3. (D) Changes in normalized and scaled gene expression from low BACH2 accessible CD4+ TRM Th1 cells (CD4 TRM Th1_2) to high BACH2 accessible CD4+ TRM Th1 cells (CD4 TRM Th1_1). FDR-adjusted p < 0.05, min.pct. > 0.1, log₂FC > 0.5. (E) Changes in global chromatin accessibility of transcription factors (measured by chromVAR bias-corrected deviations) from low BACH2 accessible CD8+ TRM_1 cells to high BACH2 accessible CD8+ TRM_1 cells. FDR-adjusted P < 0.05, average Z-score difference > 0.3. (F) Changes in normalized and scaled gene expression from low BACH2 accessible CD8+ TRM_1 cells to high BACH2 accessible CD8+ TRM_1 cells. FDR-adjusted P < 0.05, min.pct. > 0.1, log₂FC> 0.4. (G – N) Dynamic feature expression changes in TRM cellular profiles with increase in BACH2 transcription factor accessibility. Accessibility of IRFI (G) and IRF4 (H) transcription factor binding motifs decreased with increase in BACH2 motif accessibility in CD4+ TRM Th1 cells (TRM Th1_1 and TRM Th1_2 combined). Cytokine IFNG (I) and TNF (J) RNA expression decreased with increase in BACH2 motif accessibility in CD4+ TRM Th1 cells (TRM Th1_1 and TRM Th1_2 combined). Accessibility of IRFI (K) and IRF4 (L) transcription factor binding motifs decreased with increase in BACH2 motif accessibility in CD8+ TRM cells. IFNG (M) and GZMA (N) RNA expression decreased with increase in BACH2 motif accessibility in CD8+ TRM cells. See also Figure S5.

Figure 4.. TNF and IFN-γ communication from BACH2-low ligand-expressing sender cells to BACH2-high receptor-expressing receiver CD4+ TRM.

(A) WNN UMAP plot of distinct CD4+ and CD8+ T cell subsets in human gut (n = 43,113 and 45,475 in PLWH, 6,092 and 6,064 in HIV− individuals, respectively). (B) BACH2 transcription factor accessibility (measured by chromVAR bias-corrected deviations) in CD8 activated, CD4 TRM Th1_2, CD4 TRM Th1_1, and CD4 TRM Th17 cells. (C) Scriabin interaction program discovery identified IFNG ligand to IFNGR1 and IFNGR2 receptor interactions (left) and TNF ligand to TNFRSF1B and TNFRSF1A receptor interactions (right) between sender (ligand expressing) CD8 activated and CD4 TRM Th1_2 cells and receiver (receptor expressing) CD4 TRM Th1_1 and CD4 TRM Th17 cells. Dot plots showed average normalized and scaled RNA expression of ligand and receptor genes. (D) Dot plot depicting NicheNet-predicted ligand activity in receiver TRM-Th1_1 (top) and TRM Th17 (bottom) in PLWH and HIV− individuals. Dot size depict percentage of receiver cells having ligand activity weight of > 0.1. Color scales represent ligand expression from TRM-Th1_2 sender cells (measured by RNA expression fold change in TRM-Th1_2 relative to other cell clusters). (E) Alluvial plot depicting target genes predicted to be upregulated in receiver TRM Th1_1 cells in response to IFNG and TNF. (F) Alluvial plot depicting target genes predicted to be upregulated in receiver TRM Th17 cells in response to IFNG and TNF. See also Figure S5.

Figure 5.. IRF drives clonal expansion of effector CD8+ T cells in the gut.

(A) Distribution of log-transformed T cell clone size per 1,000,000 cells for all unique T cell clones identified per PLWH participant. (B) WNN UMAP plot showing CD4+ T cell clones in PLWH (1485/13,012) and in HIV− individuals (875/1,104). Proportions of CD4+ T cell in clones. (D) Cell subset proportions of CD4+ T cell clones and non-clones. (E,F) Proportions of CD4+ T cell in clones in each subset in HIV− individuals gut (E) and PLWH gut (F). (G) WNN UMAP plot showing CD8+ T cell clones PLWH (5,510/10,868) and in HIV− individuals (308/875). (H) Proportions of CD8+ T cell in clones. (I) Cell subset proportions of CD4+ T cell clones and non-clones. (J,K) Proportions of CD8+ T cell in clones in each subset in HIV− individuals gut (J) and PLWH gut (K). (L) Differential transcription factors binding motif accessibility (measured by chromVAR bias-corrected deviations) between CD8+ T cell clones and non-clones. FDR-adjusted P < 0.05, average Z-score difference > 0.1. (M) Differentially expressed genes (normalized and scaled) between CD8+ T cell clones and non-clones. FDR-adjusted P < 0.05, min.pct > 0.25, log2FC > 0.25. (N) Differentially expressed surface proteins (normalized and scaled) between CD8+ T cell clones and CD8+ T cells that were not clonal. FDR-adjusted P < 0.05, min.pct > 0.1, log2FC > 0.25. The mean expression of all protein features shown were tested to be greater than the mean expression of their specific isotype controls (Z > 2; two-sample Z test) in CD8+ T cells. (O) To identify enriched pathways in CD8+ T cell clones, all 36,601 genes were ranked by log2-fold change in normalized gene expression between CD8+ T cell clones and CD8+ cells that were not clonal. Significantly enriched cytotoxicity pathway was identified using GSEA with leading-edge genes shown. All comparisons were made for CD8+ T cells whose CDR3β junction sequences were captured. See also Figure S6 – S8.

Figure 6.. HIV-1 preferentially infects and persists in TRM CCR7+ KLRB1+ CD4+ T cells in the gut.

(A) WNN UMAP plot depicting a total of 99 HIV-1-infected cells, including 54 HIV-1+ DNA+ RNA− cells (blue) and 45 HIV-1 RNA+ cells (magenta). (B) Cell subset proportions of HIV-1-infected cells. (C) Distribution of log-transformed HIV-1 RNA copies detected in HIV-1 RNA+ cells in each CD4+ T cell subset. (D) Differential transcription factors binding motifs accessibility (measured by chromVAR bias-corrected deviations) between HIV-1+ cells and HIV-1− CD4+ T cells in PLWH. FDR-adjusted P < 0.05, average Z-score difference > 0.25. (E) Differentially expressed genes (normalized and scaled) between HIV-1+ cells and HIV-1− CD4+ T cells in PLWH. FDR-adjusted P < 0.05, min.pct > 0.3, log2FC > 0.33. (F) Differentially expressed surface proteins (normalized and scaled) between HIV-1+ cells and HIV-1− CD4+ T cells in PLWH. FDR-adjusted P < 0.05, min.pct > 0.15, log2FC > 0.3. The mean expression of all protein features shown were tested to be greater than the mean expression of their specific isotype controls (Z > 2; two-sample Z test) in CD4+ T cells. For fair comparisons between groups, HIV-1− cells were downsized to match the same number of cells in HIV-1+ cells (n = 99) with 1,000 bootstrap replicates. The 1,000 P values were transformed to follow a standard Cauchy distribution and a combined P value was calculated as the weighted sum of transformed p values, followed by correction for multiple comparisons using the Benjamini-Hochberg (FDR) procedure. (G) Genome tracks showing CCR6 gene locus chromatin accessibility, and violin plots showing average normalized and scaled chromatin accessibility at CCR6 locus, CCR6 gene expression, and CCR6 protein expression. (H) To identify enriched pathways in HIV-1+ cells, all 36,601 genes were ranked by log2-fold change in normalized gene expression between HIV-1+ cells and HIV-1− cells. Significantly enriched TNF signaling was identified in HIV-1+ cells by GSEA with leading-edge genes shown. (I,J,K,L,M) Flow cytometry of HIV-1-infected CD4+ TRM cells and HIV-1-infected CD4+ non-TRM cells at 2 days post infection (top) and 7 days post infection (bottom). The lamina propria layer of human colon was isolated, activated with CD3/CD28, and infected with HIV-dEnv-dNef-GKO pseudotyped with an R5 envelope (JR-FL). Each line represents a biological replicate from uninfected donors. TRM was defined as CD49+CD69+CD4+ T cells. p < 0.05, p < 0.01, p < 0.001, Wilcoxon rank-sum test. See also Figure S9 – S10.