HIV-1-induced cytokines deplete homeostatic innate lymphoid cells and expand TCF7-dependent memory NK cells

Yetao Wang, Lawrence Lifshitz, Kyle Gellatly, Carol L Vinton, Kathleen Busman-Sahay, Sean McCauley, Pranitha Vangala, Kyusik Kim, Alan Derr, Smita Jaiswal, Alper Kucukural, Patrick McDonel, Peter W Hunt, Thomas Greenough, JeanMarie Houghton, Ma Somsouk, Jacob D Estes, Jason M Brenchley, Manuel Garber, Steven G Deeks, Jeremy Luban, Yetao Wang, Lawrence Lifshitz, Kyle Gellatly, Carol L Vinton, Kathleen Busman-Sahay, Sean McCauley, Pranitha Vangala, Kyusik Kim, Alan Derr, Smita Jaiswal, Alper Kucukural, Patrick McDonel, Peter W Hunt, Thomas Greenough, JeanMarie Houghton, Ma Somsouk, Jacob D Estes, Jason M Brenchley, Manuel Garber, Steven G Deeks, Jeremy Luban

Abstract

Human immunodeficiency virus 1 (HIV-1) infection is associated with heightened inflammation and excess risk of cardiovascular disease, cancer and other complications. These pathologies persist despite antiretroviral therapy. In two independent cohorts, we found that innate lymphoid cells (ILCs) were depleted in the blood and gut of people with HIV-1, even with effective antiretroviral therapy. ILC depletion was associated with neutrophil infiltration of the gut lamina propria, type 1 interferon activation, increased microbial translocation and natural killer (NK) cell skewing towards an inflammatory state, with chromatin structure and phenotype typical of WNT transcription factor TCF7-dependent memory T cells. Cytokines that are elevated during acute HIV-1 infection reproduced the ILC and NK cell abnormalities ex vivo. These results show that inflammatory cytokines associated with HIV-1 infection irreversibly disrupt ILCs. This results in loss of gut epithelial integrity, microbial translocation and memory NK cells with heightened inflammatory potential, and explains the chronic inflammation in people with HIV-1.

Conflict of interest statement

Declaration of interests

The authors declare no competing financial interests.

Figures

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Fig. 1 |. HIV-1 infection decreases ILCs…
Fig. 1 |. HIV-1 infection decreases ILCs in blood and colon lamina propria.
a, Percent ILCs (Lin−CD56−CD16−CD127+ PBMCs; Extended Data Fig. 1a) from 40 HIV-1−, 36 HIV-1+ viremic, 39 HIV-1+ on ART, and 38 HIV-1+ spontaneous controllers (Supplementary Table 1). b-d, ILCs as in a, correlation with CD4s (b), CD4/CD8 ratio (c), and plasma sCD14 (d). Correlation coefficient (R) by Pearson, zero slope p value determined by the F-test (n=80). e,f, Percent Lin−CD127+ILCs (e) and Lin−CD127+RORγT+ILC3s (f), among colon lamina propria lymphoid cells from 6 HIV-1− and 7 HIV-1+ on ART with undetectable viremia (Supplementary Table 2). g, CD3−CD117+ ILC3s in rectosigmoid tissue from HIV-1− or HIV-1+ individuals on ART (Supplementary Table 1). White arrowheads, ILCs. Scale bar = 100 μm. h,i, As in g, percent ILC3s (h) and CD3−CD117+/CD3+CD117− (i) from 8 HIV-1− and 16 HIV-1+ on ART. j, MX1+ cells as in g. k, As in j, percent MX1+ as in h. l, MPO+ cells as in g. m, As in l, percent MPO+ cells as in h. n, CD127+ among Lin−CD56−CD16− PBMCs (HIV-1−), 16 hrs IL-15 with ruxolitinib (JAK1/2i, n=5), CP-690550 (JAK3i, n=11), rapamycin (RAPA, n=7), or no IL-15 (n=11). Data are mean ± s.e.m., a, e, f, h, i, k, m two-tailed unpaired t-test; n, two-tailed paired t-test. ns, not significant, *p<0.05, **p<0.01, ***p<0.001. g, j, l, three independent blocks for each donor with similar results.
Fig. 2 |. HIV-1 infection increases the…
Fig. 2 |. HIV-1 infection increases the proportion of CD94+ NK cells.
a, Lin−TBX21+ NK cells from HIV-1−, HIV-1+ viremic, HIV-1+ ART suppressed, and HIV-1+ spontaneous controllers (cohort description in Supplementary Table 1). b, Percent NK cells as in a (n=20 for each group). c, CD94 on NK cells (Lin−CD56+) in PBMCs from HIV-1−, HIV-1+ viremic, HIV-1+ ART suppressed, and HIV-1+ spontaneous controllers (Supplementary Table 1). d, As in c, percent of CD94+NK cells among NK cells from HIV-1− (n=37), HIV-1+ viremic (n=38), HIV-1+ on ART (n=38), and HIV-1+ spontaneous controllers (n=38). e, Percent CD94+ cells after 16 hrs PMA/ionomycin stimulation of Lin−CD56+CD94−NK cells sorted from HIV-1− PBMCs. f, As in e, percent CD94+NK cells after 16 hrs stimulation with PMA/ionomycin or IL-12 and IL-15 (n=6). g, Heatmap of differentially expressed genes by RNA-Seq, sorted Lin−CD56+CD94− versus Lin−CD56+CD94+NK cells, from PBMCs of two HIV-1− donors (log2 fold change >1, p<0.05 determined by DESeq2). h, Percent indicated proteins encoded by differentially expressed genes in Lin−CD56+CD94− and Lin−CD56+CD94+ NK cells from HIV-1− PBMCs, as in g, GZMK (n=21), TCF7 (n=15), CXCR3 (n=27), CD44 (n=26), CD2 (n=11), SELL (n=27), KIR2DL1 (n=18). Data are mean ± s.e.m. b, d, two-tailed unpaired t-test; f, h, two-tailed paired t-test. ns, not significant, *p<0.05, **p<0.01, ***p<0.001.
Fig. 3 |. TCF7 expression correlates with…
Fig. 3 |. TCF7 expression correlates with pseudotime trajectory from CD94−NK cells to CD94+NK Cells.
a, Two-dimensional tSNE plot of single cell RNA-Seq of 986 Lin−CD56+CD94−NK cells (pink) and 767 Lin−CD56+CD94+NK cells (aqua), sorted from HIV-1− blood. Data are representative of 2 donors. b, Spectral clustering of single cell transcriptomes from all cells in a, independent of CD94, k-nearest neighbor, search=2. c, Heatmap of 1,729 CD94−NK cells (blue) and 1,548 CD94+NK cells (yellow), sorted from 2 HIV-1− anonymous blood donors, using all differentially expressed genes from the spectral cluster analysis. d, Minimum spanning tree based on the transcriptome of individual cells from a, showing pseudotime trajectory (black line). e, TCF7 expression along the pseudotime trajectory. f, Expression and density of the indicated genes within t-SNE plots. g, Flow cytometry for CD56 and TCF7 on Lin− PBMCs. h, Sorted CD94−NK cells were untreated (No Stim) or treated with IL-15 (5 ng/ml) for 5 days, and IL-12 (50 ng/ml) and IL-15 (50 ng/ml) for 16 hrs (Stimulated). TCF7, CD44, CXCR3, and CXCR6 were detected by flow cytometry. TCF7, representative of 8 donors; CD44, CXCR3, and CXCR6, representative of 4 HIV-1− donors. All data were generated using blood from HIV-1− donors.
Fig. 4 |. TCF and WNT signaling…
Fig. 4 |. TCF and WNT signaling pathway enrichment in CD94+CD56hiNK cells.
a, Sorting strategy for CD94−CD56dim, CD94+CD56dim, and CD94+CD56hi NK cell subsets. b, Heatmap of differentially expressed genes by RNA-Seq (fold change of normalized counts, log2 >1, p<0.05 determined by DESeq2) for the indicated NK cell subsets sorted from four HIV-1− blood donors. c-e, Pairwise comparison of the indicated NK cell subsets based on differentially expressed genes. f, PCA based on RNA-Seq data from the indicated NK cell subsets. g, Reactome pathway analysis based on 152 differentially expressed genes in CD94+CD56hi NK cells. GENEONTOLOGY produces p value from Fisher’s exact test, followed by BH false discovery rate (in parentheses). All data were generated using blood from HIV-1− donors.
Fig. 5 |. Distinct chromatin landscape in…
Fig. 5 |. Distinct chromatin landscape in CD94+CD56hi NK cells.
a-c, Heatmap showing differential enrichment (>2 fold change of normalized counts) for H3K4me1 (a) and H3K4me3 (b) by CUT&RUN, or accessible chromatin by ATAC-Seq (c), in sorted CD94−CD56dim, CD94+CD56dim, and CD94+CD56hi NK cell subsets. Data is representative of two HIV-1− blood donors. d,e, H3K4me1, H3K4me3, and ATAC-Seq signal on TCF7 (d) and CD6 (e), in the indicated NK cell subsets. f,g,De novo analysis of transcription factor binding motifs enriched in open chromatin from CD56hi NK cells (f), or CD56dim NK cells (g), using HOMER. h, ATAC-Seq and TCF7 CUT&RUN signal at the indicated loci from the three NK cell subsets. All data were generated using blood from HIV-1− anonymous donors.
Fig. 6 |. Chromatin, transcriptome, and phenotype…
Fig. 6 |. Chromatin, transcriptome, and phenotype define CD94+CD56hi NK cells as memory cells.
a, CUT&RUN and ATAC-Seq showing memory gene-associated loci in sorted CD94−CD56dim, CD94+CD56dim, and CD94+CD56hi NK cells. b,c, Gene set enrichment analysis comparing memory CD8+ T cell expression signature (GSE9650) with sorted CD94+CD56hi and CD94−CD56dim NK cells (b), or with sorted CD94+CD56hi and CD94+CD56dim NK cells (c). p value and FDR determined using GSEAPreranked module from GenePattern with 1000 permutations. d, Sorted NK cells labelled with CFSE and cultured 5 days in IL-15 (5 ng/ml). CFSE and TCF7 detected by flow cytometry. e, Percent IFN-γ+ cells by flow cytometry after stimulation of sorted NK cell populations with IL-12 and IL-15 (n=4). f, Fold increase of surface CD107a on bead-enriched NK cells after incubation with K562 cells (n=4). g, bead-enriched NK cells incubated with IL-15 (5ng/ml) for 5 days, then with NL4-3-nef-GFP infected CD4+ T cells. Fold increase of surface CD107a on indicated NK populations, as in f (0h, n=5; 0.5h, 1h, 2h, n=3; 3h, n=5). h, IFNG-AS1 expression relative to GAPDH by RT-qPCR for the indicated NK cell populations sorted from 3 donors. Each population contains 2 technical replicates. i, IFNG-AS1 chromatin analysis as in a. Data are mean ± s.e.m. e-g, two-way ANOVA; h, two-tailed unpaired t-test. *p<0.05, **p<0.01, ***p<0.001. All data were generated using blood from HIV-1− anonymous donors.
Fig. 7 |. WNT inhibition blocks cytokine…
Fig. 7 |. WNT inhibition blocks cytokine induced NK memory response.
a, Experimental scheme for generation of memory NK cells ex vivo from sorted CD94−CD56dimNK cells. b, IFNG-AS1 expression relative to GAPDH by RT-qPCR using RNA extracted from sorted CD94−CD56dim NK cells just prior to secondary stimulation, after primary stimulation, and resting in low dose IL-15 (n=4). c,d, Gene set enrichment analysis (GSEA) comparing CD8+ memory T cell expression signature (GSE9650) by RNA-Seq from sorted CD94−CD56dim NK cells using RNA harvested as in b. p value and FDR determined using GSEAPreranked module from GenePattern with 1000 permutations. e, Heatmap of memory related genes by RNA-Seq that were also found in GSEA list (Supplementary Table 9,10). Fold change of normalized counts Log2>0.5, n=2. f, Heatmap of genes by RNA-Seq that distinguish memory from effector or naive cells reported by literature but that were not identified in GSEA list. Fold change of normalized counts Log2>0.5, n=2. g, Percent IFN-γ+ from sorted CD94−CD56dimNK cells after secondary stimulation. Data are mean ± s.e.m.; two-tailed paired t-test; *p<0.05, **p<0.01. All samples used were from HIV-1− anonymous blood donors.
Fig. 8 |. Expansion of TCF7 +…
Fig. 8 |. Expansion of TCF7+NK cells during HIV-1 infection.
a, Percent TCF7+NK cells among Lin−CD56+ PBMCs in HIV-1− (n=60), HIV-1+ viremic (n=46), HIV-1+ on ART (n=50), and HIV-1+ spontaneous controllers (n=40). Cohort characteristics described in Supplementary Table 1,2. b, Percent TCF7+NK cells from Lin−CD56+CD94−NK subsets of HIV-1− (n=61), HIV-1+ viremic (n=49), HIV-1+ on ART (n=57), and HIV-1+ spontaneous controllers (n=40) (Supplementary Table 1,2) c, Correlation of ILCs with CD56hiNK cells. Samples are detected from HIV-1+ viremic individuals, ART suppressed HIV-1+ individuals (ART), and HIV-1+ individuals who spontaneously control viremia without ART (Supplementary Table 1) (n=113). The correlation coefficient (R) was determined by Pearson, p value for the slope being zero was determined by the F-test. d, Percent NKG2C on Lin−CD56+ PBMCs from HIV-1− (n=9) and HIV-1+ (n=10, under ART) individuals (Supplementary Table 2). e, Model for effect of HIV-1 infection on NK cell subsets. Data are mean ± s.e.m.; a, b and d, two-tailed unpaired t-test. ns, not significant, *p<0.01, **p<0.001.

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