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

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.

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Fig. 1: HIV-1 infection decreases ILCs in blood and colon lamina propria.
Fig. 2: HIV-1 infection increases the proportion of CD94+ NK cells.
Fig. 3: TCF7 expression correlates with pseudotime trajectory from CD94 NK cells to CD94+ NK cells.
Fig. 4: TCF and WNT signaling pathway enrichment in CD94+CD56hi NK cells.
Fig. 5: Distinct chromatin landscape in CD94+CD56hi NK cells.
Fig. 6: Chromatin, transcriptome and phenotype define CD94+CD56hi NK cells as memory cells.
Fig. 7: WNT inhibition blocks cytokine-induced NK memory response.
Fig. 8: Expansion of TCF7+ NK cells during HIV-1 infection.

Data availability

The data that support the findings of this study are available within the manuscript and its Supplementary Information, and from the corresponding author upon request. Source data for Figs. 1, 2 and 68 and Extended Data Figs. 13 and 57 are provided with the paper. Bulk and single-cell RNA-seq, CUT&RUN and ATAC-seq datasets can be found under SuperSeries GSE122326 at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE97727 (CD94 and CD94+ NK cell bulk and single-cell RNA-seq (GSE97727); CD94CD56dim, CD94+CD56dim and CD94+CD56hi NK cell RNA-seq (GSE122324); CD94CD56dim NK cells, 1° stim and 5-d culture RNA-seq (GSE122325); CD94CD56dim, CD94+CD56dim and CD94+CD56hi NK cell ATAC-seq (GSE122548); and CD94CD56dim, CD94+CD56dim and CD94+CD56hi NK cell CUT&RUN (GSE122549)).

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Acknowledgements

We thank the study participants who provided blood and colon biopsy samples, as well as their caretakers, J. Daly, S. Cheeseman and M. Wessolossky of the UMMS. C. Mannarino, A. Foley, M. McManus (UMMS) and M. Krone (UCSF) provided Institutional Review Board regulatory assistance, sample preparation and record keeping. K. Luzuriaga (UMMS) supported the patient sample database and repository. A. Ratner, S. Boswell and A. Klein (Harvard Medical School) contributed technical assistance and barcoded hydrogel beads. T. Fazzio and T. Wu provided technical support and protein A-MNase for CUT&RUN. D. Artis, L. Berg, M. Colonna, J. Huh, J. Kang, R. Rutishauser and S. Swain offered invaluable advice. This research was supported by NIH grants U01HG007910 (to M.G. and J.L.), R37AI147868 (to J.L.), R01AI111809 (to J.L.), DP1DA034990 (to J.L.), R21AI119885 (to M.G.), R01DK105837 (to M.G.) and P51OD01192 (to J.D.E. at the Oregon National Primate Research Center), and the Translational Medicine Core of the University of Massachusetts Center for AIDS Research (P30 AI042845). The UCSF-based SCOPE cohort was supported by the UCSF/Gladstone Institute of Virology and Immunology CFAR (P30 AI027763) and the CFAR Network of Integrated Systems (R24 AI067039). Additional support was provided by the Delaney AIDS Research Enterprise (AI096109 and AI127966). Funding for this study was provided in part by the Division of Intramural Research/NIAID/NIH (to J.M.B.). The content of this publication does not necessarily reflect the views or policies of DHHS, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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Authors and Affiliations

Authors

Contributions

Y.W. and J.L. designed the experiments. Y.W. performed the experiments with assistance from C.L.V., K.B.-S., S.J., K.G., A.D., L.L., S.M., K.K., P.V., P.W.H., S.G.D., J.M.B., J.D.E. and P.M. Y.W. and J.L. analyzed the experimental data. Y.W., L.L., K.G., P.V., A.D., A.K., M.G. and J.L. analyzed the expression data. T.G., J.H., M.S. and S.G.D. obtained and provided the clinical samples. Y.W. and J.L. wrote the manuscript, which was revised and approved by all authors.

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Correspondence to Jeremy Luban.

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Extended data

Extended Data Fig. 1 ILC_gating_and_effect_of_HIV-1.

a, Lymphoid, singlet, live, CD45+ PBMCs from HIV-1 individuals were stained with lineage antibodies (see Methods), CD56, and CD16, and the percent ILCs were LinCD56CD16CD127+ cells. b, CD117 and CRTH2 on LinCD56CD16CD127+ cells from HIV-1 PBMCs (n = 11). c, ILCs from HIV-1, HIV-1+ viremic, HIV-1+ on ART, and HIV-1+ spontaneous controllers, as in a (Supplementary Table 1). d, Correlation of ILCs (LinCD56CD16CD127+PBMCs) with CD4+ nadir (Supplementary Table 1). Correlation coefficient (R) by Pearson, zero slope p value determined by F-test (n = 80). e, CD127, CD117, and RORγt in Lin colon lamina propria from HIV-1. f, IL-22 and CD127 on Lin colon lamina propria from HIV-1, 3 hrs PMA/ionomycin. g, Lin colon lamina propria, CD127 versus RORγT from HIV-1 and HIV-1+ (Supplementary Table 2). h, Colon lamina propria CD4+, 3 HIV-1 and 5 HIV-1+ (Supplementary Table 2). i, Percent CD3+CD117 cells in rectosigmoid tissue from 8 HIV-1 and 16 HIV-1+ on ART (Supplementary Table 1). j, Trimmed mean of M-values (DEBrowser), RNA-Seq from sorted LinCD127+ILCs of 4 HIV-1. k, Percent ILCs within LinCD56CD16 PBMCs from HIV-1 donors after incubation with IL-2 or IL-4 for 16 hrs (n = 5). l, IL-22 produced by gut lamina propria ILC3s maintains epithelium integrity (left). Irreversible decrease in ILC3s with HIV-1 infection (right). Data are mean ± s.e.m.; h,i, two tailed unpaired t-test; b,k, two tailed paired t-test. ns, not significant, *p < 0.05, **p < 0.001.

Source data

Extended Data Fig. 2 HIV-1_infection_increases_CD94+NK_cells.

a, Fraction of CD94+NK cells among LinTBX21+ PBMCs after stimulation with PMA and ionomycin (n = 10) or with IL-15 (n = 10), or with IL-12 + IL15 (n = 4). b, Sorting strategy for CD94 and CD94+NK cells. c, Percent CD107a among CD94 and CD94+NK cells after PBMCs were stimulated with PMA/iono (n = 5). d, Percent specific lysis of K562 or Jurkat cells by sorted CD94NK cells and CD94+NK cells (n = 8).e, Percent Ki67 and Annexin V among CD94 or CD94+NK cells after the indicated treatment (n = 4). f, Representative flow cytometry for indicated genes as detected in Fig. 2h. Data are mean ± s.e.m. Each dot represents a unique sample. two-tailed paired t-test, lines connect cells from common donor. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001. All data are from HIV-1 anonymous blood donors.

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Extended Data Fig. 3 Single_cell_analysis_of_CD94-_and_CD94+NK_cells.

a, Heatmap of 1,729 CD94 (blue) and 1,548 CD94+NK cells (yellow) sorted from 2 donors using all differentially expressed genes based on CD94 positivity. b, Plot of predictive strength as a function of the number of clusters in Fig. 3b shows that 2 clusters yield stable and significant groupings, while separation into additional clusters artificially segregates the cells. The predictive strength based on 1753 single cells was calculated using spectral clustering on the ICA components. c, Heatmap from Fig. 3c was reconstructed utilizing the pseudotime ordering of single cells based on the minimum spanning tree. d, Flow cytometry for CD44, CXCR3 and SELL on TCF7 and TCF7+ NK cells. e, Flow cytometry for GZMK after sorted LinCD56+CD94NK cells were treated as in Fig. 3h. f,g, PBMCs were treated with or without IL-15 for 5 days, LinCD56+ cells were gated on CD56 and CD94 (f) and percent CD56hi NK cells (g) (n = 10). Data are mean ± s.e.m. two-tailed paired t-test, *p < 0.001. All data are from HIV-1 anonymous blood donors.

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Extended Data Fig. 4 Distinct_chromatin_landscape_of_CD94+CD56hiNK_cells.

a, PCA based on H3K4me3 CUT&RUN of the indicated NK cell subsets (n = 2). b, Correlation between differentially expressed genes and enriched H3K4me3 regions by CUT&RUN (log2 fold change) p < 0.001. The correlation coefficient (R) was determined by Pearson, p value for the slope being zero was determined by the F-test. c, Differential signals for H3K4me3 CUT&RUN and ATAC-Seq at the indicated loci in the indicated NK cell subsets. d, Overlapping signal for TCF7 CUT&RUN and ATAC-Seq at the indicated loci. Data are from HIV-1 blood donors.

Extended Data Fig. 5 Memory_associated_gene_loci_are_accessible_in_the_CD94+CD56hiNK_cells.

a, H3K4me1 and H3K4me3 CUT&RUN and ATAC-Seq signal on genes associated with memory T and NK cells, except for effector marker KLRG1, on the indicated NK cell subsets. b, IFN-γ production among CD56dim and CD56hiNK cells after stimulation with IL-12 + IL-15 for 16 hr (n = 4). mean ± s.e.m.; two tailed paired t-test, *p < 0.01. c, H3K4me1 and H3K4me3 CUT&RUN and ATAC-Seq signal at loci for IFN-γ signaling related genes. Data are from HIV-1 blood donors.

Source data

Extended Data Fig. 6 Surface_markers_and_WNT-associated_gene_loci_comparing_CD56dim_and_CD56hiNK_cells.

a,b, Detection of CD16 (n = 4), KIR2DL1 (n = 7), KIR2DL2/3 (n = 8), KIR3DL1 (n = 8) and CD57 (n = 8) in CD56dim and CD56hiNK cells from PBMCs, mean ± s.e.m.; two tailed paired t-test, *p < 0.001. c, Sorted, CFSE labelled CD94+CD56hiNK cells were cultured in IL-12 (10 ng/ml) and IL-15 (10 ng/ml) for 5 days. Proliferation, CXCR6, and CD57 were detected as indicated. CD56dim and CD56hi NK cells from fresh, unstimulated PBMCs were used as control. d, H3K4me3 CUT&RUN and ATAC-Seq signals for gene loci of WNT signaling components and WNT target genes in the indicated NK cell subsets. AXIN1, in contrast, is a WNT inhibitory gene. Data are from HIV-1 blood donors.

Source data

Extended Data Fig. 7 WNT_inhibition_blocks_cytokine_induced_NK_cell_memory.

a, PBMCs were treated with or without LGK974 for 16 hrs. Percentage of LinTBX21+ cells, and the CD94 and CD94+ cells among the LinTBX21+ population, are indicated (left); data are representative of 4 anonymous HIV-1 blood donors. PBMCs were stimulated with IL-12 and IL-15 for 16 hrs in the absence or presence of LGK974. Live cells and LinCD56+ cells were examined (right); data are representative of 10 anonymous HIV-1 blood donors. b, NK cells without primary stimulation as in Fig. 7a were stimulated with IL-12 + IL-15, then IFN-γ production of cells with or without LGK974 was detected (n = 8), samples are from HIV-1 donors. c, Magnetic beads enriched NK cells from HIV-1 donors were transduced with lentivectors expressing GFP and shRNAs targeting either TCF7 or control, the TCF7 level in GFP+ cells was detected by flow cytometry. d, Control or TCF7 knockdown NK cell in c were treated as in Fig. 7a, percent IFN-γ+ among GFP+ cell were detected after secondary stimulation (n = 3). e, Correlation of ILCs with TCF7+NK cells. Samples are from HIV-1+ viremic individuals, ART suppressed HIV-1+ individuals (ART), and HIV-1+ individuals who spontaneously control viremia without ART. Cohort characteristics are described in Supplementary Table 1 (n = 53). The correlation coefficient (R) was determined by Pearson, p value for the slope being zero was determined by the F-test. Data are mean ± s.e.m; b,d, two tailed paired t-test, ns, not significant, *p < 0.01.

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Wang, Y., Lifshitz, L., Gellatly, K. et al. HIV-1-induced cytokines deplete homeostatic innate lymphoid cells and expand TCF7-dependent memory NK cells. Nat Immunol 21, 274–286 (2020). https://doi.org/10.1038/s41590-020-0593-9

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  • DOI: https://doi.org/10.1038/s41590-020-0593-9

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