Abstract
Virtual memory T (TVM) cells are a T cell subtype with a memory phenotype but no prior exposure to foreign antigen. Although TVM cells have antiviral and antibacterial functions, whether these cells can be pathogenic effectors of inflammatory disease is unclear. Here we identified a TVM cell-originated CD44super-high(s-hi)CD49dlo CD8+ T cell subset with features of tissue residency. These cells are transcriptionally, phenotypically and functionally distinct from conventional CD8+ TVM cells and can cause alopecia areata. Mechanistically, CD44s-hiCD49dlo CD8+ T cells could be induced from conventional TVM cells by interleukin (IL)-12, IL-15 and IL-18 stimulation. Pathogenic activity of CD44s-hiCD49dlo CD8+ T cells was mediated by NKG2D-dependent innate-like cytotoxicity, which was further augmented by IL-15 stimulation and triggered disease onset. Collectively, these data suggest an immunological mechanism through which TVM cells can cause chronic inflammatory disease by innate-like cytotoxicity.
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Data availability
RNA-seq data generated in this study have been deposited in the Gene Expression Omnibus under accession code GSE229631. Source data are provided with this paper. All other data that support the findings of this study are present in the article and Supplementary files or are available from the corresponding author (S.-H.P.) upon reasonable request.
Code availability
All the custom codes used in this study are available from the corresponding author (S.-H.P.) upon reasonable request.
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Acknowledgements
This work was supported by the 2020 Joint Research Project of Institutes of Science and Technology and a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT; NRF-2022R1A2C3007292 and 2021R1A4A1032094, to S.-H.P). This work was also supported by the 2022 Basic Research Funds from The Korean Hair Research Society (to J.S.) and by a grant of the MD-Phd/Medical Scientist Training Program through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (to S.-D.C). GFP-expressing retroviral vectors were kindly provided by Y.-M. Kim (KAIST, Daejeon, Korea).
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Study design: J.S., E.-C.S. and S.-H.P.; experiment and data collection: J.S., S.-D.C., J.L., Y.C., S.-Y.K., S.-M.L., S.-H.K., S.J., M.J., H.L., A.R.K. and B.C.; data analysis and interpretation: J.S., S.-D.C., J.L., B.C., S.-J.H., I.J., K.-J.Y., J.-E.P., J.H.K., B.J.K., E.-C.S., and S.-H.P.; writing: J.S., S.-D.C. and S.-H.P. with comments from all authors.
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Nature Immunology thanks Stephen Jameson, Nicole La Gruta, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: N. Bernard, in collaboration with the Nature Immunology team.
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Extended data
Extended Data Fig. 1 CD44s-hiCD49dlo CD8+ T cells are exclusively found in AA SDLNs, and CD44s-hiCD49dlo CD8+ T cells from skin exhibit higher expression of genes relevant for cell motility and inflammatory response compared to CD44s-hiCD49dlo CD8+ T cells from SDLNs.
a, The proportion of CD44highCD49dlo CD8+ T cells in SDLN CD8+ T cells (n = 16, naive; n = 29, AA). b, CD44s-hiCD49dlo CD8+ T cells were nearly absent in the spleens, liver, and skin non-draining mesenteric lymph nodes of alopecic mice (n = 5, liver; n = 4, others). c, Heatmap of differentially expressed genes (DEGs) in CD44s-hiCD49dlo CD8+ T cells from SDLNs relative naive TN cells, naive TVM cells, and AA TVM cells (pairwise comparison). Numbers in overlapping regions indicate gene transcripts shared by the overlapping DEG, whereas numbers in non-overlapping regions indicate unique DEGs to each cell subset. d, e, Heatmap of DEGs from skin CD44s-hiCD49dlo CD8+ T cells compared to SDLN CD44s-hiCD49dlo CD8+ T cells (d) and enriched GO biological process gene sets (padj < 0.05) (e). Data were acquired from AtAA mice. mLN, mesenteric lymph node. Data are presented as mean values ± SD. A Mann-Whitney test was performed for comparisons between two groups. All tests were two sided. *P < 0.05, ****P < 0.0001.
Extended Data Fig. 2 CD44s-hiCD49dlo CD8+ T cells had high enrichment of genes related to T-cell effector functions.
a, Heatmap of the gene set33. b, GSEA confirmed that CD44s-hiCD49dlo CD8+ T cells exhibited significant enrichment of the effector CD8+ T cell gene set33 compared to TVM cells from naive or AA mice and TN cells from naive mice. GSEA was performed for pairwise comparison between two groups. All tests were two sided. Data were acquired from AtAA mice. SDLN, skin draining lymph node; NES, normalized enrichment score.
Extended Data Fig. 3 Single-cell RNA-seq of CD45+ cells from the skin and SDLNs of AA and naïve mice.
a, UMAP visualization of CD45+ cell clusters detected in skin and SDLNs from AA and naive mice. b, UMAP visualization of cellular subsets obtained by ADT-labeling of the integrated CITE-Seq. c, CD44 and CD62L of ADT-labeling in CD8+ T cells. Overlays of CD44s-hiCD49dlo CD8+ T cells, TVM cells, TTM cells, and TN cells on total CD8+ T cells with effector memory T cell and central memory T cell gating (CD44hiCD62Llo and CD44hiCD62Lhi, respectively) in flow cytometric analysis. d, Different CD3+ T-cell clusters and their gene expression levels, with brightness indicating average expression and circle size indicating the percent expression. Data were acquired from AtAA mice.
Extended Data Fig. 4 Conventional memory CD8+ T cells primed by skin infection do not contribute to the induction of CD44s-hiCD49dlo CD8+ T cells.
a, Experimental scheme for testing whether skin infection could contribute to the induction of AA. After 4 weeks of vaccinia virus (VV) infection by skin scarification, VV-infected mice (VV-AT) or naïve mice (Control-AT) were induced AA by adoptive transfer method. b,c, Representative flow cytometry plots (b) and the proportion of true memory (TTM) T cells in CD8+ T cells 7 days post-VV infection (n = 3) (c). d, Disease free ratio between the VV-AT (n = 8), the control-AT (n = 10), and the historical control group (n = 74). e, Representative flow cytometry plots of CD8+ T cells after 12 weeks of adoptive transfer. f, Frequency of each cell populations in CD8+ T cells (n = 7, Control-AT; n = 5, VV-AT). g, gMFI of the NKG2D level in each cell population (n = 7, Control-AT; n = 5, VV-AT). Data are presented as mean values ± SD. A Log-rank (Mantel-Cox) test was used for comparison of survival curves. A Mann-Whitney test was performed for comparisons between two groups. All tests were two sided.
Extended Data Fig. 5 CD44s-hiCD49dlo CD8+ T cells are not generated by antigen-driven expansions.
a, Experimental scheme for evaluating the proportion of CD44s-hiCD49dlo CD8+T cells during the AA induction process in vitro (6 days) and in vivo ( > 6 weeks). b, The expressions of Nr4a1, Nr4a2 and Nr4a3, which are upregulated by TCR signalling, in the SDLNs of adoptively transferred C3H mice, measured by real-time PCR at 4 weeks after AT (before AA induction) and at a time-point post-AA onset (n = 4, naïve control; n = 3, naïve + α-CD3; n = 5 (n = 4 in Nr4a2), W4; n = 7, Post-AA). Data are presented as mean values ± SD.
Extended Data Fig. 6 CD44s-hiCD49dlo CD8+ T cells originate from TVM cells, not TN cells.
a, Representative flow cytometry plots of in vitro TN cell stimulation with various cytokines. b, Flow cytometric analysis of SDLNs from cultured naïve TN and TVM cell-adopted mice (n = 5 mice per group). The proportion of CD44s-hiCD49dlo cells in CD8+ T cells (closed square = AA developed mouse). Data are presented as mean values ± SD.
Extended Data Fig. 7 TVM cells may play a crucial role in human AA pathogenesis.
a, IL-12, IL-15, and IL-18 staining of hair follicles from a healthy volunteer and AA patient (n = 3 independent samples). Scale bars, 100μm. b, UMAP visualization of CD45+ cell clusters detected in skin from AA patients and healthy volunteers. c, Different CD3+ T-cell clusters and their gene expression levels, with brightness indicating average expression and circle size indicating the percent expression. d, CD8, pan-KIR2DL + KIR2DS, and NKG2A staining of hair follicles from a healthy volunteer and AA patient (n = 3 independent samples). Scale bars, 50μm. e, f, PBMC CD8+ T cells from healthy doners (n = 10) and patients with AA (n = 10) were analyzed by flow cytometry. The percentage of CD45RA+KIR+NKG2A+ cells among CD8+ T cells (e) and expression level of NKG2D in TVM cells (f). Data are presented as mean values ± SD. Between-group comparisons were made using the Mann-Whitney test. All tests were two sided.
Extended Data Fig. 8 CD44s-hiCD49dlo CD8+ T cells from skin exhibited residential features.
a, Heatmap of the gene expression according to the TRM gene set24. b, GSEA confirmed that skin CD44s-hiCD49dlo CD8+ T cells exhibited significant enrichment of the TRM gene set24 compared to CD44s-hiCD49dlo CD8+ T cells and TVM cells from SDLNs. GSEA was performed for pairwise comparison between two groups. All tests were two sided. Data were acquired from AtAA mice. NES, normalized enrichment score.
Extended Data Fig. 9 CD44s-hiCD49dlo CD8+ T cells exhibit activation in a TCR-independent manner and enhanced proliferation capacity compared to other cell populations.
a-c, Following cytokine stimulation, various cells from SDLNs were incubated for 48 h and flow cytometric analysis performed (50 ng/ml IL-2, 50 ng/ml IL-7, 50 ng/ml IL-12, 50 ng/ml IL-15, or 50 ng/ml IL-18). a, The percentage of GzmB+ and perforin+ cells in AA and naïve TVM cells, respectively (n = 16, AA TVM; n = 17, naïve TVM). b, The percentage of IFNγ+ cells (n = 11, AA TVM; n = 8, naïve TVM) and TNF+ cells in AA TVM cells and naive TVM cells (n = 11, AA TVM; n = 12, naïve TVM). c, Comparison of the percentage of GzmB+ cells, perforin+ cells, IFNγ+ cells, and TNF+ cells among each cell population in the presence of IL-15 (n = 12, naïve; n = 11, AA) or IL-12/18 (n = 8, naïve TVM IFNγ+; n = 12, naïve TVM TNF+; n = 11, AA). d, e, Following cytokine stimulation, various cells from SDLNs were incubated for 96 h. Representative flow cytometry plot of CTVlow cells (d) and the percentage of CTVlow cells in AA TVM cells (n = 7) and naïve TVM cells (n = 4) (e). f, gMFI of NKG2D in each cell population (n = 4, AA TVM; n = 4, naïve TVM). g, Comparison of the gMFI of NKG2D among each cell population without stimulation or in the presence of IL-15 (n = 4 per group). Data were acquired from AtAA mice. Data are presented as mean values ± SD. A Mann-Whitney test was performed for comparisons between two groups. All tests were two sided. ****P < 0.0001.
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Supplementary Source Data 1
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Supplementary Source Data 2
Source data for TCR-seq.
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Seok, J., Cho, SD., Lee, J. et al. A virtual memory CD8+ T cell-originated subset causes alopecia areata through innate-like cytotoxicity. Nat Immunol 24, 1308–1317 (2023). https://doi.org/10.1038/s41590-023-01547-5
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DOI: https://doi.org/10.1038/s41590-023-01547-5
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