Tissue-resident memory T cells (TRM cells) provide rapid and superior control of localized infections. While the transcription factor Runx3 is a critical regulator of CD8+ T cell tissue residency, its expression is repressed in CD4+ T cells. Here, we show that, as a direct consequence of this Runx3-deficiency, CD4+ TRM cells lacked the transforming growth factor (TGF)-β-responsive transcriptional network that underpins the tissue residency of epithelial CD8+ TRM cells. While CD4+ TRM cell formation required Runx1, this, along with the modest expression of Runx3 in CD4+ TRM cells, was insufficient to engage the TGF-β-driven residency program. Ectopic expression of Runx3 in CD4+ T cells incited this TGF-β-transcriptional network to promote prolonged survival, decreased tissue egress, a microanatomical redistribution towards epithelial layers and enhanced effector functionality. Thus, our results reveal distinct programming of tissue residency in CD8+ and CD4+ TRM cell subsets that is attributable to divergent Runx3 activity.
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The code generated and used for the analysis of sequencing data are available from the corresponding author on reasonable request.
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We thank the Flow Cytometry Unit and Bioresources Facility at Peter Doherty Institute (University of Melbourne) for technical assistance. This work was supported by a Howard Hughes Medical Institute and Bill and Melinda Gates International Research Scholarship OPP1175796, National Health and Medical Research Council (NHMRC) AP1113293 to F.RC. and L.K.M. S.L.P. was supported by a NHMRC EL1 Investigator Grant GNT1175626. N.G.Z. was supported by FAPESP (BEPE 2019/12431-2). A.T.S. was supported by the National Institutes of Health (U01CA260852 and UM1HG012076), the Parker Institute for Cancer Immunotherapy and a Pew-Stewart Scholars for Cancer Research Award. L.K.M is a Senior Medical Research Fellow supported by the Sylvia and Charles Viertel Charitable Foundation.
The authors declare no competing interests. A.T.S. is a founder of Immunai and Cartography Biosciences and receives unrelated research funding from Merck Research Laboratories, Allogene Therapeutics, and Arsenal Biosciences, L.K.M. receives unrelated research funding from Pfizer.
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(a-d) Microscopy showing CD4+ SMARTA and CD8+ P14 cells in the small intestine (SI) 7 and 30 d.p.i. with LCMV (IEL cells highlighted by teal (CD4+) and yellow (CD8+) circles) (a), kinetics of CD4+ SMARTA and CD8+ P14 cells (**P=0.0079, 0.0043, 0.0079 respectively, two-tailed Mann-Whitney’s test) (b), CD69 and CD103 expression in SI-IEL at 7, 14 or 30 d.p.i. (***P=0.007, <0.0001 respectively, multiple paired t-tests) (c) and representative flow plots 30 d.p.i. (d). (e-i) CD69 and CD103 expression in CD4+ gDT-II and CD8+ gBT-I cells from skin at 7, 14 or 30d post-HSV infection (***P=0.0028, <0.0001 respectively, multiple paired t-tests) (e) and representative flow plots in skin 30 d.p.i. (f), representative histograms of Runx3 expression in spleen or skin 30 d.p.i. (g), representative histograms of ThPOK and Runx3 expression (h) and Runx3 expression (gMFI) in spleen 7 d.p.i. normalized to naïve (i). (j) Correlation plots of Runx1 or Runx3 and CD103 gMFI on endogenous Foxp3−CD69+CD4+ and CD69+CD8+ isolated from skin 14 d.p.i. with HSV (linear regression line with 95% confidence interval, P showing slope is non-zero). (k, l) Runx1 and Runx3 expression in CD8+ gBT-I cells (k) and CD4+ gDT-II cells (l) in vitro prior to transfer into recipient mice following CRISPR-Cas9 mediated editing of CD19 (Ctrl), Runx1 (sgRunx1), or Runx3 (sgRunx3). (m) Log2-fold change of sgCtrl and sgRunx1 CD4+ SMARTA cells (as in k, **P=0.002, paired t-test) in the spleen and SI-IEL >20d post-LCMV infection. Data are representative of 2 independent experiments with (a) n=3, (b-i) n=5, (j) 1 experiment, n=5, (k) 2 independent biological replicates, (m) pooled from 2 independent experiments, with n=5 mice. Symbols represent (c, e, j, m) mice, (b, i) mean; error bars indicate SEM. Box plots represent the median, interquartile range, and minimum/maximum whiskers.
(a, b) Schematic and frequency of CD8+ P14 cells transduced with control (CD8-Ctrl) or Runx3 (CD8-Runx3) retroviruses in the spleen, skin and SI-IEL 14d post-LCMV infection and DNFB skin treatment (a) and enumeration of total or CD69+CD103+ cells in the spleen, skin (**P=0.0022, two-tailed paired t-test) and SI-IEL (***P=0.0002 and 0.0007 respectively) (b). Data pooled from (a, b) 2 independent experiments, n=4 mice. Symbols represent individual mice; bars represent mean; error bars indicate SEM.
(a-c) Schematic and frequency of CD4+ gDT-II cells transduced with control (CD4-Ctrl) or Runx3 (CD4-Runx3) retroviruses in the spleen and skin (a), epidermis/dermis proportion (**P=0.0048, two-tailed paired t-test) (b) and CD69 and CD103 expression in the epidermis (**P=0.0021, two-tailed paired t-test) and dermis (***P=0.0001, two-tailed paired t-test) (c) 14d post-HSV infection. (d-f) Schematic (d), frequency of CD4+ SMARTA cells transduced with control (CD4-Ctrl) or Runx3 (CD4-Runx3) retroviruses (e) and number of total and CD69+CD103+ cells in spleen, skin (*P=0.043, two-tailed paired t-test) and SI-IEL (*P=0.0432 and 0.0332) (f) 14d post-LCMV infection in mice treated on the skin with DNFB. (g, h) Representative histograms of Runx3 (g) and expression (MFI) by endogenous CD4+, CD8+ and CD4+CD8αα+ T cells (h) in the spleen and SI-IEL >90d post-LCMV infection. (i) Runx3 expression by endogenous CD8+ T cells (CD8-Endog) and transduced Ctrl- (CD4-Ctrl) or Runx3-transduced (CD4-Runx3) CD4+ gDT-II (spleen and skin) or CD4+ SMARTA (SI-IEL) cells as in (a-f) 14 d.p.i. with HSV or LCMV respectively. Data pooled from (a-c) 2 independent experiments with n=10 (d-f) 2 independent experiments, n=5 or representative of (g, h) 2 independent experiments, n=5 and (i) 2 independent experiments, n=3 mice. Symbols represent individual mice; bars indicate mean; error bars indicate SEM.
(a) Heatmap showing expression of the top TRM cell DEGs (derived from the comparison between skin CD8+ TRM cells vs spleen CD8+ TCIRC cells from GSE70813) in CD4-Runx3 and CD8-Ctrl samples relative to CD4-Ctrl cells, by CD8+ gBT-I cells transduced with control (CD8-Ctrl) and CD4+ gDT-II cells transduced with control (CD4-Ctrl) or Runx3-expressing (CD4-Runx3) retroviruses isolated from the skin 14 d.p.i. (columns represents independent samples, color scale is based on Z-score). (b) GSEA in CD8-Ctrl vs CD4-Ctrl (top) or CD4-Runx3 vs CD8-Ctrl cells (bottom) for the top 100 DEGs in skin CD8+ TRM vs splenic CD8+ TCIRC cells. (c) Scatter plot showing DEGs between skin CD8+ TRM vs splenic CD8+ TCIRC cells and CD8-Ctrl vs CD4-Ctrl or CD8-Ctrl vs CD4-Runx3 (dots represent up- (red) or down-regulated (blue) genes in skin CD8+ TRM vs splenic CD8+ TCIRC cells, quadrants represent up- (red) or down-regulated (blue) genes in CD8-Ctrl vs CD4-Ctrl or CD8-Ctrl vs CD4-Runx3 comparisons). (d) Scatter plots of standardized log fold-change of CD8-Ctrl vs CD4-Ctrl (x-axis) and CD4-Runx3 vs CD4-Ctrl (y-axis; orange dots highlight top skin TRM cell genes (left panel), blue and red dots highlight top 200 down- (middle panel) and up-regulated (right panel) genes in skin TRM cells) (d). Data pooled from 2 independent experiments, n=2 biological replicates pooled from 10 mice. Green line represents least-squares regression line fitted to orange points.
(a) Scatter plots showing peak accessibility changes in CD8-Ctrl cells treated with TGF-β vs Ctrl and CD4-Ctrl (top) or CD4-Runx3 (bottom) treated with TGF-β vs Ctrl. (b) Scatter plots showing peak accessibility changes in CD8-Runx3 vs CD8-Ctrl cells and CD4-Runx3 vs CD4-Ctrl treated with TGF-β (bottom) or untreated (top; orange dots highlight TGF-β regulated genes). (c) Itga1, Cd244, S1pr1 and Ly6c genome tracks (height normalized) in CD4+ gDT-II and CD8+ gBT-I cells transduced with control (CD4-Ctrl and CD8-Ctrl) or Runx3-expressing (CD4-Runx3 and CD8-Runx3) retroviruses cultured ± TGF-β for 48 hours. Data representative of 2 independent experiments, with n=2 technical replicates.
(a) Heatmap showing the top TGF-β regulated genes (derived from the comparison between skin wild-type (WT) CD8+ T cells vs Tgfbr2−/− CD8+ T cells 14d post-HSV infection from GSE178769) in CD4-Runx3 and CD8-Ctrl samples relative to CD4-Ctrl expression, by CD8+ gBT-I cells transduced with control (CD8-Ctrl) and CD4+ gDT-II cells transduced with either control (CD4-Ctrl) or Runx3-expressing (CD4-Runx3) retroviruses isolated from the skin 14d post-HSV infection (columns represents independent samples, color scale is based on Z-score). (b) GSEA in CD8-Ctrl vs CD4-Ctrl (top) or CD4-Runx3 vs CD8-Ctrl cells (bottom) for the top 100 DEGs in skin WT vs Tgfbr2−/− cells. (c) Scatter plot showing DEGs between WT vs Tgfbr2−/− cells and CD8-Ctrl vs CD4-Ctrl (left) or CD8-Ctrl vs CD4-Runx3 (right, dots represent up- (red) and down-regulated (blue) genes in WT vs Tgfbr2−/− cells comparison, quadrants represents up- (red) and down-regulated (blue) genes in CD8-Ctrl vs CD4-Ctrl or CD8-Ctrl vs CD4-Runx3 comparisons). (d) Scatter plots of standardized log-fold changes of CD8-Ctrl vs CD4-Ctrl (x-axis) and CD4-Runx3 vs CD4-Ctrl (y-axis) for all genes (orange dots highlight top TGF-β regulated genes (left panel), blue and red dots highlight top 200 down-regulated (middle panel) and up-regulated (right panel) genes in WT cells). Data pooled from 2 independent experiments, n=2 biological replicates pooled from 10 mice. Green line represents least-squares regression line fitted to orange dots.
(a) Intracellular cytokine production of IFNγ and TNFα by CD4+ gDT-II cells transduced with either control (CD4-Ctrl) or Runx3-expressing (CD4-Runx3) retroviruses 4 hours after PMA+ionomycin stimulation. (b, c) Viral titer (*P=0.0111, two-tailed unpaired t-test) 6d post-HSV infection (b), and enumeration of transduced cells, inflammatory monocytes, NK cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, B cells and dendritic cells 3 d.p.i (*P=0.0455, 0.0301 and 0.021 respectively, two-tailed unpaired t-test) (c) in the skin of C57BL/6 mice transferred i.d. with CD4+ gDT-II cells transduced with control (CD4-Ctrl) or Runx3-expressing (CD4-Runx3) retroviruses 14d prior to HSV infection. Data pooled from 2 independent experiments with (a) n=2 biological replicates, (b, c) n=5 mice. Symbols represent (a) biological replicates or (b, c) mice. Bars represent mean; error bars indicate SEM. Box plots show the median, interquartile range and minimum/maximum whiskers.
Supplementary methods, references and gating strategy.
CD4+ T cells from uGFP-reporter mice were transduced with an empty-Ametrine-encoding retrovirus (CD4-Ctrl) and CD4+ T cells from ubiTomato-reporter mice were transduced with a Runx3-GFP-encoding retrovirus (CD4-Runx3). Transduced cells were cotransferred i.d. into recipient mice and intravital two-photon microscopy performed at day 14 post-i.d. transfer. SHG..
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Fonseca, R., Burn, T.N., Gandolfo, L.C. et al. Runx3 drives a CD8+ T cell tissue residency program that is absent in CD4+ T cells. Nat Immunol 23, 1236–1245 (2022). https://doi.org/10.1038/s41590-022-01273-4