Abstract
Local cytokines, including TGFβ, drive CD8+ tissue-resident memory T (TRM) cell differentiation and long-term persistence within tissues. However, the signals that prevent CD8+ TRM cell formation are not well defined. Here we found that IL-4 suppressed CD8+ T cell acquisition of an epithelial TRM cell phenotype. IL-4 inhibited the expression of TGFβ-induced CD103 and CD49a and increased the expression of Eomes by activated CD8+ T cells in vitro and in vivo. This change in phenotype was correlated with prolonged downregulation of TGFβRII, decreased expression of pSmad2/3 and increased expression of inhibitory Smad7. Naive CD8+ T cells exposed to IL-4 during activation exhibited impaired cutaneous CD103+CD8+ TRM cell formation. Additionally, IL-4 produced within atopic dermatitis lesions decreased the expression of CD103 in infiltrating CD8+ T cells and reduced CD8+ TRM cell formation, resulting in reduced protection from cutaneous herpes simplex virus infection. Together, these findings reveal that IL-4 decreases the responsiveness of CD8+ T cells to TGFβ, resulting in impaired formation of CD8+ TRM cells and impaired CD8+ TRM cell-mediated protection from local infection.
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Acknowledgements
We thank M. Conroy, S. Islam, A. Luster and C. Sokol for helpful discussions. We thank F. Tabatabaei for critical reading of the manuscript. We thank K. Fairfax (University of Utah) for providing KN2 mice, S. Islam (MGH) for providing Ilfrafl/fl mice with permission from F. Brombacher (University of Cape Town) and F. Finkelman (University of Cincinnati) for Il4ra−/− mice. This work was supported by NIH grant no. R01 AI163517 (to S.K.B.) and National Eczema Association Catalyst Research Grant no. NEA19-CRG121 (to R.M.-B.).
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R.M.-B., M.E.L., A.S., A.Y.C., H.A., V.M. and S.K.B. performed experiments. R.M.-B., M.E.L., A.S., H.A. and S.K.B. analyzed and interpreted data. R.M.-B. and S.K.B. designed experiments and wrote the manuscript.
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Extended data
Extended Data Fig. 1 IL-4 impairs TGF-β-induced CD49a expression by CD8+ T cells in vitro.
(a) Relative CD49a expression in CD8+ T cells that were purified from cultures of day 4.5 in vitro-activated (1 μg/mL CD3 and CD28 antibodies) C57Bl/6 spleen and LN cells, and then stimulated with TGF-β with or without supernatant from in vitro-activated, TH2 polarized CD4+ T cells (Spn) at dilutions ranging from 1:10-1:400 and with 0, 0.1, 1, 10, or 100 μg/mL of IL-4 antibody for 3 days. Relative expression CD49a was determined by flow cytometric analysis and is calculated as [(% CD49a expression in CD8+ T cells) / (% CD49a expression of cells cultured in TGF-β (10 ng/mL) alone)]/100. *p = 0.0293; **p = 0.0109; ***p = 0.0024; ****p = 0.001; *****p = 0.0004 vs TGF-β stimulation. #p = 0.0116; ##p = 0.0110; ###p = 0.004 vs TH2 spn 1:10 dilution, two-tailed paired t-test. Data from three independent experiments are shown. Mean + standard error (SEM). (b) Percent of CD49a expression in CD45.1+CD44hiCD8+ OT-I T cells isolated from the spleen and skin draining lymph node (dLN) of CD45.1+CD45.2+ wild-type (WT) C57Bl/6 mice that were injected intravenously (i.v.) with naïve CD45.1+CD44−CD8+ OT-I T cells (TN cells) and infected with herpes simplex virus-OVA (HSV-OVA) by skin scarification at day 1 post-transfer, and cultured ex vivo for 3 days with 10 ng/mL of TGF-β, IL-4, TGF-β + IL-4, or no cytokine. CD49a expression was determined by flow cytometry. Data are pooled from six independent experiments. *p = 0.0003; **p = 0.0001, two-tailed paired t-test. Mean ± SEM. (c) Percent CD49a+ T cells of WT, Il4ra−/−, and Stat6−/− CD8+ T cells activated in vitro as in a and treated with TGF-β (10 ng/mL) with or without IL-4 (10 ng/mL) for three days (left), and percent change in CD49a expression calculated as [(%CD49a expression in TGF-β + IL-4 cultures -%CD49a expression in TGF-β cultures) / ([(%CD49a expression in TGF-β cultures)]×100). Data are pooled from five or six independent experiments per genotype: WT (n = 5), Il4ra−/− (n = 5) and Stat6−/− (n = 6). *p = 0.0298; **p = 0.0007, Right, two-tailed one sample t-test. Mean ± SEM. *p < 0.0001, Left, 2way ANOVA with Šídák’s correction for multiple comparisons. Mean ± SEM. (d) Flow cytometric analysis determined mean percent CD49a+ cells of WT (n = 6), Il4ra−/− (n = 5), and Stat6−/−(n = 6) CD44hiCD8+ T cells that were purified from cultures of day 4.5 in vitro-activated CD8+ T cells as in a and cultured in the presence or absence of TGF-β (10 ng/mL) for three days. Data are pooled from 4-6 independent experiments: WT (n = 6), Il4ra−/− (n = 4), and Stat6−/− (n-6). *p = 0.00428; **p = 0.0415; ***p = 0.0389; ****p = 0.0121, two-tailed t-test. Mean ± SEM. (e) RT-qPCR determined expression of Itga1 (which encodes CD49a) in WT, Il4ra−/− or Stat6−/− CD8+ T cells activated as in a and cultured with TGF-β (10 ng/mL) with or without IL-4 (10 ng/mL) for 3 days. Values are expressed as copies of Itga1 relative to B2m. Data are pooled from four or five independent experiments; WT (n = 5), Il4ra−/− (n = 4) or Stat6−/− (n = 5). *p < =0.0082, paired t-test. Mean ± SEM.
Extended Data Fig. 2 CD8+ T cell-specific-deletion of Il4ra does not change the frequencies of CD4+ or CD8+ T cells in lymphoid tissues.
(a) Representative flow cytometry of CD4 and CD8 staining in CD3+ cells isolated from thymus, brachial lymph node (bLN), and spleen of Il4rafl/− and CD8creIl4rafl/− mice. (b) Percent CD4+ and CD8+ cells of CD3+ T cells recovered from the thymus, bLN, and spleen as in a. Representative (a) or pooled (b) data for CD4+ T cells from thymus and spleen Il4rafl/− (n = 7), CD8Cre+Il4rafl/− (n = 5); CD4+ T cells from bLN Il4rafl/− (n = 6), CD8Cre+Il4rafl/− (n = 5). Pooled data for CD8+ T cells from thymus Il4rafl/− (n = 7), CD8Cre+Il4rafl/− (n = 5); CD8+ T cells from bLN and spleen Il4rafl/− (n = 9), CD8Cre+Il4rafl/− (n = 8). Median with IQR.
Extended Data Fig. 3 DNFB model of non-specific skin inflammation and regulation of CD103+CD8+ TRM cell conditioning by IL-4.
(a) Number of transferred CD45.1+CD44hiCD8+ OT-I T cells recovered from the flank skin of congenic wild-type (WT) CD45.1+CD45.2+ C57Bl/6 recipient mice that were injected intravenously (i.v.) with in vitro-activated (1 μg/mL CD3 and CD28 antibodies) WT CD45.1+CD44hiCD8+ OT-I T cells and immediately painted on the flank skin with DNFB. The cells were isolated at 3, 5, 8, 11 or 14 days post-DNFB treatment. Pooled data from 4 independent experiments; day 3 (n = 4), day 5 (n = 7), day 8 (n = 6), day 11 (n = 7) and day 14 (n = 5) mice. Mean ± SEM. (b) Mean percent CD103+ cells of CD45.1+CD8+CD44hi OT-I T cells isolated from the flank skin as in a. Pooled data from 4 independent experiments; day 3 (n = 4), day 5 (n = 7), day 8 (n = 6), day 11 (n = 7) and day 14 (n = 5) mice. Mean ± SEM. (c) Representative contour plots of CD103 expression in CD45.1+CD44hiCD8+ OT-I T cells isolated from the skin as in a. (d) Representative contour plots of CD103 expression in WT CD45.1+CD44hiCD8+ OT-I and Il4−/− CD45.2+CD44hiCD8+ OT-I T cells isolated from the flank skin of congenic WT CD45.1+CD45.2+ C57Bl/6 recipient mice that were injected i.v. with a mix of in vitro-activated WT CD45.1+CD44hiCD8+ OT-I and Il4−/− CD45.2+CD44hiCD8+ OT-I T cells and painted with DNFB as in a, at day 5 and day 30 post-transfer. (e) CD103 gMFI in (top) and mean percent (bottom) CD103+ cells of WT CD45.1+CD44hiCD8+ OT-I and Il4−/− CD45.2+CD44hiCD8+ OT-I T cells isolated from the flank skin as in d. (f) Representative contour plots of WT CD45.1+CD44hiCD8+ OT-I (black gates) and Il4−/− CD45.2+CD44hiCD8+ OT-I (orange gates) T cells isolated from the flank skin and spleen as in d. (g) Ratios of WT compared to Il4−/− CD44hiCD8+ OT-I T cells isolated from the bLN, spleen and skin as in d. Median with IQR. (h) Numbers of transferred WT and Il4−/− CD44hiCD8+ OT-I T cells recovered from the bLN, spleen and skin as in d. *p = 0.0371; **p = 0.0039; ***p = 0.002, two-tailed Wilcoxon test. Representative (d, f) or pooled (e, g, h) data. Day 5, one experiment (n = 3); day 30, three independent experiments (n = 10).
Extended Data Fig. 4 IL-4 present during CD8+ T cell activation impairs CD103 expression and CD8+ TRM cell formation/persistence in vivo.
(a) Numbers of CD45.1+CD44hiCD8+ and CD45.2+CD44hiCD8+ T cells that were in vitro-activated with IL-4 (IL4Act OT-I) or without (noIL4Act OT-I), adoptively co-transferred intravenously into congenic CD45.1+CD45.2+ C57Bl/6 mice that were immediately painted on the flank with DNFB, and recovered from the spleen, bLN and flank skin at day 5 post-transfer. *p = 0.0234, **p = 0.0039, two-sided Wilcoxon test. (b) Numbers of IL4Act OT-I and noIL4Act OT-I CD8+ T cells recovered from spleen, bLN and flank skin as in a at day 30 post-transfer. *p = 0.0039, two-sided Wilcoxon test. (a, b) Data are pooled from three independent experiments on day 5 (n = 9) and on day 30 (n = 9). (c) Numbers of IL4Act OT-I and noIL4Act OT-I CD8+ T cells T cells that were adoptively co-transferred intradermally into congenic CD45.1+CD45.2+ C57Bl/6 and recovered from the bLN and flank skin at day 2 post-transfer. (d) Numbers of IL4Act OT-I and noIL4Act OT-I CD8+ T cells recovered from spleen, bLN and flank skin as in c at day 5 post-transfer. (e) Numbers of IL4Act OT-I and noIL4Act OT-I CD8+ T cells recovered from spleen, bLN and flank skin as in c at day 30 post-transfer. *p = 0.0391; **p = 0.0156, two-tailed Wilcoxon test. (f) Percent CD49a+, CD69+ and CD127+ cells of noIL4Act or Il4Act OT-I CD8+ T cells isolated from the flank skin as in c on day 30 post-transfer. (g) gMFI of CD49a, CD69 and CD127 expression in noIL4Act and Il4Act OT-I CD8+ T cells isolated from the flank skin as in c on day 30 post-transfer. (c-f) Data are pooled from three independent experiments on day 2 (n = 6), day 5 (n = 7), and day 30 (n = 8).
Extended Data Fig. 5 In vivo CD8+ T cell activation following OVA plasmid tattoo and MC903-induced IL-4 expression.
(a) Numbers of CD45.1+CD44hiCD8+ OT-I T cells isolated from the bLN and spleen of recipient congenic CD45.1+CD45.2+ C57Bl/6 mice that were injected intravenously with naive CD44−CD8+ OT-I T cells (TN cells) and immediately tattoo-immunized on the flank skin with OVAp. The cells were isolated at day 5, 8, 14, and 30 post-transfer. Data are pooled from two experiments on day 5 (n = 6), day 8 (n = 7), and day 14 (n = 6), or data are from one experiment on day 30 (n = 7), Median with IQR (b) Representative contour plots of CD44 expression by CD8+OT-I T cells recovered from the bLN and spleen on day 5 as in a. (c) Numbers of CD45.1+CD44hiCD8+ OT-I T cells recovered from the flank skin as in a. Median with IQR. (d) CD103 gMFI in CD45.1+CD44hiCD8+ OT-I T cells recovered from the flank skin as in a. Median with IQR. (d, e) Data are from pooled from two experiments on day 5 (n = 6), day 8 (n = 7), and day 14 (n = 6), or data are from one experiment on day 30 (n = 7). (e) Representative images of the flank skin of C57Bl/6 mice treated daily with Ethanol (EtOH) or MC903 from day 0–9. Images were taken on day 10. Data are representative of one experiment (n = 4 mice per group). (f) Skin thickness (mm) of C57Bl/6 mice treated as in e and measured on day 10. Data are from of one experiment (n = 4 mice per group). *p = 0.0286, two-tailed Mann-Whitney test. Median with IQR. (g) RT-qPCR measurement of Il4 mRNA expression on day 10 in the skin of C57Bl/6 mice treated as in e. Values are expressed as copies of Il4 relative to B2m. Data are from of one experiment (n = 4 mice per group). * p = 0.0286, two-tailed Mann-Whitney test. Median with IQR. (h) Representative contour plots of IL4-GFP+ cells isolated on day 10 from the bLN and skin of 4Get mice treated as in e. Gated on CD45+ cells. (i) Numbers of IL4-GFP+ cells of CD45+ cells isolated on day 10 from the bLN and skin of 4Get mice treated as in e. *p = 0.0286, two-tailed Mann-Whitney test. Median with IQR. Data are from of one experiment (n = 4 mice per group). (j) Representative pseudocolor plots of human CD2 (hCD2) expression by cells recovered on day 10 from the bLN and skin of C57Bl/6 and KN2 mice painted from day 0–9 with MC903. Gated on CD45+B220−CD3+ cells. (k) Numbers of hCD2+CD4+T cells isolated from the bLN and skin of C57Bl/6 and KN2 mice treated as in j. *p = 0.0095, two-tailed Mann-Whitney test. Median with IQR. Data are pooled from two experiments with C57Bl6 (n = 4) and KN2 (n = 6) mice.
Extended Data Fig. 6 IL-4 impedes CD103 expression by CD8+ T cells and CD8+ TRM cell formation in vivo.
Numbers of wild-type (WT) CD45.1+ and Il4ra−/− CD45.2+ CD44hiCD8+ OT-I T cells and numbers of WT CD45.1 and Il4ra−/− CD45.1+ CD44hiCD103+CD8+ OT-I T cells isolated on day 8, 10, 15 and 30 from the bLN, spleen and skin of congenic Il4ra−/−CD45.1+CD45.2+ recipient mice that were injected i.v. with WT and Il4ra−/− naïve CD44−CD8+ OT-I T cells on day 0, immediately tattooed with OVAp, and then painted with MC903 on days 5–14. *p = 0.0322; **p = 0.0156; ***p = 0.0078; ****p = 0.0049; *****p = 0.0039; ******p = 0.002, two-tailed Wilcoxon test. Data are from two independent experiments on day 8 (n = 11), day 15 (n = 8), and day 30 (n = 9), and three independent experiments on day 11 (n = 10).
Extended Data Fig. 7 Impairment of CD8+ TRM cell formation in MC903-painted skin is IL-4 dependent and CD8+ T cell-intrinsic.
(a) gMFI of CD103 expression (left) and percent CD103 expression (right) of CD45.1+CD44hiCD8+ OT-I T cells isolated on day >28 from the flank skin of congenic WT CD45.2+ C57Bl/6 recipient mice that were injected i.v. with naive CD45.1+CD8+ OT-I T cells and immediately tattoo-immunized with OVAp on day 0, treated on the flank skin with MC903 from day 5–14,and injected intraperitoneally with 0.5 mg isotype control antibody or with neutralizing IL-4 antibody every 3-4 days from day 5–21 post-transfer. Data are pooled from four independent experiments (n = 14 per group). Mean ± SEM (left), Median with IQR (right). (b) Numbers of CD44hiCD8+ OT-I T cells recovered on day >28 from the spleen and skin of mice as in a. *p = 0.035, tailed Mann-Whitney test. Mean ± SEM, spleen; Median with IQR, skin. Data are pooled from four independent experiments (n = 14 per group). (c) Contour plots illustrating the gating strategy used to identify CD44hiCD8+OVA(257-264)/Kb T cells isolated on day 30 from the flank skin of CD8Cre+Il4rafl/− mice injected intramuscularly with poly(i:c) and OVA on day 0, tattooed on the flank skin with OVAp on day 6, and treated daily with MC903 on days 7–16. (d) gMFI of CD103 expression (left) and percent CD103 expression (right) of CD44hiCD8+OVA(257-264)/Kb T cells T cells isolated on day >28 from the flank skin of CD8Cre+Il4rafl/− and littermate control Il4rafl/− mice treated as in c. Data are pooled from two independent experiments with CD8Cre+Il4rafl/− (n = 18) and Il4rafl/− (n = 15) mice. Mean ± SEM, left; Median with IQR, right. (e) Representative pseudocolor plots of CD8+OVA(257-264)/Kb T cells recovered from the flank skin of CD8Cre+Il4rafl/− and littermates treated as in c. (f) Numbers of CD8+Ova(257-64)/Kb T cells recovered on day >28 from the spleen and flank skin of CD8Cre+Il4rafl/− and littermates treated as in c. Data are pooled from two independent experiments with CD8Cre+Il4rafl/− (n = 18) and Il4rafl/− (n = 15) mice. *p = 0.0273, two-tailed Mann-Whitney test. Median with IQR.
Extended Data Fig. 8 Difference in TGF-βRII expression is independent of cell proliferation.
Histogram illustrating CellTrace Violet (CTV) dilution as a measure cell proliferation in Day 4.5 in vitro-activated CD8+ T cells that were labeled with CTV and then cultured for one day with TGF-β (10 ng/mL) with or without IL-4 (10 ng/mL) (left) Histogram of TGF-βRII expression after one day of T cell culture with TGF-β (10 ng/mL) with or without IL-4 (10 ng/mL) (right). Representative of three independent experiments.
Extended Data Fig. 9 TGF-β does not decrease Eomes expression in day 4.5 in vitro-activated CD8+ T cells.
(a) Mean fluorescence intensity of Eomes expression gared on CD8+CD3+ T cells (left), median fluorescence intensity of Eomes expression gated on CD8+CD3+ T cells (middle), and mean fluorescence intensity of Eomes expression gated on Eomes+CD8+CD3+ (right) in CD8+ T cells that were activated in vitro (1 mg/mL CD3 and CD28 antibody) and then cultured with or without TGF-β (10 ng/mL) and with or without IL-4 (10 ng/mL) for 3 days. Data are pooled from five independent experiments. Eomes mean fluorescence intensity (left), *p = 0.0056; **p = 0.0033; ***p = 0.0017; ****p = 0.0014; *****p = 0.0013. Eomes median fluorescence intensity (middle), *p = 0.0065; **p = 0.003; ***p = 0.0021; ****p = 0.002; *****p = 0.0019. Eomes mean fluorescence intensity in Eomes+CD8+CD3+ T cells (right) *p = 0.0111; **p = 0.0043; ***p = 0.0037; ****p = 0.0028; *****p = 0.0006, two-tailed paired t-test. Mean ± SEM. (b) RT-qPCR-determined expression of Eomes in CD8+ T cells activated and treated with cytokines as in a. Data are pooled from five independent experiments. *p = 0.0099; **p = 0.0032; ***p = 0.0023; ****p < 0.0001, two-tailed paired t-test. Mean ± SEM.
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Mora-Buch, R., Lake, M.E., Sama, A. et al. IL-4 impairs the formation of skin-resident memory CD8+ T cells. Nat Immunol 26, 1329–1338 (2025). https://doi.org/10.1038/s41590-025-02207-6
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DOI: https://doi.org/10.1038/s41590-025-02207-6
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