Abstract
Tissue-resident memory T (TRM) cells persist indefinitely in epithelial barrier tissues and protect the host against pathogens1,2,3,4. However, the biological pathways that enable the long-term survival of TRM cells are obscure4,5. Here we show that mouse CD8+ TRM cells generated by viral infection of the skin differentially express high levels of several molecules that mediate lipid uptake and intracellular transport, including fatty-acid-binding proteins 4 and 5 (FABP4 and FABP5). We further show that T-cell-specific deficiency of Fabp4 and Fabp5 (Fabp4/Fabp5) impairs exogenous free fatty acid (FFA) uptake by CD8+ TRM cells and greatly reduces their long-term survival in vivo, while having no effect on the survival of central memory T (TCM) cells in lymph nodes. In vitro, CD8+ TRM cells, but not CD8+ TCM cells, demonstrated increased mitochondrial oxidative metabolism in the presence of exogenous FFAs; this increase was not seen in Fabp4/Fabp5 double-knockout CD8+ TRM cells. The persistence of CD8+ TRM cells in the skin was strongly diminished by inhibition of mitochondrial FFA β-oxidation in vivo. Moreover, skin CD8+ TRM cells that lacked Fabp4/Fabp5 were less effective at protecting mice from cutaneous viral infection, and lung Fabp4/Fabp5 double-knockout CD8+ TRM cells generated by skin vaccinia virus (VACV) infection were less effective at protecting mice from a lethal pulmonary challenge with VACV. Consistent with the mouse data, increased FABP4 and FABP5 expression and enhanced extracellular FFA uptake were also demonstrated in human CD8+ TRM cells in normal and psoriatic skin. These results suggest that FABP4 and FABP5 have a critical role in the maintenance, longevity and function of CD8+ TRM cells, and suggest that CD8+ TRM cells use exogenous FFAs and their oxidative metabolism to persist in tissue and to mediate protective immunity.
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Acknowledgements
We thank B. Moss (US National Institute of Health (NIH)) for providing rVACV expressing the OT-I T cell epitope OVA257–264, as well as WR-VACV. This work was supported by NIH grants R01AI041707 (T.S.K.), R01AI127654 (T.S.K.), TR01AI097128 (T.S.K. and R.A.C.) and R01AR063962 (R.A.C.). C.O.P. was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C1799).
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Y.P. and T.S.K. conceived the project, designed experiments and analysed the data. Y.P., T.T., C.O.P., S.Y.L., X.L., J.T.O. and A.G. performed the experiments and helped to analyse the data. J.T.O., A.G., J.E.T., J.G.K. and R.A.C helped with human skin sample collection, processing, experiments and data analysis. S.M. helped to analyse the microarray data. C.L. and P.P. helped with Seahorse metabolism experiments. S.J.D., R.F. and G.S.H. helped with mice experiments and analysing data. Y.P. and T.S.K. wrote the manuscript.
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Reviewer Information Nature thanks F. Carbone and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 CD8+ TRM cells show increased gene expression of Fabp4 and Fabp5.
a, Number of OT-I Thy1.1+ cells in infected skin at indicated time after infection. Graph shows mean ± s.d. of 10 mice. b, Venn diagram analysis of genes differentially expressed in pairwise comparisons between OT-I TCM, TEM and TRM cells (day 30) relative to that of TN cells (fold change cutoff, ≥ 2). c, Absolute qPCR analysis of Fabp gene expression in TRM cells (day 30). ND, not detectable. d, qPCR assessments of Fabp4 and Fabp5 levels in indicated T cell subsets. OT-I Thy1.1+ cells were intravenously transferred into Thy1.2+ recipient mice 1 day before mice were infected with 2 × 106 p.f.u. VACVOVA by intratracheal infection. After 45 days, mice were euthanized. TCM and TEM cells were sorted from spleen and TRM cells were sorted from lung. e, qPCR analysis of Pparg-knockdown efficiency by lentiviral vector encoding two specific and one scrambled siRNA in OT-I CD8+ TRM cells. f, qPCR analysis of Fabp4 and Fabp5 gene expression in OT-I CD8+ TRM cells from mice treated with or without GW9662. Graphs in c, d, e, f show mean ± s.d. from triplicates. β-actin was used as internal control and mRNA was normalized to TN samples (d), or TRM samples with scrambled siRNA transduction (e) or without GW9662 treatment (f). T cells from 15–20 mice were pooled for each sample. **P < 0.01; NS, not significant.
Extended Data Figure 2 Visualization of lipid distribution in mouse skin tissue by Bodipy staining.
Mouse skin cryosections were stained for lipids (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503), green) and nuclei (4′,6-diamidino-2-phenylindole (DAPI), blue). Images were acquired with a Leica TCS SP8 confocal microscope and analysed with ImageJ. n = 15 sections from 5 mice and 1 representative site is shown. Dashed line indicates epidermal–dermal boundary. Scale bar, 20 μm.
Extended Data Figure 3 OT-I Fabp4−/−/Fabp5−/− TRM cells show a similar surface protein expression phenotype as wild-type cells.
Thy1.2+ CD45.2+ recipient mice were given a 1:1 ratio of naive OT-I Thy1.1+ CD45.1+ wild-type and OT-I Thy1.1+ CD45.2+ Fabp4−/−/Fabp5−/− cells one day before mice were infected with 2 × 106 p.f.u. VACVOVA by skin scarification. Subsequently, 45 days after infection, infected skin sites were collected and the phenotype of TRM cells was examined by flow cytometry. Data are representative of three independent experiments (n = 5 mice per group). ESL, E-selectin ligand.
Extended Data Figure 4 Relative contribution of FABP4 and FABP5 in fatty acid acquisition and long-term maintenance of skin CD8+ TRM cells.
a, Average MFI of Bodipy FL C16 uptake by OT-I wild-type, Fabp4+/−/Fabp5+/−, Fabp4−/− and Fabp5−/− TRM cells. b–d, Number of OT-I wild-type, Fabp4+/−/Fabp5+/−, Fabp4−/− and Fabp5−/− TRM cells at different time points after VACVOVA infection. Thy1.2+ CD45.2+ recipient mice were given a 1:1 ratio of naive OT-I Thy1.1+ CD45.1+ wild-type and OT-I Thy1.1+ CD45.2+ Fabp4/Fabp5 knockout cells (Fabp4+/−/Fabp5+/− (b), Fabp4−/− (c) or Fabp5−/− (d) cells) one day before infection with 2 × 106 p.f.u. VACVOVA by skin scarification. Relative percentages of the two T cell populations were analysed longitudinally by flow cytometry. Graphs show mean ± s.d. of 5 mice per group. NS, not significant.
Extended Data Figure 5 OT-I Fabp4−/−/Fabp5−/− Teff cells have similar proliferative capacity and tissue-homing receptor expression as wild-type counterparts.
a, Quantification of Ki67+ cells in OT-I TN, Teff, TCM, TEM and TRM cells. b, Average MFI of Bodipy FL C16 uptake by OT-I TN, Teff, TCM, TEM or TRM cells. Graphs show mean ± s.d. of 5 mice per group (a, b). *P < 0.05; **P < 0.01. c, Flow cytometric analysis of T cell proliferation and homing receptor expression on OT-I wild-type and Fabp4−/−/Fabp5−/− T cells. Thy1.2+ CD45.2+ recipient mice were given a 1:1 ratio of CFSE-labelled naive OT-I Thy1.1+ CD45.1+ wild-type and OT-I Thy1.1+ CD45.2+ Fabp4−/−/Fabp5−/− cells one day before mice were infected with 2 × 106 p.f.u. VACVOVA by skin scarification. At 60 h after infection, proliferation and tissue-homing receptor expression of OT-I cells isolated from draining lymph nodes were analysed by flow cytometry. Data are representative of three independent experiments (n = 5 mice per group). ESL, E-selectin ligand; PSL, P-selectin ligand.
Extended Data Figure 6 Effect of Pparg lentiviral siRNA knockdown or PPARγ inhibition on CD8+ TRM cell maintenance in peripheral tissue.
a, Number of OT-I CD8+ TRM transduced with scrambled siRNA or siRNA targeting Pparg. OT-I cells transduced with Pparg siRNA (together with the same number of congenically scrambled siRNA transduced OT-I cells) were cotransferred into recipient mice that were previously infected with VACVOVA by skin scarification. After 40 days, mice were euthanized and the number of siRNA-transduced OT-I cells in the infected skin tissue were collected and counted by flow cytometry on the basis of the GFP marker. b, Number of OT-I CD8+ TRM cells in infected skin from mice treated with or without GW9662. Thy1.2+ CD45.2+ recipient mice were infected with 2 × 106 p.f.u. VACVOVA by skin scarification. 40 days later, mice were treated with GW9662 daily by intradermal injection for 5 days, after which mice were euthanized and TRM cells were counted and analysed by flow cytometry. Graphs show mean ± s.d. of 5 mice per group. **P < 0.01.
Extended Data Figure 7 Gene expression profile of OT-I Fabp4−/−/Fabp5−/− TRM cells.
a, Principal-component analysis of gene-expression data for skin infiltrating T cells isolated at day 10 and day 30 after infection. b, Differentially expressed genes selected from a pairwise comparison between OT-I Fabp4−/−/Fabp5−/− TRM and OT-I wild-type TRM cells. Thy1.2+ CD45.2+ recipient mice were given a 1:1 ratio of naive OT-I Thy1.1+ CD45.1+ wild-type and OT-I Thy1.1+ CD45.2+ Fabp4−/−/Fabp5−/− cells one day before mice were infected with 2 × 106 p.f.u. VACVOVA by skin scarification. At 30 days after infection, OT-I CD8+ TRM cells were sorted from infected skin sites for gene microarray. c, qPCR analysis of genes involved in anti-inflammatory responses (Il10 and Socs2), cell apoptosis (Casp3 and Bcl2113) as well as immune responses (Ccr8, Ccl21a, Il1r1, Il6, Cxcl13, Cxcl12, Ccl12, Gzmc, Gzma) in OT-I Fabp4−/−/Fabp5−/− TRM cells compared to wild-type TRM cells. Graphs show mean ± s. d. from triplicates. β-actin was used as internal control and mRNA was normalized to OT-I wild-type TRM cells. mRNA was pooled from 15 mice from 3 independent biological groups (5 mice per group). **P < 0.01.
Extended Data Figure 8 Effect of Cpt1a lentiviral siRNA knockdown or CPT1A inhibition on OT-I CD8+ TRM cell maintenance in peripheral tissue.
a, qPCR analysis of Cpt1a lentiviral siRNA knockdown efficiency in OT-I CD8+ TRM cells. b, c, Quantification of OT-I wild-type and OT-I Fabp4−/−/Fabp5−/− TRM cells in infected skin from mice treated with or without etomoxir (b) or trimetazidine (c). Thy1.2+ CD45.2+ recipient mice were given a 1:1 ratio of naive OT-I Thy1.1+ CD45.1+ wild-type and OT-I Thy1.1+ CD45.2+ Fabp4−/−/Fabp5−/− cells one day before infection with 2 × 106 p.f.u. VACVOVA by skin scarification. Mice were administered etomoxir or trimetazidine daily by intradermal injection for 5 days from day 40 after infection, after which mice were euthanized and TRM cells were analysed by flow cytometry and counted. Symbols represent individual mice. d, OCR and ECAR as measured by the Seahorse assay of skin-infiltrating T cells sorted at indicated time points after infection under basal conditions. Graphs show mean ± s.d. of triplicates. **P < 0.01; NS, not significant.
Extended Data Figure 9 Skin CD8+ TRM cells residing in distal infection sites are deficient in long-time survival and virus clearance.
a, Ratio of peripheral blood CD3 counts in FTY720 versus untreated mice, illustrating the kinetics of lymphoid sequestration by FTY720. n = 5 mice per group. b, Quantification of OT-I wild-type and OT-I Fabp4−/−/Fabp5−/− cells infiltrating distal infection sites at different time points after VACVOVA infection. Thy1.2+ CD45.2+ recipient mice were given a 1:1 ratio of naive OT-I Thy1.1+ CD45.1+ wild-type and OT-I Thy1.1+ CD45.2+ Fabp4−/−/Fabp5−/− cells one day before infection with 2 × 106 p.f.u. VACVOVA by skin scarification of the right ears. Relative percentages of the two T cell populations infiltrating left ears were analysed longitudinally by flow cytometry. Graphs show mean ± s.d. of 5 mice per group. c, qPCR analysis of distal infection skin viral load at six days after re-challenge. OT-I wild-type or OT-I Fabp4−/−/Fabp5−/− cells were adoptively transferred into μMT mice before infection with 2 × 106 p.f.u. VACVOVA by skin scarification of the right ears. 25 days later, mice were re-challenged with VACVOVA at the left ear by skin scarification. U, un-immunized mice; I, immunized mice. Symbols represent individual mice. **P < 0.01; NS, not significant.
Extended Data Figure 10 Visualization of lipid distribution in human skin from patients with psoriasis by Bodipy staining.
Cryosections of lesional scalp skin samples from patients with psoriasis were stained for lipid (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503), green) and nuclei (4′,6-diamidino-2-phenylindole (DAPI), blue). Images were acquired with a Leica TCS SP8 confocal microscope and analysed with ImageJ. Dashed lines indicate epidermal–dermal boundary. Scale bar, 50 μm.
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Pan, Y., Tian, T., Park, C. et al. Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism. Nature 543, 252–256 (2017). https://doi.org/10.1038/nature21379
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DOI: https://doi.org/10.1038/nature21379
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