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Tissue-resident memory CD8+ T cells shape local and systemic secondary T cell responses

Nature Immunology volume 21, pages 1070–1081 (2020)Cite this article

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Abstract

Tissue-resident memory CD8+ T cells (TRM cells) are crucial in protecting against reinvading pathogens, but the impact of reinfection on their tissue confinement and contribution to recall responses is unclear. We developed a unique lineage tracer mouse model exploiting the TRM-defining transcription factor homolog of Blimp-1 in T cells (Hobit) to fate map the TRM progeny in secondary responses. After reinfection, a sizeable fraction of secondary memory T cells in the circulation developed downstream of TRM cells. These tissue-experienced ex-TRM cells shared phenotypic properties with the effector memory T cell population but were transcriptionally and functionally distinct from other secondary effector memory T cell cells. Adoptive transfer experiments of TRM cells corroborated their potential to form circulating effector and memory cells during recall responses. Moreover, specific ablation of primary TRM cell populations substantially impaired the secondary T cell response, both locally and systemically. Thus, TRM cells retain developmental plasticity and shape both local and systemic T cell responses on reinfection.

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Fig. 1: Hobit identifies CD8+ TRM cells across tissues after Lm-OVA infection.
Fig. 2: Hobit+ TRM cells expand locally and in draining lymph nodes after pathogen rechallenge.
Fig. 3: Hobit+ TRM cells downregulate Hobit expression on antigen encounter and form circulating memory cells after pathogen rechallenge.
Fig. 4: Ex-Hobit+ T cells primarily acquire a TEM phenotype after pathogen rechallenge.
Fig. 5: Secondary ex-Hobit+ T cells constitute a transcriptionally and functionally distinct effector memory subset.
Fig. 6: CD8+ TRM cells generate systemic responses on pathogen rechallenge.
Fig. 7: Formation of secondary CD8+ TRM and TEM cells depends on primary Hobit+ T cells.
Fig. 8: CD8+ TRM cells substantially contribute to secondary memory responses after pathogen rechallenge.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. The RNA-seq data have been deposited at the National Center for Biotechnology Information Sequence Read Archive under the BioProject accession code PRJNA635759.

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Acknowledgements

We thank members of the van Gisbergen laboratory and Department of Hematopoiesis, as well as M. Wolkers, for fruitful discussions. F.M.B., L.P.V. and K.P.J.M.v.G. were supported by Vidi grant no. 917.13.338 from the Netherlands Organization for Scientific Research (NWO) and a fellowship from the Landsteiner Foundation of Blood Transfusion Research. R.S. was supported by a fellowship from the Alexander von Humboldt Foundation and by Veni grant no. 016.186.116 from the (NWO).

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  1. These authors contributed equally: Felix M. Behr, Loreto Parga-Vidal.

Authors and Affiliations

  1. Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands

    Felix M. Behr, Loreto Parga-Vidal, Natasja A. M. Kragten, Teunis J. P. van Dam, Thomas H. Wesselink, Rene A. W. van Lier, Regina Stark & Klaas P. J. M. van Gisbergen

  2. Department of Experimental Immunology, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands

    Felix M. Behr, Regina Stark & Klaas P. J. M. van Gisbergen

  3. Department of Molecular and Cellular Hemostasis, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands

    Teunis J. P. van Dam

  4. Department of Microbiology and Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, USA

    Brian S. Sheridan

  5. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands

    Ramon Arens

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Contributions

F.M.B., R.A.W.v.L., R.S. and K.P.J.M.v.G. designed the experiments. F.M.B., L.P.V., N.A.M.K. and R.S. performed the experiments. T.H.W., B.S.S. and R.A. contributed critical reagents and experimental help. F.M.B. analyzed the data and performed the statistical analysis. T.J.P.v.D. analyzed the RNA-seq data. F.M.B. and K.P.J.M.v.G. wrote the manuscript.

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Correspondence to Klaas P. J. M. van Gisbergen.

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Extended data

Extended Data Fig. 1 Expression of reporter protein tdTomato reflects Hobit expression.

a, Exemplary gating strategy for the analysis of OT-I T cells. b, c, HR × Rosa26eYFP naïve and memory OT-I T cells were FACS-purified from spleen liver and small intestine at >30 days after oral infection with L.m.-OVA. The expression of Hobit mRNA in CD44lo CD62L+ TN cells, CD69– CD62L+ TCM cells, tdTomato– (Tom-) CD69– CD62L– TEM cells from spleen and liver, and tdTomato+ (Tom + ) CD69+ CD62L– TRM cells from liver and small intestine (SI, SI LPL and SI LPL combined) was determined via (b) qPCR and via (c) RNA sequencing. Combined data from two independent experiments (n = 3 or 4 biological replicates) (b) and data from one experiment (n = 3 biological replicates) (c). Symbols represent biological replicates; bars represent the mean. Error bars represent mean ± SD.

Extended Data Fig. 2 Fate-mapping of Hobit-expressing cells identifies primary TRM cells.

a, Schematic representation is shown of the experimental setup using HR OT-I mice crossed onto Rosa26-flox-STOP-flox-eYFP (Rosa26eYFP) reporter mice for fate mapping of TRM cells during secondary responses. (b–e) The phenotype of adoptively transferred HR × Rosa26eYFP OT-I T cells was analyzed at >30 days after oral infection with L.m.-OVA. b, Representative flow cytometry plots show expression of YFP and tdTomato by TCM (CD69– CD62L+) and TEM (CD69– CD62L–) cells in spleen, and TRM cells (CD69+ CD62L–) in liver, SI IEL and LPL. c, The frequency of YFP+ expression was quantified within the indicated populations of memory HR × Rosa26eYFP OT-I T cells. d, The expression levels (geoMFI) of tdTomato within the indicated populations of memory HR × Rosa26eYFP OT-I T cells were quantified. Two-way ANOVA with Tukey’s multiple comparisons test, ***P < 0.0001. e, The frequencies of CD69+, CXCR6+ and CD62L+ expression within the indicated populations was quantified. One-way ANOVA with Tukey’s multiple comparisons test, *P = 0.0131, **P < 0.01, ***P < 0.0001. Combined data is shown from two independent experiments (n = 8 or 11 mice). Symbols represent individual mice; bars represent the mean. Error bars represent mean ± SEM.

Extended Data Fig. 3 Polyclonal donor TRM cells maintain resident phenotype after adoptive transfer.

Congenically marked TRM (green) and TCM cells (blue), that were FACS-purified from SI IEL and spleen of LCMV-infected mice, respectively, were co-transferred into naïve recipients, and distribution and phenotype of donor cells were analyzed 14 days later. a, Dot plot shows distribution of TRM and TCM cells within virus-specific (Db GP33+) donor cell population (left panel) and histograms display expression of phenotypic markers by Db GP33+ TRM and TCM cells (right panel) prior to transfer. b, Graphic scheme depicts setting of adoptive transfer experiments. c, Representative flow cytometry plots show expression of the congenic markers CD45.1 and CD45.2 to identify donor TRM-derived (green), donor TCM-derived (blue) and host-derived (grey) cells within the memory (CD44high) CD8+ T cell population in the indicated tissues at two weeks after transfer. d, The frequency of TRM- and TCM-derived CD44high cells was determined in indicated tissues. Dotted line indicates detection limit, as determined by analysis of non-transferred mice. e, Representative flow cytometry plots show expression of CD62L, CD69 and CD103 on CD44high TRM and TCM donor cells in liver. f, The frequency of CD69+ CD62L–, CD69+ CD103+ and CD44+ CD62L+ cells within the donor populations was quantified. Two-tailed paired t-test, ***P < 0.001. g, Representative flow cytometry plots show presence of virus-specific (Db GP33+) cells within donor populations in the liver. Combined data is shown from two independent experiments (n = 9 or 11 mice). Symbols represent individual mice; bars represent the mean. Error bars represent mean ± SEM.

Extended Data Fig. 4 Monoclonal donor TRM cells maintain resident phenotype after adoptive transfer.

Congenically marked HR OT-I TRM (green) and HR OT-I TCM cells (blue), that were FACS-purified from SI IEL and spleen of L.m.-OVA -infected mice, respectively, were co-transferred into naïve recipients, and distribution and phenotype of donor cells were analyzed 14 days later. a, Dot plot shows distribution of HR OT-I TRM and TCM cells within the total donor cell population (left panel) and histograms display expression of phenotypic markers by OT-I TRM and TCM cells (right panel) prior to transfer. b, Graphic scheme depicts setting of adoptive transfer experiments. c, Representative flow cytometry plots show expression of the congenic markers CD45.1 and CD45.2 to identify donor TRM-derived (green), donor TCM-derived (blue) and host-derived (grey) cells within the memory (CD44high) CD8+ T cell population in the indicated tissues at two weeks after transfer. d, The frequency of TRM- and TCM-derived CD44high cells was determined in indicated tissues. Dotted line indicates detection limit, as determined by analysis of non-transferred mice. e, Representative flow cytometry plots show expression of CD62L and CD69 on CD44high TRM and TCM donor cells in liver. f, The frequency of CD69– CD62L+ cells within the donor populations was quantified. Two-tailed paired t-test, *P = 0.0173. g, Representative flow cytometry plots show expression of tdTomato and CD69 on CD44high TRM and TCM donor cells in liver. h, The frequency of CD69+ tdTomato+ cells within the donor populations was quantified. Two-tailed paired t-test, ***P = 0.0005. Combined data from two independent experiments (n = 4 mice). Symbols represent individual mice; bars represent the mean. Error bars represent mean ± SEM.

Extended Data Fig. 5 Monoclonal CD8+ TRM cells generate systemic responses upon pathogen rechallenge.

a, Graphic scheme depicts settings of rechallenge experiments with adoptively transferred HR OT-I TCM and TRM cells. In brief, congenically marked HR OT-I TRM and TCM cells were FACS-purified from SI IEL and spleen of L.m.-OVA -infected mice, respectively, and co-transferred into naïve recipients, which were challenged orally with L.m.-OVA 14 days later. The offspring of the donor OT-I T cells was analyzed at >30 days p.i. b, Representative flow cytometry plots show expression of the congenic markers CD45.1 and CD45.2 to identify donor TRM-derived (green), donor TCM-derived (blue) and host-derived (grey) cells within the CD8+ T cell population in the indicated tissues at >30 days p.i. c, The number of TRM- and TCM-derived OT-I T cells was determined in spleen and liver at the indicated time points before and after L.m.-OVA challenge. (d–f) Representative flow cytometry plots show (d) expression of CD62L and CD44 and (e) CX3CR1 and KLRG1 on HR OT-I TRM- and TCM-derived memory T cells in spleen, and (f) expression of tdTomato and CD69 by HR OT-I TRM- and TCM-derived memory T cells in liver at >30 days after L.m.-OVA challenge. (g–i) The frequency of (g) CD62L expression, (h) KLRG1 and CX3CR1 co-expression and (i) CD69 and tdTomato co-expression within the offspring of donor TRM and TCM populations was quantified. Two-tailed paired t-test, *P = 0.0195 (g), *P = 0.0241 (h). Combined data from two independent experiments (n = 4 or 5 mice). Symbols represent the mean (c) or individual mice (g–i); bars represent the mean. Error bars represent mean ± SEM.

Extended Data Fig. 6 Intestinal and liver CD8+ TRM cells generate systemic responses upon pathogen rechallenge.

a, Graphic scheme depicts settings of rechallenge experiments with adoptively transferred TRM cells from liver and intestine. In brief, congenically marked TRM cells were FACS-purified from liver and SI IEL of LCMV-infected mice, respectively, and co-transferred into naïve recipients, which were challenged with LCMV two weeks later. The offspring of the virus-specific (Db GP33+) donor T cells was analyzed at 8 and >30 days p.i. b, Representative flow cytometry plots show expression of the congenic markers CD45.1 and CD45.2 to identify donor SI IEL TRM-derived (green), donor liver TRM-derived (turquoise) and host-derived (grey) cells within the Db GP33+ T cell population in the indicated tissues at 8 and >30 days p.i. c, d, Representative flow cytometry plots show (c) expression of CD44 and CD62L and (d) expression of CX3CR1 and KLRG1 on Db GP33+ SI IEL TRM- (left) and liver TRM-derived memory T cells (right) in spleen at >30 days after LCMV challenge. e, f, The frequency of (e) CD62L expression and (f) co-expression of KLRG1 and CX3CR1 within the offspring of donor TRM populations and host Db GP33+ cells were quantified. Two-tailed paired t-test, *P < 0.05; ***P < 0.001. g, Representative flow cytometry plots show expression of CXCR6 and CD69 on Db GP33+ secondary memory cells developing from donor SI IEL TRM (left) or liver TRM (right) cells after LCMV challenge. h, The frequency of TRM cells (CD69+ CXCR6+) within the donor populations was quantified. Two-tailed paired t-test. Combined data from two independent experiments (n = 9 or 10 mice). Symbols represent individual mice; bars represent the mean. Error bars represent mean ± SEM.

Extended Data Fig. 7 Efficient depletion of TRM cells by DT administration.

Wild-type mice containing congenically marked naïve WT and HR OT-I T cells were infected orally with L.m.-OVA. DT was administered in the memory phase after infection to specifically deplete HR TRM cells. One day after the last DT administration, presence and phenotype of donor WT and HR OT-I T cells were analyzed. a, Representative flow cytometry plots show expression of CD8α and tdTomato by HR OT-I T cells in liver under control conditions (left) and after DT treatment (right). b, Hobit+ HR OT-I cells were enumerated in control (Ctrl) and DT-treated mice. Two-tailed unpaired t-test, **P = 0.0023, ***P = 0.0007. (c–i) The contribution of WT and HR OT-I T cells to the formation of primary memory populations was analyzed in control and DT-treated mice. (c–f) Representative flow cytometry plots show expression of CD45.1 and CD45.2 to identify the presence of WT (CD45.1+) and HR (CD45.2+) OT-I T cells within (c) CD69+ TRM cells in liver, (d) CD69+ TRM cells in SI IEL, (e) CD62L+ TCM cells in spleen, and (f) KLRG1+ TEM cells in spleen under control conditions (left) and after DT-treatment (right). (g–i) The ratio between HR and WT OT-I T cells was determined under control conditions and after DT treatment for (g) the CD69+ TRM population of liver and SI IEL, (h) the CD62L+ TCM population of spleen and mLN, and (i) the KLRG1+ TEM population of spleen and liver. Ratios were normalized to controls. Two-tailed Mann-Whitney U-test, *P = 0.0286. Data from one experiment (n = 4 mice). Symbols represent individual mice; bars represent the mean.

Extended Data Fig. 8 NAD administration results in efficient and selective depletion of WT TRM cells.

Wild-type mice containing congenically marked naïve WT and P2rx7–/– OT-I T cells were infected orally with L.m.-OVA. NAD was administered in the memory phase after infection to specifically deplete WT TRM cells. After two weeks, presence of donor WT and P2rx7–/– OT-I T cells, and of host T cell populations was analyzed. (a–d) The contribution of WT and P2rx7–/– OT-I T cells to the formation of primary memory populations was analyzed in control and NAD-treated mice. Representative flow cytometry plots show expression of CD45.1 and CD45.2 to identify the contribution of WT (CD45.1+) and P2rx7–/– (CD45.2+) OT-I T cells to the formation of (a) CD69+ TRM cells in liver, (b) CD69+ TRM cells in SI IEL, (c) CD62L+ OT-I TCM cells in spleen, and (d) KLRG1+ TEM cells in spleen under control conditions (left) and after NAD-treatment (right). (e) Representative flow cytometry plots show expression of Helios and Foxp3 by host CD4+ T cells in spleen in control (left) and NAD-treated mice (right). (f, g) The number of (f) Foxp3+ Helios+ CD4+ T cells and (g) TCRγδ+ T cells in the indicated tissues of control and NAD-treated mice is shown. Two-tailed Mann-Whitney U-test. Data is representative of two independent experiments (n = 4 mice). Symbols represent individual mice; bars represent the mean.

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Behr, F.M., Parga-Vidal, L., Kragten, N.A.M. et al. Tissue-resident memory CD8+ T cells shape local and systemic secondary T cell responses. Nat Immunol 21, 1070–1081 (2020). https://doi.org/10.1038/s41590-020-0723-4

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