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

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.

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.

References

  1. 1.

    Lefrançois, L. Development, trafficking, and function of memory T-cell subsets. Immunol. Rev. 211, 93–103 (2006).

    PubMed  Google Scholar 

  2. 2.

    Mueller, S. N., Gebhardt, T., Carbone, F. R. & Heath, W. R. Memory T cell subsets, migration patterns, and tissue residence. Annu. Rev. Immunol. 31, 137–161 (2013).

    CAS  PubMed  Google Scholar 

  3. 3.

    Sallusto, F., Lenig, D., Förster, R., Lipp, M. & Lanzavecchia, A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708–712 (1999).

    CAS  PubMed  Google Scholar 

  4. 4.

    Masopust, D., Vezys, V., Marzo, A. L. & Lefrançois, L. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291, 2413–2417 (2001).

    CAS  PubMed  Google Scholar 

  5. 5.

    Wherry, E. J. et al. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4, 225–234 (2003).

    CAS  PubMed  Google Scholar 

  6. 6.

    Olson, J. A., McDonald-Hyman, C., Jameson, S. C. & Hamilton, S. E. Effector-like CD8+ T cells in the memory population mediate potent protective immunity. Immunity 38, 1250–1260 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Sallusto, F., Geginat, J. & Lanzavecchia, A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu. Rev. Immunol. 22, 745–763 (2004).

    CAS  PubMed  Google Scholar 

  8. 8.

    Mackay, L. K. et al. The developmental pathway for CD103+CD8+ tissue-resident memory T cells of skin. Nat. Immunol. 14, 1294–1301 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Mackay, L. K. et al. Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes. Science 352, 459–463 (2016).

    CAS  PubMed  Google Scholar 

  10. 10.

    Steinert, E. M. et al. Quantifying memory CD8 T cells reveals regionalization of immunosurveillance. Cell 161, 737–749 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Mueller, S. N. & Mackay, L. K. Tissue-resident memory T cells: local specialists in immune defence. Nat. Rev. Immunol. 16, 79–89 (2016).

    CAS  PubMed  Google Scholar 

  12. 12.

    Behr, F. M., Chuwonpad, A., Stark, R. & van Gisbergen, K. Armed and ready: transcriptional regulation of tissue-resident memory CD8 T cells. Front. Immunol. 9, 1770 (2018).

    PubMed  PubMed Central  Google Scholar 

  13. 13.

    Park, S. L. et al. Local proliferation maintains a stable pool of tissue-resident memory T cells after antiviral recall responses. Nat. Immunol. 19, 183–191 (2018).

    CAS  PubMed  Google Scholar 

  14. 14.

    Beura, L. K. et al. Intravital mucosal imaging of CD8+ resident memory T cells shows tissue-autonomous recall responses that amplify secondary memory. Nat. Immunol. 19, 173–182 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Beura, L. K. et al. T cells in nonlymphoid tissues give rise to lymph-node-resident memory T cells. Immunity 48, 327–338.e5 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Masopust, D., Ha, S.-J., Vezys, V. & Ahmed, R. Stimulation history dictates memory CD8 T cell phenotype: implications for prime-boost vaccination. J. Immunol. 177, 831–839 (2006).

    CAS  PubMed  Google Scholar 

  17. 17.

    Vezys, V. et al. Memory CD8 T-cell compartment grows in size with immunological experience. Nature 457, 196–199 (2009).

    CAS  PubMed  Google Scholar 

  18. 18.

    Jabbari, A. & Harty, J. T. Secondary memory CD8+ T cells are more protective but slower to acquire a central-memory phenotype. J. Exp. Med. 203, 919–932 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Nolz, J. C. & Harty, J. T. Protective capacity of memory CD8+ T cells is dictated by antigen exposure history and nature of the infection. Immunity 34, 781–793 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Fraser, K. A., Schenkel, J. M., Jameson, S. C., Vezys, V. & Masopust, D. Preexisting high frequencies of memory CD8+ T cells favor rapid memory differentiation and preservation of proliferative potential upon boosting. Immunity 39, 171–183 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Van Braeckel-Budimir, N., Varga, S. M., Badovinac, V. P. & Harty, J. T. Repeated antigen exposure extends the durability of influenza-specific lung-resident memory CD8+ T cells and heterosubtypic immunity. Cell Rep. 24, 3374–3382.e3 (2018).

    CAS  PubMed  Google Scholar 

  22. 22.

    Huster, K. M. et al. Unidirectional development of CD8+ central memory T cells into protective Listeria-specific effector memory T cells. Eur. J. Immunol. 36, 1453–1464 (2006).

    CAS  PubMed  Google Scholar 

  23. 23.

    Graef, P. et al. Serial transfer of single-cell-derived immunocompetence reveals stemness of CD8+ central memory T cells. Immunity 41, 116–126 (2014).

    CAS  PubMed  Google Scholar 

  24. 24.

    Masopust, D., Vezys, V., Wherry, E. J., Barber, D. L. & Ahmed, R. Cutting edge: gut microenvironment promotes differentiation of a unique memory CD8 T cell population. J. Immunol. 176, 2079–2083 (2006).

    CAS  PubMed  Google Scholar 

  25. 25.

    Sheridan, B. S. et al. Oral infection drives a distinct population of intestinal resident memory CD8+ T cells with enhanced protective function. Immunity 40, 747–757 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    van Stipdonk, M. J. B. et al. Dynamic programming of CD8+ T lymphocyte responses. Nat. Immunol. 4, 361–365 (2003).

    CAS  PubMed  Google Scholar 

  27. 27.

    Joshi, N. S. et al. Inflammation directs memory precursor and short-lived effector CD8+ T cell fates via the graded expression of T-bet transcription factor. Immunity 27, 281–295 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Sarkar, S. et al. Functional and genomic profiling of effector CD8 T cell subsets with distinct memory fates. J. Exp. Med. 205, 625–640 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Xin, A. et al. A molecular threshold for effector CD8+ T cell differentiation controlled by transcription factors Blimp-1 and T-bet. Nat. Immunol. 17, 422–432 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Nishimura, M. et al. Dual functions of fractalkine/CX3C ligand 1 in trafficking of perforin+/granzyme B+ cytotoxic effector lymphocytes that are defined by CX3CR1 expression. J. Immunol. 168, 6173–6180 (2002).

    CAS  PubMed  Google Scholar 

  31. 31.

    Böttcher, J. P. et al. Functional classification of memory CD8+ T cells by CX3CR1 expression. Nat. Commun. 6, 8306 (2015).

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Gerlach, C. et al. The chemokine receptor CX3CR1 defines three antigen-experienced CD8 T cell subsets with distinct roles in immune surveillance and homeostasis. Immunity 45, 1270–1284 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Stark, R. et al. TRM maintenance is regulated by tissue damage via P2RX7. Sci. Immunol. 3, eaau1022 (2018).

    PubMed  Google Scholar 

  34. 34.

    Skon, C. N. et al. Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells. Nat. Immunol. 14, 1285–1293 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Walzer, T. et al. Natural killer cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor. Nat. Immunol. 8, 1337–1344 (2007).

    CAS  PubMed  Google Scholar 

  36. 36.

    Wirth, T. C. et al. Repetitive antigen stimulation induces stepwise transcriptome diversification but preserves a core signature of memory CD8+ T cell differentiation. Immunity 33, 128–140 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Fernandez-Ruiz, D. et al. Liver-resident memory CD8+ T cells form a front-line defense against malaria liver-stage infection. Immunity 45, 889–902 (2016).

    CAS  PubMed  Google Scholar 

  38. 38.

    Behr, F. M. et al. Blimp-1 rather than Hobit drives the formation of tissue-resident memory CD8+ T cells in the lungs. Front. Immunol. 10, 400 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Schenkel, J. M., Fraser, K. A., Vezys, V. & Masopust, D. Sensing and alarm function of resident memory CD8+ T cells. Nat. Immunol. 14, 509–513 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Schenkel, J. M. et al. Resident memory CD8 T cells trigger protective innate and adaptive immune responses. Science 346, 98–101 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Ariotti, S. et al. Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert. Science 346, 101–105 (2014).

    CAS  PubMed  Google Scholar 

  42. 42.

    Schenkel, J. M., Fraser, K. A. & Masopust, D. Cutting edge: resident memory CD8 T cells occupy frontline niches in secondary lymphoid organs. J. Immunol. 192, 2961–2964 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Fonseca, R. et al. Developmental plasticity allows outside-in immune responses by resident memory T cells. Nat. Immunol. 21, 412–421 (2020).

    CAS  PubMed  Google Scholar 

  44. 44.

    Schenkel, J. M. & Masopust, D. Tissue-resident memory T cells. Immunity 41, 886–897 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    van Gisbergen, K. P. J. M. et al. Mouse Hobit is a homolog of the transcriptional repressor Blimp-1 that regulates NKT cell effector differentiation. Nat. Immunol. 13, 864–871 (2012).

    CAS  PubMed  Google Scholar 

  46. 46.

    Kragten, N. A. M. et al. Blimp-1 induces and Hobit maintains the cytotoxic mediator granzyme B in CD8 T cells. Eur. J. Immunol. 48, 1644–1662 (2018).

    CAS  PubMed  Google Scholar 

  47. 47.

    Wakim, L. M., Woodward-Davis, A. & Bevan, M. J. Memory T cells persisting within the brain after local infection show functional adaptations to their tissue of residence. Proc. Natl Acad. Sci. USA 107, 17872–17879 (2010).

    CAS  PubMed  Google Scholar 

  48. 48.

    Enamorado, M. et al. Enhanced anti-tumour immunity requires the interplay between resident and circulating memory CD8+ T cells. Nat. Commun. 8, 16073 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Osborn, J. F. et al. Central memory CD8+ T cells become CD69+ tissue-residents during viral skin infection independent of CD62L-mediated lymph node surveillance. PLoS Pathog. 15, e1007633 (2019).

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: single-cell RNA-seq by multiplexed linear amplification. Cell Rep. 2, 666–673 (2012).

    CAS  PubMed  Google Scholar 

  51. 51.

    Simmini, S. et al. Transformation of intestinal stem cells into gastric stem cells on loss of transcription factor Cdx2. Nat. Commun. 5, 5728 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589–595 (2010).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    PubMed  PubMed Central  Google Scholar 

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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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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.

Corresponding author

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

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Editor recognition statement L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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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. (be) 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. (df) 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. (gi) 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 (gi); 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. (ci) 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. (cf) 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). (gi) 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. (ad) 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 (2020). https://doi.org/10.1038/s41590-020-0723-4

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