CD8+ T cell immunosurveillance dynamics influence the outcome of intracellular infections and cancer. Here we used two-photon intravital microscopy to visualize the responses of CD8+ resident memory T cells (TRM cells) within the reproductive tracts of live female mice. We found that mucosal TRM cells were highly motile, but paused and underwent in situ division after local antigen challenge. TRM cell reactivation triggered the recruitment of recirculating memory T cells that underwent antigen-independent TRM cell differentiation in situ. However, the proliferation of pre-existing TRM cells dominated the local mucosal recall response and contributed most substantially to the boosted secondary TRM cell population. We observed similar results in skin. Thus, TRM cells can autonomously regulate the expansion of local immunosurveillance independently of central memory or proliferation in lymphoid tissue.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
von Andrian, U. H. & Mackay, C. R. T-cell function and migration. Two sides of the same coin. N. Engl. J. Med. 343, 1020–1034 (2000).
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).
Stemberger, C. et al. Stem cell-like plasticity of naïve and distinct memory CD8+ T cell subsets. Semin. Immunol. 21, 62–68 (2009).
Wherry, E. J. et al. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4, 225–234 (2003).
Farber, D. L., Yudanin, N. A. & Restifo, N. P. Human memory T cells: generation, compartmentalization and homeostasis. Nat. Rev. Immunol. 14, 24–35 (2014).
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).
Park, C. O. & Kupper, T. S. The emerging role of resident memory T cells in protective immunity and inflammatory disease. Nat. Med. 21, 688–697 (2015).
Mueller, S. N. & Mackay, L. K. Tissue-resident memory T cells: local specialists in immune defence. Nat. Rev. Immunol. 16, 79–89 (2016).
Mowat, A. M., McInnes, I. B. & Parrott, D. M. V. Functional properties of intra-epithelial lymphocytes from mouse small intestine. IV. Investigation of the proliferative capacity of IEL using phorbol ester and calcium ionophore. Immunology 66, 398–403 (1989).
Ebert, E. C., Roberts, A. I., Brolin, R. E. & Raska, K. Examination of the low proliferative capacity of human jejunal intraepithelial lymphocytes. Clin. Exp. Immunol. 65, 148–157 (1986).
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).
Steinert, E. M. et al. Quantifying memory CD8 T cells reveals regionalization of immunosurveillance. Cell 161, 737–749 (2015).
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).
Wakim, L. M. et al. The molecular signature of tissue resident memory CD8 T cells isolated from the brain. J. Immunol. 189, 3462–3471 (2012).
Ariotti, S. et al. Skin-resident memory CD8+ T cells trigger a state of tissue-wide pathogen alert. Science 346, 101–105 (2014).
Schenkel, J. M. et al. Resident memory CD8 T cells trigger protective innate and adaptive immune responses. Science 346, 98–101 (2014).
Gebhardt, T. et al. Different patterns of peripheral migration by memory CD4+ and CD8+ T cells. Nature 477, 216–219 (2011).
Zaid, A. et al. Persistence of skin-resident memory T cells within an epidermal niche. Proc. Natl. Acad. Sci. USA 111, 5307–5312 (2014).
Miller, M. J., Wei, S. H., Parker, I. & Cahalan, M. D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).
Casey, K. A. et al. Antigen-independent differentiation and maintenance of effector-like resident memory T cells in tissues. J. Immunol. 188, 4866–4875 (2012).
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).
Beura, L. K. et al. Lymphocytic choriomeningitis virus persistence promotes effector-like memory differentiation and enhances mucosal T cell distribution. J. Leukoc. Biol. 97, 217–225 (2015).
Nakanishi, Y., Lu, B., Gerard, C. & Iwasaki, A. CD8+ T lymphocyte mobilization to virus-infected tissue requires CD4+ T-cell help. Nature 462, 510–513 (2009).
Jiang, X. et al. Skin infection generates non-migratory memory CD8+ TRM cells providing global skin immunity. Nature 483, 227–231 (2012).
Sun, J. C. & Bevan, M. J. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300, 339–342 (2003).
Shedlock, D. J. & Shen, H. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300, 337–339 (2003).
Hickman, H. D. et al. Anatomically restricted synergistic antiviral activities of innate and adaptive immune cells in the skin. Cell Host Microbe 13, 155–168 (2013).
Gaylo, A., Schrock, D. C., Fernandes, N. R. J. & Fowell, D. J. T cell interstitial migration: motility cues from the inflamed tissue for micro- and macro-positioning. Front. Immunol. 7, 428 (2016).
Weninger, W., Biro, M. & Jain, R. Leukocyte migration in the interstitial space of non-lymphoid organs. Nat. Rev. Immunol. 14, 232–246 (2014).
Steinbach, K. et al. Brain-resident memory T cells represent an autonomous cytotoxic barrier to viral infection. J. Exp. Med. 213, 1571–1587 (2016).
Glennie, N. D. et al. Skin-resident memory CD4+ T cells enhance protection against Leishmania major infection. J. Exp. Med. 212, 1405–1414 (2015).
Stary, G. et al. A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells. Science 348, aaa8205 (2015).
Khan, T. N., Mooster, J. L., Kilgore, A. M., Osborn, J. F. & Nolz, J. C. Local antigen in nonlymphoid tissue promotes resident memory CD8+ T cell formation during viral infection. J. Exp. Med. 213, 951–966 (2016).
Mackay, L. K. et al. Long-lived epithelial immunity by tissue-resident memory T (TRM) cells in the absence of persisting local antigen presentation. Proc. Natl. Acad. Sci. USA 109, 7037–7042 (2012).
Wu, T. et al. Lung-resident memory CD8 T cells (TRM) are indispensable for optimal cross-protection against pulmonary virus infection. J. Leukoc. Biol. 95, 215–224 (2014).
Çuburu, N. et al. Intravaginal immunization with HPV vectors induces tissue-resident CD8+ T cell responses. J. Clin. Invest. 122, 4606–4620 (2012).
Kang, S. S. et al. Migration of cytotoxic lymphocytes in cell cycle permits local MHC I-dependent control of division at sites of viral infection. J. Exp. Med. 208, 747–759 (2011).
Klein, I. & Crispe, I. N. Complete differentiation of CD8+ T cells activated locally within the transplanted liver. J. Exp. Med. 203, 437–447 (2006).
Wakim, L. M., Waithman, J., van Rooijen, N., Heath, W. R. & Carbone, F. R. Dendritic cell-induced memory T cell activation in nonlymphoid tissues. Science 319, 198–202 (2008).
Posavad, C. M. et al. Enrichment of herpes simplex virus type 2 (HSV-2) reactive mucosal T cells in the human female genital tract. Mucosal Immunol. 10, 1259–1269 (2017).
Zhu, J. et al. Immune surveillance by CD8αα+ skin-resident T cells in human herpes virus infection. Nature 497, 494–497 (2013).
Clark, R. A. Resident memory T cells in human health and disease. Sci. Transl. Med. 7, 269rv1 (2015).
Anderson, K. G. et al. Intravascular staining for discrimination of vascular and tissue leukocytes. Nat. Protoc. 9, 209–222 (2014).
Thompson, E. A., Beura, L. K., Nelson, C. E., Anderson, K. G. & Vezys, V. Shortened intervals during heterologous boosting preserve memory CD8 T cell function but compromise longevity. J. Immunol. 196, 3054–3063 (2016).
Mohammed, J. et al. Stromal cells control the epithelial residence of DCs and memory T cells by regulated activation of TGF-β. Nat. Immunol. 17, 414–421 (2016).
Fife, B. T. et al. Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR-induced stop signal. Nat. Immunol. 10, 1185–1192 (2009).
We thank the members of the Masopust laboratory for helpful discussions. This work was funded by the Howard Hughes Medical Institute Faculty Scholars program (D.M.) and the US National Institutes of Health (grants R01AI111671 and R01AI084913 to D.M.; grant R21AI123600 to B.J.B.). H.D.H. was funded by the Intramural Research Program of the US National Institute of Allergy and Infectious Diseases.
The authors declare no competing financial interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Figure 1 LCMV-specific CD8+ T cells are present throughout the uterus and upregulate Ki67 after local reactivation.
CD90.1+ P14 CD8+ T cells were transferred to C57BL/6J mice one day prior to infection with LCMV Armstrong. a) 60 days later, longitudinal sections of the murine uterine horn were stained for collagen-IV (blue) and CD90.1 (cyan), scale bars = 250 μm. b). P14 CD8+ T cells were enumerated by QIM in the three indicated layers of the uterus. Data are representative of two independent experiments, with 4 mice/experiment. c-d) P14 immune chimeras were t.c. challenged with gp33 peptide and the FRT was harvested 40h later. The uterus was stained for collagen-IV (blue), P14 (cyan) and Ki67 (red), scale bars = 250 μm, and the proportion of P14 CD8+ T cells that were Ki67+ was enumerated. Data are representative of two independent experiments, with 3 mice/experiment. * p<0.05, ** p<0.01, One way ANOVA (b), Kruskal–Wallis ANOVA (d). Box plots with individual data points shown. On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the minimum and maximum data points.
a) Transverse sections of the murine uterine horn were stained for collagen-I (magenta) and the three major layers of the uterine horn are highlighted (scale bars, 200 μm). b) Collagen-I staining intensity across the region highlighted by the yellow line (representing 200 μm) indicates that collagen density is higher in the perimetrium and subjacent connective tissue (top 50 μm) than myometrium (bottom 150 μm). c&d) GFP+ P14 CD8+ T cells were transferred to C57BL/6J mice one day before recipients were infected with LCMV Armstrong. 40 days later, intravital imaging of uterine horn was performed. c) A 3D isometric plot depicts superimposed tracks of several GFP+ P14 CD8+ T cells from the top 50 μm (cyan) and bottom 100 μm (magenta) of the uterus after normalizing starting coordinates to the origin. d) Average track speeds. **** p<0.0001 Mann-Whitney U-test, bars indicate mean ± S.E.M.
Supplementary Figure 3 Proliferation of endogenous LCMV-specific CD8+ T cells within the FRT after local challenge.
C57BL/6 mice were infected with LCMV Armstrong. >90 days later, rechallenged with gp33 t.c. 46h after peptide challenge, mice were injected i.p. with BrdU. Two hours later, H-2Db/gp33 MHC I tetramer+ CD8+ T cells that were isolated from the FRT were stained with anti-Ki67 and anti-BrdU antibodies to determine that cells were proliferating. SIINFEKL constituted a control peptide that did not reactivate TRM cells. a) representative flow cytometry. b) Summary of data from one of two similar experiments (n=4 per group per experiment). ** p<0.01, Mann-Whitney U-test, box plots with individual data points shown. On each box, the central mark indicates the median, and the bottom and top edges of the box indicate the 25th and 75th percentiles, respectively. The whiskers extend to the minimum and maximum data points.
Supplementary Figure 4 CD4+ T cell help is dispensable for CD8+ T cell migration and retention in the FRT, and depletion of dendritic cells does not impair in situ proliferation.
Wild type (WT) or MHC II– mice received 5X104 P14 CD8+ T cell and were infected with LCMV Armstrong one day later. a) Representative immunohistochemistry images showing P14 CD8+ T cells (stained using anti-CD90.1 antibody, cyan) in the uterine horn 30 days post-infection in both strains of mice (scale bars, 250 μm). b&c) Number of P14 CD8+ T cells enumerated by QIM on indicated days. d) P14 immune chimeras were made as described in CD11c-DTR bone chimeric mice as described in Fig. 4e. Dendritic cells were identified as CD11c+/MHC II bright CD45+ cells. Flow cytometric plots of CD45+ leukocytes (top row) and CD90.1+ P14 CD8+ T cells (bottom row) isolated from the FRT indicate that the depletion of DCs via diphtheria toxin (DT) did not impair induction of P14 proliferation program 24h after gp33 t.c. Data are representative of two separate experiments with 3 mice/group per experiment. ns= not significant, Mann-Whitney U-test, bars indicate mean ± S.E.M.
Supplementary Figure 5 Equilibration of P14 CD8+ memory T cells in lymph nodes and spleen after parabiosis.
a) Schematic for experiments in Fig. 6. Both CD45.1+ P14 and CD90.1+ P14 LCMV immune chimeras were generated in separate C57BL/6 (CD45.2+/CD90.2+) mice. 60 days after LCMV infection, mice underwent parabiosis surgery. 14-30 days later, both mice were challenged t.c. with gp33 peptide. CD45.1+ and CD90.1+ P14 were assessed in the spleens and FRTs of both parabiont pairs at day 2 and day 30 post-recall. b) Representative plots, gated on CD8+ lymphocytes, and c&d) plots indicating the ratio of CD45.1+ to CD90.1+ P14 memory CD8+ T cells in individual parabiont pairs before gp33 peptide challenge. Data are representative of two separate experiments with at least three parabiont pairs/experiment totaling 12 individual mice in individual groups. Wilcoxson signed rank test. ns-not significant.
Naïve CD45.1+ OT-I CD8+ T cells were intravenously transferred to C57Bl/6 mice. The following day, recipients were infected with VSV-OVA i.v. 120 days later, lymphocytes were isolated from the indicated tissues and analyzed by flow cytometry. Top row gated on CD8β+ lymphocytes, bottom row gated on CD45.1+ OT-I CD8+ T cells.
Migration of CD8+ T cells in the uterine stroma at the peak of viremia
Migration of CD8+ T cells at the peak of effector response
Migration of resident memory CD8+ T cells in the FRT
Reduced migrational speed of TRM cells after local antigen recognition in the FRT
Non-antigen-specific recall failed to induce deceleration of TRM cells
Cognate antigen interaction is essential for arrest of TRM cell motility in the FRT
Examples of memory T cells undergoing division in the uterine stroma
TRM cells divide in situ after local reactivation
About this article
Cite this article
Beura, L.K., Mitchell, J.S., Thompson, E.A. 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). https://doi.org/10.1038/s41590-017-0029-3
Journal of Experimental Medicine (2021)
Seminars in Immunology (2020)
Viral Immunology (2020)
Nature Reviews Immunology (2020)
Trends in Parasitology (2020)