Intravital mucosal imaging of CD8+ resident memory T cells shows tissue-autonomous recall responses that amplify secondary memory

Abstract

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.

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Fig. 1: Migration of CD8+ T cells in the FRT.
Fig. 2: Motility arrest by TRM cells after cognate antigen interaction.
Fig. 3: In situ division of CD8+ T cells in the FRT.
Fig. 4: TRM cells initiate division in mucosae after reactivation.
Fig. 5: Recruited circulating bystander memory CD8+ T cells adopt a TRM cell phenotype.
Fig. 6: TRM cell proliferation dominates expansion of local secondary memory T cell populations.
Fig. 7: TRM cell proliferation dominates expansion of local secondary memory T cell populations after FRT or skin rechallenge.

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Acknowledgements

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.

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L.K.B., J.S.M., E.A.T., J.M.S., J.M., S.W., R.F. and B.J.B. performed the experiments; H.D.H., V.V. and B.T.F. contributed critical reagents and experimental help; L.K.B., J.S.M. and E.A.T. analyzed intravital microscopy data; L.K.B. and D.M. wrote the manuscript; and D.M. was responsible for research supervision, coordination and strategy.

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Correspondence to David Masopust.

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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.

Supplementary Figure 2 CD8+ T cells in uterine perimetrium migrate slower than those in myometrium.

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.

Supplementary Figure 6 VSV infection establishes broadly distributed CD8+ T cell memory.

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.

Supplementary information

Videos

Supplementary Video 1

Migration of CD8+ T cells in the uterine stroma at the peak of viremia

Supplementary Video 2

Migration of CD8+ T cells at the peak of effector response

Supplementary Video 3

Migration of resident memory CD8+ T cells in the FRT

Supplementary Video 4

Reduced migrational speed of TRM cells after local antigen recognition in the FRT

Supplementary Video 5

Non-antigen-specific recall failed to induce deceleration of TRM cells

Supplementary Video 6

Cognate antigen interaction is essential for arrest of TRM cell motility in the FRT

Supplementary Video 7

Examples of memory T cells undergoing division in the uterine stroma

Supplementary Video 8

TRM cells divide in situ after local reactivation

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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

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