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 (T_RM cells) within the reproductive tracts of live female mice. We found that mucosal T_RM cells were highly motile, but paused and underwent in situ division after local antigen challenge. T_RM cell reactivation triggered the recruitment of recirculating memory T cells that underwent antigen-independent T_RM cell differentiation in situ. However, the proliferation of pre-existing T_RM cells dominated the local mucosal recall response and contributed most substantially to the boosted secondary T_RM cell population. We observed similar results in skin. Thus, T_RM cells can autonomously regulate the expansion of local immunosurveillance independently of central memory or proliferation in lymphoid tissue.

Fig. 1. Migration of CD8⁺ T cells in the FRT

a, CD90.1⁺GFP⁺CD8⁺ P14 T cells were transferred to female C57BL/6J mice 1 d before Infection with LCMV Armstrong. We stained the FRT with DAPI (blue), anti-CD90.1 (to label P14; cyan) and anti-LCMV nucleoprotein (magenta) 4.5, 8 or 40 d later. Scale bars, 200 μm. b,c, The average track speed (b) and motility coefficients (c) of GFP⁺CD8⁺ P14 T cells in the FRT on the indicated days after LCMV infection, as determined by intravital microscopy. d, Flower plots of 2D tracks of several GFP⁺CD8⁺ P14 T cells, superimposed after normalization of their starting coordinates to the origin. e, GFP⁺CD8⁺ OT-I T cells were transferred to female C57BL/6J mice 1 d before infection with VSV-OVA or VV-OVA. The plot shows the average track speed of GFP⁺CD8⁺ OT-I T cells in the uterine horn 60 d after the respective infections. f, Transverse sections of mouse uterine horn stained for collagen-l (magenta), E-cadherin (cyan) and nuclei (DAPI; blue). Part of the section on the left (white rectangle) is shown at higher magnification on the right, depicting three layers of uterine horn with different collagen densities. Scale bars, 200 μm. g, CD8⁺ P14 memory T cell track speeds binned into three groups (<2 μm, 2–5 μm and >5 μm) on the basis of their average distance from the collagen (SHG) surface. Data in b,c,e,g are shown as mean ± s.e.m.; circles represent individual cells. **P<0.01, ***P< 0.001, Kruskal-Wallis one-way analysis of variance (ANOVA) with Dunn’s multiple comparison test. Data are representative of two independent experiments with n = 4 mice per infection (a,f) or three separate experiments with n = 3 mice per group per experiment (b,c,e,g).

Fig. 2. Motility arrest by T_RM cells after cognate antigen interaction

a, To assess in situ T_RM cell reactivation, we transferred naive GFP⁺CD8⁺ P14 T cells into female C57BL/6J mice 1 d before infection with LCMV Armstrong, and 60 d later we challenged the mice trans-cervically with PBS, P14 reactivating peptide gp33 or control peptide SIINFEKL. Shown are representative snapshots, from Supplementary Videos 4 and 5, of the maximal projection of 3D z-stack images of P14 immune chimeras 12 h after trans-cervical exposure to peptide. Green, GFP⁺CD8⁺ P14 T cells; yellow, CD11c⁺ dendritic cells. Scale bars, 40 μm. b, Average track speed and motility coefficients of GFP⁺CD8⁺ P14 T cells 12 h or 48 h after trans-cervical challenge with the indicated peptide. c,d, To assess the motility of a bystander population of memory T cells, we transferred CFP⁺CD8⁺ P14 T cells into naive mice 1 d before LCMV infection. Thirty days later, we transferred GFP⁺CD8⁺ OT-I T cells into these LCMV immune chimeras, and then infected them with VSV-OVA. Thirty days after VSV-OVA infection (60 d after LCMV infection), we challenged these double-immune chimeras trans-cervically with gp33 peptide, and carried out intravital microscopy 12 h after challenge. The average track speed (c) and motility coefficients (d) of P14 and OT-I CD8⁺ T cells 12 h after gp33 peptide challenge are shown. Data are representative of two separate experiments with 4 mice per group per experiment. ***P< 0.001; ns, not significant; Kruskal-Wallis ANOVA with Dunn’s multiple comparison test (b) or Mann-Whitney U-test (c,d). Data in b-d are shown as the mean ± s.e.m.; circles represent individual cells.

Fig. 3. In situ division of CD8⁺ T cells in the FRT

a,b, Accumulation of P14 CD8⁺ T cells after local T_RM cell reactivation in FRT. a, Representative maximal projections of 3D z-stack images of GFP⁺CD8⁺ P14 T cells (cyan) after trans-cervical challenge with gp33. Scale bars, 30 μm. b, The abundance of P14 CD8⁺ T cells enumerated by QIM in 7-μm sections of FRT. c, Top, representative time-lapse images (time stamps in minutes and seconds are shown above the images) of GFP⁺CD8⁺ P14 T cells (cyan) undergoing division beginning ~36 h after gp33 challenge. Scale bars, 40 μm. Bottom, magnified views (4× magnification compared with the images above) of dividing cells, indicated by white arrowheads (or two yellow arrowheads for cells after cytokinesis). Magnified cells are outlined by white rectangles in the images above. d, P14 immune chimeras were challenged with peptides (gp33 or SIINFEKL) or mock-challenged (with PBS), and tissues were harvested 48 h after challenge. Animals received 2 mg of BrdU i.p. before cell collection. e, OT-I immune chimeras were challenged with viruses (VV-OVA and VV-gp33) or mock-challenged, and tissues were harvested 48 h after recall. In d and e, numbers in corners indicate the percentage of cells in each quadrant. f,g, The frequency of Ki67⁺ cells in FRT of the mice described in d (see f) and e (see g). SIIN, SIINFEKL. h, Instantaneous track speed versus time, beginning 15min before cytokinesis, for four representative dividing and four nondividing P14 CD8⁺ T cells. i, The mean track speed of dividing and nondividing cells 36h after recall. *P< 0.05, ***P< 0.001, Mann-Whitney U-test. Data in b,f-i are shown as the mean ± s.e.m. Data are representative of two separate experiments with 3 (a,b) or 4 (f,g) mice per group per experiment or are pooled from 6 individual movies and 3 animals (i).

Fig. 4. T_RM cells initiate division in mucosae after reactivation

a, The presence of remaining P14 memory CD8⁺ T cells in the indicated tissues 4 d after i.p. anti-CD90.1 treatment in P14 LCMV immune chimeric mice. Numbers indicate the percentage of cells in the gate. b, P14 immune chimeras were treated as in a. Their blood was assessed for successful depletion of P14 CD8⁺ T cells 4 d later, and they received anti-VCAM-1 and anti-CD49d i.p. on day 4. On day 5, mice were rechallenged trans-cervically with gp33 peptide or PBS (Mock), and they were assessed 24h or 36 h after rechallenge. Mice received 2 mg of BrdU i.p. 2 h before they were killed, and P14 CD8⁺ T cells isolated from FRT were stained for Ki67 and BrdU incorporation by flow cyotmetry. Numbers in corners indicate the percentage of cells in the gate. c, The percentage of Ki67⁺ or BrdU⁺ cells among P14 CD8⁺ T cells isolated from the FRT of mice treated with anti-CD90.1, anti-VCAM-1, anti-CD49d and gp33 as indicated. Data points represent individual mice. d, Ki67⁺CD8⁺ P14 T cell frequency as enumerated by flow cytometry 24h after challenge in wild-type (WT) and MHC-II-deficient P14 immune chimeric mice treated as in b. e, P14 immune chimeras were generated in CD11c-DTR chimeric mice. Sixty days later, mice were either treated with diphtheria toxin (every 48 h starting 2 d before recall) or left untreated. The mice were also treated with antibodies to CD90.1, VCAM-1, CD49d and gp33 peptide as in b and c. The plot shows the percentage of Ki67⁺CD8⁺ P14 T cells isolated from FRT as determined by flow cytometry 24 h after challenge. ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Kruskal-Wallis ANOVA (c) or Mann-Whitney U-test (d,e). Data in c–e are shown as the mean ± s.e.m. Data are representative of two separate experiments with 3 (a) or 4 (d,e) mice per group per experiment, or three separate experiments with 4 mice per group per experiment (b,c).

Fig. 5. Recruited circulating bystander memory CD8⁺ T cells adopt a T_RM cell phenotype

a, To pull non-antigen-specific recirculating OT-I memory cells to the FRT, we transferred OT-I memory CD8⁺ T cells i.v. into P14 immune chimeras. The next day, we challenged recipients trans-cervically with PBS (mock) or gp33 peptide. Mice were rested until day 45 or day 75, at which time we used QIM to enumerate OT-I CD8⁺ T cells in the FRT. b, The frequency of FRT OT-I or P14 CD8⁺ T cells that were CD69⁺, as evaluated by flow cytometry. c, Representative flow cytometry plots of CD69 and CD103 staining on P14 and OT-I CD8⁺ T cells 45 d after recall. Numbers in corners indicate the percentage of cells in each quadrant. d,e, To confirm that recruited OT-I CD8⁺ T cells were resident, we carried out parabiotic surgery to conjoin mice treated 40 d prior with trans-cervical gp33 peptide as described above (P14 immune hosts) with naive B6 partner mice. We examined each parabiont for the presence of OT-I CD8⁺ T cells in blood and FRT 35 d after surgery. *P<0.05, **P< 0.01, Kruskal-Wallis ANOVA (a) or Mann-Whitney U-test (b,d,e). Box plots indicate medians (center lines), 25th and 75th percentiles (bottom and top box edges, respectively), minima and maxima (whiskers), and individual data points (circles). Data are representative of three separate experiments with 4 mice per group per experiment (a–c) or two separate experiments with four parabiont pairs per experiment (d,e).

Fig. 6. T_RM cell proliferation dominates expansion of local secondary memory T cell populations

a, Equilibration of recirculating memory CD8⁺ T cells in parabiotically conjoined CD45.1⁺ and CD90.1⁺ P14 immune chimeras challenged trans-cervically with gp33 peptide 14–30 d after surgery. Scale bars, 50 μm. a–g, We analyzed spleen and FRT by flow cytometry 2 d (a) or 30 d (e) after gp33 challenge to evaluate the relative contributions of host and donor P14 CD8⁺ T cells to the secondary response. Tissues from a CD45.1⁺ immune chimera are shown, and histograms indicate phenotypes of gated CD45.1⁺ and CD90.1⁺ P14 CD8⁺ T cells at day 2 (a) or day 30 (e) post-recall. CD45.1⁺ and CD90.1⁺ P14 CD8⁺ T cells were also analyzed in each parabiont by immunofluorescence staining 2 d (b) and 30 d (f) after t.c. gp33 challenge. d, Host and parabiotic partner P14 CD8⁺ T cells enumerated by QIM on the indicated days after trans-cervical gp33 challenge. c,g, The ratio of host and parabiotic partner P14 CD8⁺ T cells in each parabiont 2 d (c) and 30 d (g) after trans-cervical peptide recall. All data are representative of two separate experiments with at least four parabiont pairs/time points, with a total of > 16 individual mice in individual groups. **P<0.01, Mann-Whitney U-test. In a and e, numbers adjacent to outlines indicate the percentage of cells in the gate. Box plots indicate medians (center lines), 25th and 75th percentiles (bottom and top box edges, respectively), minima and maxima (whiskers), and individual data points (circles).

Fig. 7. T_RM cell proliferation dominates expansion of local secondary memory T cell populations after FRT or skin rechallenge

a, Tissues from VSV-OVA-immunized CD45.1⁺ and CD90.1⁺ OT-I immune chimeras that were surgically conjoined by parabiosis to achieve equilibration of recirculating memory CD8⁺ T cells. a–f, Both parabionts were challenged with MVA-OVA 14–30 d after surgery via either the (a–c) trans-cervical or (d–f) epicutaneous route. OT-I CD8⁺ T cells were analyzed by immunofluorescence staining and enumerated by QIM in either FRT (b,c) or skin epidermis (e,f) and in the spleen 30 d after rechallenge. Magenta, CD45.1; cyan, CD90.1; blue, DAPI (nuclei). Scale bars, 50 μm. Ratios of host and donor-derived OT-I CD8⁺ T cells were calculated after normalization to the respective ratios in spleen. Absolute numbers of OT-I CD8⁺ T cells in FRT (c) and skin epidermis (f) in unchallenged and MVA-OVA-challenged (30 d post-recall) mice are shown. All data are representative of two separate experiments with three parabiont pairs per experiment, with a total of 12 individual mice in individual groups. *P< 0.05, **P<0.01, Mann-Whitney U-test (b) or unpaired t-test (e). Box plots indicate medians (center lines), 25th and 75th percentiles (bottom and top box edges, respectively), minima and maxima (whiskers), and individual data points (circles).