Developmental plasticity allows outside-in immune responses by resident memory T cells

Abstract

Central memory T (T_CM) cells patrol lymph nodes and perform conventional memory responses on restimulation: proliferation, migration and differentiation into diverse T cell subsets while also self-renewing. Resident memory T (T_RM) cells are parked within single organs, share properties with terminal effectors and contribute to rapid host protection. We observed that reactivated T_RM cells rejoined the circulating pool. Epigenetic analyses revealed that T_RM cells align closely with conventional memory T cell populations, bearing little resemblance to recently activated effectors. Fully differentiated T_RM cells isolated from small intestine epithelium exhibited the potential to differentiate into T_CM cells, effector memory T cells and T_RM cells on recall. Ex-T_RM cells, former intestinal T_RM cells that rejoined the circulating pool, heritably maintained a predilection for homing back to their tissue of origin on subsequent reactivation and a heightened capacity to redifferentiate into T_RM cells. Thus, T_RM cells can rejoin the circulation but are advantaged to re-form local T_RM when called on.

Fig 1.. Local reactivation of T_RM precipitates egress to circulation.

a. Experimental design. b. Pooled draining and non-draining SLOs were used to phenotype the graft-derived CD90.1⁺ OT-I T cells post reactivation. Gated on live CD90.1⁺CD8α⁺ T cells c&d. Experimental design and representative flow plots of H-2K^b/SIINFEKL tetramer⁺ cells in the blood of mice after indicated days post-tattooing with SIINFEKL. Flow plots are gated on live CD8α⁺ cells (top row) and H-2K^b/SIINFEKL tetramer ⁺, CD8α⁺ T cells (middle row). Expression of CD103, CD49a, Ly6C, KLRG1 and CD62L was compared between CD45.1⁺ (circulating memory derived, orange) and CD90.1⁺ (resident memory derived, blue) cells 10 days post-recall in the bottom row. e. Bar graph depicting frequency of CD103⁺ and Ly6C^lo cells between CD90.1⁺ and CD45.1⁺ cells. Bars represent mean ± s.e.m and symbols represent individual animals. Two-tailed Mann-Whitney U test. f&g. Experimental design and representative flow plots of H-2D^b/gp33 tetramer⁺ cells in the blood of mice after indicated days post t.c. challenge. Gated on live CD8α⁺ T cells (top row) and live H-2D^b/gp33 tetramer⁺, CD8α⁺ T cells (middle row). Superimposed histograms of CD62L, Ly6C, and CD44 expression on CD90.1⁺ (blue) and CD45.1⁺ cells (orange) within the H-2D^b/gp33⁺ specific population from blood 10 days post challenge. b-g. n=5 mice per experiment and one experiment shown of 3 independent experiments.

Fig 2.. Epigenetic profiling of T_RM reveals memory state with potential developmental plasticity.

a. Representative phenotype of memory CD90.1⁺ CD8+ P14 cells isolated from the blood (PBL), epithelium (SI IEL) or lamina propria (SI LP) of the small intestine, salivary gland, or female reproductive tract (FRT) of LCMV immune chimeras 90 days after infection. Representative data of n=10 from two independent experiments. b. Principal Component Analysis (PCA) of previously published CD8⁺ naive, T_CM, T_EM, and gut T_RM transcriptome. n=2 per group. c. CD8⁺ CD90.1⁺ P14 cells were isolated 4, 8, or 90 days after LCMV infection, and naive P14 cells were used for comparison. Spleen day 4 and 8 effector populations were sorted into CD127^hiKLRG1^lo (memory precursor, MP) or CD127^loKLRG1^hi (terminal effector, TE) populations. Memory populations were sorted into T_CM (CD44⁺CD127⁺CD62L⁺, spleen), T_EM (CD44⁺CD127⁺CD62L^-, spleen), T_RM (CD62L^-CD103⁺CD69⁺Ly6C^-, SI IEL), and naive (CD44^-CD127^-CD62L⁺, spleen). Whole genome bisulfite sequencing was performed to determine allelic frequency of cytosine methylation. Day 4 and day 8 effector P14 subset methylation data were obtained from a previously published study. PCA of the top 3000 variably methylated CpGs in CD8⁺ naive, day 4 and day 8 MP and TE, T_CM, T_EM and T_RM, each dot represents one sample from 50,000 cells. d. Hierarchical summary graph of CD8⁺ T cell subset differentiation potential derived from a DNA methylation-based T cell multipotency index. CpG sites were identified from a machine learning algorithm using naive and exhausted gp33-specific CD8 T cells as a training data set. Plasticity indices were derived from the methylation status of 598 CpG sites. Each bar represents an individual sample.

Fig 3.. Transdifferentiation of T_RM into circulating memory T cell subsets.

CD45.1⁺ P14 cells were isolated from LCMV immune chimeras 90 days after LCMV infection and sorted into T_CM (CD8α⁺CD45.1⁺CD44⁺CD127⁺CD62L⁺, isolated from pooled lymph nodes), T_EM (CD8α⁺CD45.1⁺CD44⁺CD127⁺CD62L^-, isolated from spleen), and T_RM (CD8α⁺CD45.1⁺CD62L^-CD103⁺CD69⁺Ly6C^-, isolated from SI IEL). 20,000 T_CM, T_EM, T_RM, and naive P14 cells were transferred into separate C57BL/6J recipients, which were subsequently infected with LCMV Armstrong. The recall response was monitored in blood until 100 days post infection, after which mice were sacrificed and tissues were analyzed. a. Post sort analysis of T_RM cells and experimental design. Number (b) and percent of CD62L⁺ (c) of each transferred P14 CD8⁺ T cell subset in blood over time. d. Representative FACS analysis of CD127, CD62L and Ly6C expression on transferred cells in blood or mesenteric LN 100 days after recall. e. Representative histograms of CD103, CD69, CD127 and Ly6C expression on transferred cells in spleen 100 days post transfer. f. Longitudinal Ly6C and g. CCR9 expression and representative histograms on P14 CD8⁺ T cells. One-way ANOVA with Tukey’s multiple comparison test. b, f. Symbols represent mean ± s.e.m. c. Symbols represent mean with linear regression line. g. Bars represent mean ± s.e.m. and symbols represent individual mice. b-g. n=4 mice per group per experiment and data are representative of 1 of 4 independent experiments with similar results.

Fig 4.. Developmental plasticity and tissue redistribution of T_CM and T_RM

. a. Recovery of P14 cells from transferred T_CM, T_RM and naive (as in Fig. 3) in spleen, SI IEL, SI LP, salivary gland (SG) and female reproductive tract (FRT) 100 days after recall. b. Ratio of P14 cells in spleen to either SI IEL, SI LP, SG and FRT. c. CD69, CD103, Ly6C and Granzyme B expression on transferred cells recovered from SI epithelium after 100 days after recall. Gated on i.v.^-, CD8β⁺, CD44⁺, CD45.1⁺, H-2D^b/gp33 tetramer⁺. d. Representative immunofluorescence images of SI. Scale bars, 50 microns. a-c. One-way ANOVA with Tukey’s multiple comparison test. Bars represent mean ± s.e.m. and symbols represent individual mice. a-d. n=5 mice per group per experiment and one shown of four independent experiments with similar results.

Fig 5.. Ex-T_RM remain epigenetically poised for migration and T_RM re-differentiation.

a. 1º memory T_CM from LN and T_RM from SI IEL were sorted (as in Fig. 3) and transferred to naive recipients, followed by LCMV infection. 100 days later, splenocytes containing 10⁵ 2º memory P14 were transferred to naive recipients, again followed by LCMV infection. 50 days later, 3º memory P14 were analyzed for phenotype and distribution. 1º and 2º responses were assessed for comparison in d and e. b. 3º memory P14 isolated from spleen and SI IEL. Multiple unpaired two-tailed Student’s t-test. c. Ratio of 3º memory P14 isolated from SI IEL to spleen. Unpaired two-tailed Student’s t-test. d, e. Percentage of CD69⁺CD103⁺ 3º memory P14 cells and representative plots of CD69, CD103 and Ly6C expression on P14 isolated from SI IEL. 3º Ex-T_CM and Ex-T_RM were compared to 1º and 2º Ex- T_CM mucosal memory. Gated on i.v.^-, CD8β⁺, CD44⁺, CD45.1⁺, H-2D^b/gp33 tetramer⁺. One-way ANOVA with Tukey’s multiple comparison test. b-e. Bars represent mean ± s.e.m. and symbols represent individual mice. n=5 mice per group per experiment and one shown of two independent experiments with similar results.

Fig 6.. Ex-T_RM are poised to reacquire T_RM characteristics in response to cytokines.

1º memory T_CM from LN, T_EM from spleen, and T_RM from SI epithelium were sorted (as in Fig. 3) and transferred to naive recipients which were subsequently infected with LCMV-Armstrong. 50–70 days post infection, splenocytes were isolated and progenies of each cell subtype were stimulated in vitro with IL-15 and TGFβ to induce T_RM-biased differentiation program. IL-2 was included in all cultures. Cells were analyzed after four days in culture. a. Representative flow plots and b. summary of IL-15, TGFβ and IL-2 stimulated cultures and gated on H-2D^b/gp33 tetramer⁺, CD45.1⁺, CD8α⁺ T cells. T_CM, T_RM (n=6 technical replicates per group) and T_EM (n=5 technical replicates) from 3 pooled mice per group are shown from one of two independent experiments with similar results.