Lymphatic vessel transit seeds cytotoxic resident memory T cells in skin draining lymph nodes

Abstract

Lymphatic transport shapes the homeostatic immune repertoire of lymph nodes (LNs). LN-resident memory T cells (T_RMs) play an important role in site-specific immune memory, yet how LN T_RMs form de novo after viral infection remains unclear. Here, we tracked the anatomical distribution of antiviral CD8⁺ T cells as they seeded skin and LN T_RMs using a model of vaccinia virus-induced skin infection. LN T_RMs localized to the draining LNs (dLNs) of infected skin, and their formation depended on the lymphatic egress of effector CD8⁺ T cells from the skin, already poised for residence. Effector CD8⁺ T cell transit through skin was required to populate LN T_RMs in dLNs, a process reinforced by antigen encounter in skin. Furthermore, LN T_RMs were protective against viral rechallenge in the absence of circulating memory T cells. These data suggest that a subset of tissue-infiltrating CD8⁺ T cells egress from tissues during viral clearance and establish a layer of regional protection in the dLN basin.

Figure 1.. Lymph node resident memory T cells form in draining lymph nodes following skin infection

(A-G) P14 TCR-tg T cells (CD90.1⁺) were transferred into naïve mice (CD90.1⁻) and the following day, ear skin was infected by scarification with vaccinia virus expressing GP33_33–41 (VV-GP33). (A) Representative flow plots (left, gated on live, CD8α⁺, CD45 IV⁻, lymphocytes) and quantification (B) of P14 T cells in lymph nodes (LN) 28 days post-infection (d.p.i.). DLN (dLN, ipsilateral parotid); non-dLN (ndLN, contralateral parotid). (C) Representative flow plots (left, gated on CD44⁺CD90.1⁺ cells in (A)) and quantification (D) of CD69⁺CD62L⁻ P14 T cells in LNs 28 d.p.i. (E) Representative histograms at 28 d.p.i. comparing P14 T cells. Top is CD69⁻CD62L⁺ P14 T cells in the dLN; middle and bottom are CD69⁺CD62L⁻ in dLN (middle) or skin (bottom). (F-G) Phenotype of P14 T cells in ipsilateral and contralateral parotid (Parot), brachial (Brac), and inguinal (Inguin) LNs as well as mesenteric (Mes) LNs and spleen 28 d.p.i. (H) Number of CD69⁺CD103⁺ P14 T cells in skin and draining parotid LN over time. (A-E) Data are combined from 4 experiments for a total of n=13 mice. (F) Data are representative of two experiments with 4 mice per experiment. (H) Data are combined from at least two experiments per time point with 3–4 mice per experiment. Day 102 time point represents one experiment at day 101 and another at day 103 post-infection. Bars represent average + SEM. Each point represents an individual mouse. Statistical significance was determined using Wilcoxon matched-pairs signed rank test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Figure 2.. Dermal lymphatic vessels are necessary for LN T_RM formation after skin infection

(A) Number of CD69⁺CD62L⁻ P14 T cells in the skin 49–50 days post-infection with vaccinia virus expressing GP33_33–41 (VV-GP33). (B) Representative flow plots gated on live, CD44⁺CD8α⁺CD90.1⁺ lymphocytes and (C) quantification of percent and (D) total number of CD69⁺CD62L⁻ in draining lymph node (dLN). Data are combined from 2 experiments with 3–5 mice per group per experiment. (E) Experimental design for (F-H). (F) Number of CD45 IV⁻CD90.1⁺CD69⁺CD62L⁻ P14 T cells in skin. (G) Percent and (H) number of CD45 IV⁻CD90.1⁺CD69⁺CD62L⁻ P14 T cells in dLN. Data are representative of three experiments with 3–5 mice per group per experiment. (I) Experimental design for (J-M). (J) Representative flow plot of tetramer staining for H2-K^b-B8R-specific T cells. Gated on live, CD8a⁺ lymphocytes. (K) Representative flow plots and (L) quantification of percent and (M) number of Kaede-Red fluorescence in tetramer⁺ cells. Data are combined from two experiments with 5 mice per experiment. Statistical significance was determined using unpaired student’s t test (A,G,H), Mann Whitney test (C,D,F) or Wilcoxon matched-pairs signed rank test (L,M). Bars represent average + SEM. Each point represents an individual mouse. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Fig. 3. Egressing T cells share transcriptional similarities with LN T_RM

Kaede-tg ears were infected with VV-GP33 by scarification and photoconverted (KaedeGreen à KaedeRed) 11 days later. 24 hours after photoconversion, KaedeGreen⁺ and KaedeRed⁺ cells were sorted by FACS (Live, CD45 IV⁻ CD8⁺CD44⁺) from draining lymph nodes and analyzed by scRNA-seq. Prior to sorting, viral specific cells were stained with 2 APC conjugated tetramers (H2-K^b-B8R_20–27 and H2-D^b-GP_33–41). APC was then tagged with an oligonucleotide to identify a subset of viral-specific cells by scRNA-Seq. (A) UMAP projection of cells colored according to cluster. (B) CITE-seq detection of tetramers visualized by UMAP projection. (C) UMAP projection showing distribution of KaedeGreen⁺ or KaedeRed⁺ and (D) quantification of their proportions within each cluster. (E) Expression of select genes across clusters normalized from −1 to 1. Size of dot corresponds to percentage of cells in cluster expressing the transcript. (F) UMAP projection of select transcripts or (G) signature score for LN resident memory T cells (Molodtsov et al. 2021).

Figure 4.. T cell transit through skin is required for the formation of LN T_RM

(A) Experimental design for (B-F). (B) Frequency and (C) number of P14 T cells in untreated (14 d.p.i.) or depleted mice (16 d.p.i., 3 days post depletion) in draining lymph node (dLN). (D) Prevalence of P14 T cells in LNs of depleted mice 15 days post depletion (contralateral, non-dLN, ndLN). (E) Percent of P14 T cells with a CD69⁺CD62L⁻ residence phenotype and (F) representative surface phenotype of P14 T cells in dLNs of depleted (orange) or IgG treated (black). Data are combined from two experiments (B,C,E,F) or representative of two experiments (D) with 3–4 mice per group per experiment. (G) Experimental design with a single ear infection for (H-L). Data are combined from two experiments with 3–5 mice per group per experiment. (H) Total number of P14 T cells per spleen 28 d.p.i. (I) Representative flow plots and (J) quantification of CD69⁺CD62L⁻ P14 T cells in dLNs. (K) Representative flow plots and (L) quantification of CD69⁺CD103⁺ P14 T cells in dLNs. (M) Experimental design for (N-Q). Quantification of P14 T cells in dLNs (N-Q). Data are combined from two experiments (N-Q) with 3–4 mice per experiment. Bars represent average + SEM. Statistical significance was determined using Mann-Whitney (B,C), paired student’s t test (D,N,O,P), Wilcoxon matched pairs signed rank test (E-Q), one way ANOVA (H), or Kruskal-Wallis test with multiple comparisons (J,L). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Figure 5.. LN T_RM establishment is dependent upon antigen recognition in skin

(A) Experimental design for (B-E). (B) Representative flow plots and (C) quantification of frequency of CD45.1⁺ OT-1 T cells 27–28 d.p.i. in draining lymph nodes (dLN). Flow plots are gated on live, CD8a⁺ lymphocytes. (D) Representative flow plots gated on CD45.1⁺ OT-1 T cells in dLN and (E) quantification of number of CD69⁺CD62L⁻ OT-I T cells in dLNs. Data are combined from two experiments with 4 mice per experiment. (F) Quantification of GFP expression in CD45.1⁺ OT-1 Nur77-GFP T cells transferred to mice one day prior to infection with VV-OVA. (G) Experimental design for (H and I) where CD45.1⁺ OT-1 Nur77-GFP T cells were transferred into naïve mice and the ear was infected the following day with VV-OVA. 80 d.p.i mice either received a secondary VV-OVA challenge (Challenged) or did not (Unchallenged). (H) Representative flow plots and (I) quantification of GFP expression in CD45.1⁺ OT-I T cells. Data are combined from 2 experiments. Each point represents an individual mouse. Statistical significance was determined using Wilcoxon matched-pairs signed rank test (C,E), one way ANOVA (F), or Kruskal-Wallis test with multiple comparisons (I). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Figure 6.. LN T_RM are poised for cytotoxicity and localized protection

(A) UMAP representation of single-cell RNA-seq data from CD45 IV⁻ P14 TCR-tg T cells isolated from draining lymph nodes (dLN) 45 d.p.i. with VV expressing GP33_33–41 (VV-GP33). (B) Cells scored against a published gene signature for LN resident memory T cells (T_RM). (C) Single-cell regulatory network inference and clustering (SCENIC) analysis inferring gene regulatory networks in each cluster. (D) Differentially expressed genes between resident (cluster 2) and circulating memory (clusters 0,1,3,5) cells. (E) Expression of Gzmb, Gzmk, and Prf1 in T_CIRC (clusters0,1,3,5) and T_RM (cluster 2). (F) Expression of Granzyme B by OT-1 TCR-tg T cells at least 60 days after ear skin infection with VV expressing SIINFEKL (VV-OVA) as measured by flow cytometry. Data are representative of two experiments with 5 mice per experiment. (G) Experimental design for (H and I). (H) Representative image of plaques and (I) quantification of plaque forming units (PFU) measured 3 days following challenge dLN (ipsilateral) or contralateral, non-dLN (ndLN). Data are combined from 3 experiments with 3–4 mice per experiment. Each point represents an individual mouse. Statistical significance was determined using Wilcoxon matched-pairs signed rank test (D,E,F,I). ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001