ABC transporters and NR4A1 identify a quiescent subset of tissue-resident memory T cells - PubMed (original) (raw)

. 2016 Oct 3;126(10):3905-3916.

doi: 10.1172/JCI85329. Epub 2016 Sep 12.

Shiny Nair, Simon M Gray, Heba N Nowyhed, Rakesh Verma, Joanna A Gibson, Clara Abraham, Deepak Narayan, Juan Vasquez, Catherine C Hedrick, Richard A Flavell, Kavita M Dhodapkar, Susan M Kaech, Madhav V Dhodapkar

ABC transporters and NR4A1 identify a quiescent subset of tissue-resident memory T cells

Chandra Sekhar Boddupalli et al. J Clin Invest. 2016.

Abstract

Immune surveillance in tissues is mediated by a long-lived subset of tissue-resident memory T cells (Trm cells). A putative subset of tissue-resident long-lived stem cells is characterized by the ability to efflux Hoechst dyes and is referred to as side population (SP) cells. Here, we have characterized a subset of SP T cells (Tsp cells) that exhibit a quiescent (G0) phenotype in humans and mice. Human Trm cells in the gut and BM were enriched in Tsp cells that were predominantly in the G0 stage of the cell cycle. Moreover, in histone 2B-GFP mice, the 2B-GFP label was retained in Tsp cells, indicative of a slow-cycling phenotype. Human Tsp cells displayed a distinct gene-expression profile that was enriched for genes overexpressed in Trm cells. In mice, proteins encoded by Tsp signature genes, including nuclear receptor subfamily 4 group A member 1 (NR4A1) and ATP-binding cassette (ABC) transporters, influenced the function and differentiation of Trm cells. Responses to adoptive transfer of human Tsp cells into immune-deficient mice and plerixafor therapy suggested that human Tsp cell mobilization could be manipulated as a potential cellular therapy. These data identify a distinct subset of human T cells with a quiescent/slow-cycling phenotype, propensity for tissue enrichment, and potential to mobilize into circulation, which may be harnessed for adoptive cellular therapy.

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Figures

Figure 1

Figure 1. Enrichment of Tsp cells in human tissues with Trm phenotype.

(A) FACS analysis of Hoechst dye effluxing Tsp cells from human blood, BM, IEL, and skin (right panel), with SP inhibitor verapamil (left panel). (B) Bar graph represents frequency of Tsp cells from blood, BM, IEL, and skin. (C) Graph represents CD4+CD8+ subsets in Tsp cells from blood (n = 20), BM (n = 7), IEL (n = 6), and skin (n = 4). (D) Pie diagram represents Vβ repertoire in CD8+ Tsp and MAIT CD8+ T cells. (E) FACS analysis documenting human influenza-matrix peptide HLA A*0201-Tetramer+ cells (left) and SP fraction on influenza Tet+CD8+ T cells. (F) Representative FACS plot gated on CD8+ Tsp and NSP cells from IEL (representative of 6 independent experiments); the same is plotted in bar graph. (G) FACS analysis on CD8+ Tsp and NSP cells from skin; bar graph represents compiled data from 4 independent skin samples. (H) FACS analysis on blood Tsp, NSP cells that are gated on CD45RO+CD62L–CCR7–CD8+ T cells; bar graph represents compiled data (n = 6). *P < 0.05, ***P < 0.001, by Student’s t test.

Figure 2

Figure 2. SP phenotype marks LCMV-specific CD8+ Trm cells.

(A) Representative CD8+ Tsp analysis on LCMV-Arm–infected mouse spleen and IEL at day 8 (left) and day 30 (right). p.i., post infection. (B) Graph representing the analysis in A (n = 4 mice). Experiment was repeated twice; graph here represents one of the experiments. (C) Top panel shows percentage of CD8+ Tsp and NSP gp33+CD8+ T cells at day 35 after infection in different organs. Bottom panel is gated on gp33+CD8+ Tsp cells. (D) Percentage of gp33+ Trm cells in CD8+ Tsp and NSP fractions. Bar graphs represent data from 7–10 mice. Data were compiled from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, by Student’s t test.

Figure 3

Figure 3. Cell-cycle analysis of Tsp cells.

(A) Hoechst–Pyronin Y cell-cycle analysis on Tsp and NSP fraction from human blood CD8+ T cells (top panel), IEL (middle panel), and skin CD8+ Trm cells (bottom panel). Result is representative of 3 independent experiments. (B) Bar graph represents combined data of 3 independent experiments. (C) Bar graph displays percentage of G0 and G1 fractions in CD8+ Tsp and NSP gated on Trm compartment from PP, IEL in mice at day 35 (p.i.) with LCMV-Arm. (D) Schematic diagram showing the protocol for measuring GFP label retention in H2B-GFP mice upon LCMV-Arm infection. (E) FACS analysis on memory CD8+ T cells from BM, spleen, liver, IEL, and PP after 5 weeks of chase; the same is documented in the bar graph (n = 4–5 mice). (F) FACS analysis of GFP label retention in SP and NSP CD8+ Trm compartment at 5 weeks chase in H2B GFP mice infected with LCMV-Arm. (G) Graph represents percentages of GFP+ cells in SP and NSP CD8+ Trm cells. *P < 0.05. Mouse experiments were repeated twice; graphs represent analysis from 1 experiment (n = 4 mice).

Figure 4

Figure 4. Human CD8+ Tsp cells express a distinct gene-expression profile.

(A) PCA plot comparing CD8+ naive, CD8+ Tem, CD8+ Tcm, and CD8+ Tsp cells. (B) Venn diagram of differentially regulated transcripts (>4-fold) in CD8+ Tsp compared with naive, Tem, and Tcm CD8+ T cells. (C) Top 10 GO terms from MetaCore pathway analysis of genes enriched in CD8+ Tsp cells. (D) Relative expression of human skin Trm genes in blood CD8+ Tsp cells compared with naive, Tem, and Tcm CD8+ T cells.

Figure 5

Figure 5. Impact of Nur77 on CD8+ Trm cells.

Flow cytometry on Vα2 CD8+ T cells obtained from chimeras generated by reconstitution of WT mice with 1:1 mixture of WT OT-1 and Nur77–/– OT-1 cells and then assessed at day 35 after infection with influenza X-31 OVA. (A) FACS analysis showing CD45.2 (Nur77–/– OT-1) and CD45.1 (WT OT-1) cells in spleen, BM, liver, lung, PP, and IEL. (B) Bar graphs represent absolute number of Nur77–/– OT-1 and WT OT-1 CD8+ T cells from different organs. Experiments were repeated twice with 3 to 4 mice in each group; graphs represent data from 1 independent experiment (n = 4 mice). *P < 0.05, by Student’s t test.

Figure 6

Figure 6. ABCB1a/b ABCG2 KO mice display increased inflammatory Trm compartment.

(A) Bar graph comparing the CD4+, CD4+CD8+, and CD8+ Trm compartments in the gut (PP, IEL, and LPL) of WT and ABCB1a/b ABCG2 KO mice. (B) Intracellular cytokine flow cytometry documenting IFN-γ production upon PMA plus ionomycin activation of WT and ABCB1a/b ABCG2 KO mouse IELs. (C) Bar graph represents IFN-γ production in different T cell populations from WT and ABCB1a/b ABCG2 KO mice. Experiments were repeated twice with 3 to 4 mice in each group; graphs represent data from 1 experiment (n = 4 mice). *P < 0.05, **P < 0.01, ***P < 0.001, by Student’s t test.

Figure 7

Figure 7. Human Tsp cells cause greater tissue pathology following adoptive transfer in mice and can be mobilized into circulation by plerixafor.

Sorted Tsp cells and control T cells (total T cells); 100,000 of each were retroorbitally transferred into individual NSG mice and analyzed after 5 to 6 weeks. (A) FACS plot showing engraftment of human CD45+ T cells; the same is plotted in the bar graph. (B) Photographs compare skin pathology, liver, and skin histopathology in NSG mice receiving Tsp and control T cells. Original magnification ×200. (C) Graph representing GVHD score for liver and skin. Experiments were repeated 3 times with 4 mice/group. (D) FACS plot represents Tsp phenotype on pre- and postmobilized CD8+ T cells and CD34+ cells from human peripheral blood. (E) Graph denotes percentage of Tsp fraction in CD8+ and CD34+ cells at baseline and after mobilization (n = 6). *P < 0.05, by Student’s t test.

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