G-Protein Coupled Receptor 18 Contributes to Establishment of the CD8 Effector T Cell Compartment

Abstract

The requirements for effector and memory CD8 T cell development are incompletely understood. Recent work has revealed a role for G-protein coupled receptor 18 (GPR18) in establishment of the intestinal CD8αα intraepithelial lymphocyte compartment. Here, we report that GPR18 is also functionally expressed in conventional CD8αβ T cells. When the receptor is lacking, mice develop fewer CD8⁺ KLRG1⁺ Granzyme B⁺ effector-memory cells. Bone marrow chimera studies show that the GPR18 requirement is CD8 T cell intrinsic. GPR18 is not required for T-bet expression in KLRG1⁺ CD8 T cells. Gene transduction experiments confirm the functional activity of GPR18 in CD8 T cells. In summary, we describe a novel GPCR requirement for establishment or maintenance of the CD8 KLRG1⁺ effector-memory T cell compartment. These findings have implications for methods to augment CD8 effector cell numbers.

Keywords: CD8 T cells; G-protein coupled receptor 18; KLRG1; effector-memory; granzyme B.

Figure 1

Reduction of KLRG1⁺ CD8 EM T cells in G-protein-coupled receptor 18 (GPR18)-deficient peripheral blood lymphocytes (PBL). (A) Gpr18 transcript abundance in the indicated cell subsets relative to Hprt. Each point indicates cells sorted from an individual mouse and lines indicate mean ± SEM. n = 3 or 4 in each populations. EM, effector-memory; CM, central memory; IEL, intraepithelial lymphocytes. (B) Flow cytometric analysis of CD44 and CD62L expression in CD8⁺ TCRβ⁺ PBL from the indicated mature (6 months old) mice. Numbers show percentage of cells in the indicated gate. (C) Frequency of EM (CD44^hi CD62L^lo), CM (CD44^hi CD62L^hi), and naive (CD44^lo CD62L^hi) CD8⁺ or CD4⁺ TCRβ⁺ cells in PBL from young (2 months old, left panel) or mature (6 months old, right panel) Gpr18^+/−and Gpr18^−/− mice. Left panel: Gpr18^+/−, n = 8; Gpr18^−/−, n = 8. Right panel: Gpr18^+/−, n = 21; Gpr18^−/−, n = 16. (D) Flow cytometric analysis of KLRG1 expression in CD8 EM PBL from the indicated mature (6 months old) mice. Numbers show percentage of cells in the indicated gate. (E) Frequency of KLRG1⁺ CD8 EM PBL in indicated young (2 months old) or mature (6 months old) mice. Young adults: Gpr18^+/−, n = 8; Gpr18^−/−, n = 8. Mature adults: Gpr18^+/−, n = 21; Gpr18^−/−, n = 16. Each point represents data from an individual mouse and lines represent means ± SEM (C,E). ***p < 0.001, **p < 0.01, *p < 0.05, “n.s.” p > 0.05 by Student’s t-test (C,E).

Figure 2

Reduction of KLRG1⁺ CD8 effector memory (EM) T cells in lymphoid tissues of G-protein coupled receptor 18 (GPR18)-deficient naïve mice (A) Frequency of EM (CD44^hi CD62L^lo), central memory (CM) (CD44^hi CD62L^hi), and naive (CD44^lo CD62L^hi) populations in CD8⁺ or CD4⁺ TCRβ⁺ splenocytes in mature mice (6 months old) of the indicated type. Gpr18^+/−, n = 11; Gpr18^−/−, n = 12. (B) Number of CD8⁺ TCR β⁺ splenocytes in the indicated mature (6 months old) mice. Each population was pre-gated on CD45⁺TCRβ⁺ cells. Gpr18^+/−, n = 13; Gpr18^−/−, n = 14. (C) Frequency of KLRG1⁺ cells in CD8 EM splenocytes in indicated mature (6 months old) mice. Gpr18^+/−, n = 9; Gpr18^−/−, n = 8. (D) Numbers of KLRG1⁺ CD8 EM splenocytes in indicated mature (6 months old) mice. Gpr18^+/−, n = 8; Gpr18^−/−, n = 8. (E) Frequency of EM, CM, and naive populations in CD8⁺ or CD4⁺ TCRβ⁺ lymphocytes from mesenteric lymph nodes (mLN) in indicated mature (6 months old) mice. Gpr18^+/−, n = 7; Gpr18^−/−, n = 6. (F) Frequency of KLRG1⁺ populations in CD8 EM from mLN in indicated mature (6 months old) mice. Gpr18^+/−, n = 8; Gpr18^−/−, n = 7. (G) Percentages of Gpr18^−/− or control Gpr18^−/− EM cells in gp33 tetramer⁺ CD8 T cells in peripheral blood lymphocytes (PBL) at day 8 and day 30 after lymphocytic choriomeningitis virus (LCMV) Armstrong infection. (H) Percentages of early effector cell (EEC) (KLRG1^lo CD127^lo), memory precursor effector cells (MPEC) (KLRG1^lo CD127^hi), and short-lived effector cells (SLEC) (KLRG1^hi CD127^lo) in gp33 tetramer⁺ CD44^hi CD8 PBL at day 8 and day 30 after LCMV Armstrong infection. (G,H) Gpr18^+/−, n = 4; Gpr18^−/−, n = 5. Each point represents data from an individual mouse and lines represent means ± SEM (A–H). ***p < 0.001, **p < 0.01, *p < 0.05, “n.s.” p > 0.05 by Student’s t-test.

Figure 3

Cell intrinsic defects of KLRG1⁺ CD8 T cells in G-protein coupled receptor 18 (GPR18)-deficiency. B6-CD45.1⁺ mice were reconstituted with CD45.1/2⁺ WT and CD45.2⁺ Gpr18^+/− or Gpr18^−/− bone marrow (BM) 10 weeks before analysis (n = 6). (A) Flow cytometric analysis of CD44 and CD62L expression in CD8⁺ TCRβ⁺ and congenic (CD45) marker⁺ splenocytes from the indicated donor cells in the same animal. Numbers show percentage of cells in the indicated gate. (B) Naïve (CD44^loCD62L^hi), effector memory (EM) (CD44^hiCD62L^lo), and CM (CD44^hiCD62L^hi) T cells in Gpr18^+/− or Gpr18^−/− CD8⁺ TCRβ⁺ cells, identified as in (A), were presented as a ratio to the WT control donor cells from the same animal in peripheral blood lymphocytes (PBL) (upper panel) or spleen (lower panel). (C) Flow cytometric analysis of KLRG1 expression in CD8 EM splenocytes from the indicated donor cells in the same animal. (D) Percentage of KLRG1⁺ cells in Gpr18^+/− or Gpr18^−/− CD8⁺ EM TCRβ⁺ cells, determined as in (C), presented as a ratio to the WT control donor cells from the same animal in PBL (upper panel) or spleen (lower panel). Each symbol in (B,D) represents an individual mouse, and lines represent means ± SEM. *p < 0.05 by Student’s t-test (B,D). Data from one of two or three independent experiments are shown.

Figure 4

Granzyme B, T-bet, CXCR3, and Ki67 expression in Gpr18^−/− CD8 effector memory (EM) cells. (A) Intracellular staining of splenic CD8 EM (left panel) or KLRG1⁺ (right panel) cells for Granzyme B. Data are plotted as ratio of Granzyme B⁺ Gpr18^+/− or Gpr18^−/− cells and WT cells in the same mixed bone marrow (BM) chimeric animal. (B) Representative histograms of T-bet staining in KLRG1⁺ (left panels) or KLRG1⁻ (right panels) CD8 EM splenocytes. Upper panels, stained with isotype control antibodies, middle and lower panels stained with T-bet antibodies. Cells were from mixed BM chimeras of the indicated types. y-axis of histogram overlays was normalized to mode. (C) Mean fluorescence intensity (MFI) of T-bet staining in KLRG1⁺ or KLRG1⁻ CD8 EM splenocytes, plotted as ratio of MFI in Gpr18^+/− or Gpr18^−/− compared to control WT in mixed BM chimeras (n = 6). (D) Flow cytometry analysis of CXCR3 expression in KLRG1⁺ and KLRG1⁻ CD8 EM cells from mixed BM chimeras. Upper panel; histograms from Gpr18^+/− plus WT mixed BM chimera. Lower panel; histograms from Gpr18^−/− plus WT mixed BM chimera. y-axis of histogram overlays was normalized to mode. (E) Ratio of percentage of Ki67⁺ CD8 EM or KLRG1⁺ CD8 EM cells in Gpr18^+/− or Gpr18^−/− compared to control WT in mixed BM chimeras (n = 6). (F) In vivo labeling with CD8α-PE antibody. Three minutes after antibody injection into the indicated mice, splenocytes were harvested and stained ex vivo with antibodies to identify KLRG1⁺ or KLRG1⁻ CD8 EM cells. Representative plots gated on KLRG1⁺ (upper panels) and KLRG1⁻ (lower panels) are shown. Numbers show percentage of cells in indicated CD8α-PE⁺ gate. Each symbol in (A,C,E) represents an individual mouse, and lines represent means ± SEM. *p < 0.05, “n.s.” p > 0.05 by Student’s t-test (A,C,E). Data from one of two independent experiments are shown.

Figure 5

Rescue effects of G-protein-coupled receptor 18 (GPR18) expression on CD8 effector memory (EM) cells in Gpr18^−/− mice. (A,B) CD45.2⁺ Gpr18^−/− bone marrow transduced with empty vector or Gpr18 retrovirus was used to reconstitute CD45.1⁺ recipients (n = 5). Donor cells were gated on CD45.1⁻CD45.2⁺ and then gated on GFP⁺ (transduced donor cells) or GFP⁻ (untransduced donor cells). (A) CD8⁺ TCRβ⁺ cells from peripheral blood lymphocytes (PBL) (left panel) or spleen (right panel) were stained for naïve (CD44^loCD62L^hi), EM (CD44^hiCD62L^lo), and CM (CD44^hiCD62L^hi). Percentage of EM, CM, and naïve populations in GFP⁺ donor cells were presented as a ratio to those in GFP⁻ donor cells from the same animal. (B) CD8 EM cells from PBL (left panel) or spleen (right panel) were stained for KLRG1. Percentage of KLRG1⁺ populations in GFP⁺ donor cells were presented as a ratio to those in GFP⁻ donor cells from the same animal. Each symbol represents an individual mouse, and lines represent means ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 by Student’s t-test.