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

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.

inTrODUcTiOn CD8 T cells that have responded to antigenic stimuli have been classically divided into CD44 hi CD62L lo effector memory (EM) and CD44 hi CD62L hi central memory (CM) cells (1). Early studies on the CD8 T cell response following lymphocytic choriomeningitis virus (LCMV) and Listeria infection showed that CD8 T cells expand and differentiate through an early effector cell (EEC) stage into distinct effector populations, including short-lived effector cells (SLEC) and memory precursor effector cells (MPEC) (2,3). SLECs are distinguished by high expression of KLRG1 and low expression of the IL7Rα chain (CD127), while MPEC have the reciprocal marker pattern (4,5). Both types of cell express effector molecules such as Granzyme B and IFNγ, but only MPECs are efficient at giving rise to memory responses. Subsequent studies in a number of systems have shown a less clear correlation between expression of KLRG1 and a short-lived effector state. In some cases, the KLRG1 + cells persisted to the memory phase and provided effective control of the infection despite weak recall proliferative responses (6,7). Other studies have noted that the amount of KLRG1 expressed by the effector-memory population may be determined by the amount of exposure to inflammatory signals during CD8 cell differentiation (8,9). While all the factors responsible for determining the size of the KLRG1 + effector-memory population have not been defined, it has been established that the size of this compartment can be promoted by the pro-survival activity of IL-15 and restricted by the proapoptotic effect of TGFβ (4,10). Several studies have shown a role for high expression of the transcription factor T-bet in establishing the KLRG1 + effector cell compartment (11)(12)(13).
The G-protein coupled receptor G-protein coupled receptor 18 (GPR18) is abundantly expressed in lymphocytes, with particularly high expression in CD8αα γδT intraepithelial lymphocytes (IELs) (14). Two recent studies using independently generated GPR18-deficient mouse lines found that this receptor plays a role in establishing an IEL compartment of normal size (14,15). However, whether this receptor has functions in conventional T cells has been unknown.
In the course of our work to characterize how GPR18 contributes to IEL function, we noticed that GPR18-deficient mice had a lower frequency of CD44 hi CD62L lo effector-memory type CD8 T cells. Here, we have characterized this deficiency and find that GPR18 knockout (KO) mice have lower numbers of spontaneously forming KLRG1 + CD8 effector-memory cells.
described (14) To generate bone marrow (BM) chimeras, CD45.1 + B6 mice were irradiated by exposure to 1,100 rad of γ-irradiation in two doses 5 h apart and i.v. injected with at least 2 × 10 6 total BM cells from each genotype of mice as indicated and analyzed after 2-3 months. All chimeras appeared healthy at the time of analysis. For LCMV infection, mice were infected with 2 × 10 5 plaque-forming units of LCMV Armstrong administered i.v. Animals were housed in a specific pathogen-free environment in the Laboratory Animal Research Center at the UCSF, and all experiments conformed to ethical principles and guidelines approved by the UCSF Institutional Animal Care and Use Committee.

cell Preparations
For peripheral blood lymphocytes (PBL) preparation, blood was collected into EDTA-coated tubes and red cell lysis was performed. Splenocyte and mesenteric lymph node cell suspensions were prepared by mashing the organs through 70-µm cell strainers and then suspended with RPMI-1640 medium supplemented with 5% FCS.

intravascular staining
A total of 3 µg anti-CD8α-PE (clone 53-6.7 from Biolegend) antibody was injected i.v. At 3 min after injection, the animals were sacrificed and analyzed as described (18).

statistical analysis
Prism (GraphPad, ver. 5.0a) software was used for all statistical analyses. Two-tailed, unpaired Student's t-tests were performed when comparing two groups. p-Values less than 0.05 were considered significant. In graphs, horizontal lines indicate means, and error bars indicate SEM.

resUlTs
Analysis of GPR18 transcript expression in T cell subsets confirmed the high expression in CD8αα IELs and revealed considerable expression in CD8 T cells and slightly lower expression   in CD4 T cells (Figure 1A). Expression was retained in CD44 hi CD62L lo EM and CD44 hi CD62L hi CM CD8 T cells. Flow cytometric analysis of CD44 hi CD62L lo EM CD8 T cell frequencies in blood revealed a slight reduction in young (2-month-old mice) and a significant reduction in mature (6-month old) GPR18-deficient mice (Figures 1B,C). The frequencies of blood CD4 T cells and of naïve and CM CD8 T cells were unaltered in GPR18-deficient mice ( Figure 1C; Figure S1A in Supplementary Material). Examination of marker expression within the CD8 EM compartment revealed a strong deficiency in KLRG1 + cells in the GPR18 KO mice (Figures 1D,E). Given that exposure to commensal and environmental antigens may differ between mouse cages, our studies were done with co-cocaged littermate mice generated in heterozygote by KO intercrosses. We do not exclude the possibility that GPR18 heterozygosity causes a partial effect on the CD8 compartment that may cause us to underestimate the magnitude of the KO phenotype. The reduction in CD8 EM cells, but not CD4 EM cells, was also observed in the spleen of mature-aged mice (Figure 2A; Figure  S1B in Supplementary Material). Enumeration of total spleen cells established that there was an overall deficiency in EM CD8 T cells ( Figure 2B). Staining for KLRG1 confirmed the selective deficiency of KLRG1 + cells amongst CD8 EM cells (Figures 2C,D). A deficiency in KLRG1 + CD8 EM cells was also observable in mesenteric LNs (Figures 2E,F). Regarding GPR18 expression, KLRG1 + and KLRG1 − CD8 EM cells showed comparable mRNA levels ( Figure 1A).
The KLRG1 + CD8 EM cells studied above were those arising endogenously in mice housed in our specific pathogen-free mouse facility. To test whether GPR18 was involved in the generation of KLRG1-expressing cells that appear rapidly following viral infection, GPR18 KO and control mice were infected with LCMV Armstrong and examined after 8 and 30 days. Gating on LCMVspecific gp33-tetramer + CD8 T cells revealed an equivalent percentage of EM cells in control and GPR18 KO mice (Figure 2G; Figure S2 in Supplementary Material). Gating on total CD44 hi gp33-tetramer + cells showed an equivalent fraction of these cells were KLRG1 + CD127 lo SLEC not only at day 8 but also at day 30, when the LCMV response is in the effector or effector-memory stage (Figure 2H; Figure S2 in Supplementary Material). These findings indicate that the influence of GPR18 on establishment of a KLRG1 + CD8 cell compartment varies depending on the induction conditions.
To determine if the reduction in CD8 EM reflected a cell intrinsic role for GPR18, we generated mixed BM chimeras. Analysis of these mice 10 weeks after reconstitution revealed a selective deficiency in GPR18 KO EM cells in blood and spleen (Figures 3A,B). The effect was again most prominent for KLRG1 + CD8 EM cells (Figures 3C,D). These data indicate that the GPR18 receptor acts intrinsically to favor establishment or maintenance of the KLRG1 + CD8 EM compartment.
To rule out that the reduction in CD8 EM cells was due to selective loss of the KLRG1 surface marker, we stained CD8 T cells in mixed BM chimeras for Granzyme B, since this gene is highly expressed in KLRG1 + cells (2, 3). The frequency of Granzyme B + cells was reduced in GPR18 KO CD8 EM to an extent similar to the reduction in KLRG1 + cells (Figure 4A, left panel and Figure 3A; Figure S3A in Supplementary Material). When gating on the KLRG1 + population, the fraction of Granzyme B + cells was not altered, in accord with a reduction in population size rather than selective changes in marker expression (Figure 4A right panel; Figure S3B in Supplementary Material). Since T-bet (Tbx21) is needed for KLRG1 + CD8 EM cell development, we tested whether its expression was affected by GPR18 deficiency. Intracellular flow cytometry showed similar expression in the KLRG1 + CD8 cells that were present in GPR18 KO mice compared to those present in matched controls (Figures 4B,C). Indeed, rather than being reduced, a non-statistically significant trend for increased T-bet expression was observed. T-bet expression levels in KLRG1 − cells were lower than in KLRG1 + cells as expected and were equivalent in KO and control (Figures 4B,C). These data indicate that GPR18 is not required for upregulation of the T-bet transcription factor in CD8 EM cells.
CXCR3 affects the balance between effector and memory CD8 T-cell generation (19), and we, therefore, investigated expression levels of this chemokine receptor. CXCR3 was expressed comparably in GPR18 KO and wild-type CD8 T cells (Figure 4D). Further analysis of the mixed BM chimeras showed that the fraction of cells that were in or had recently been in cell cycle, as determined by Ki-67 staining, was unaltered by GPR18-deficiency ( Figure 4E; Figures S4A,B in Supplementary Material). IFNγ staining showed that expression of this cytokine in CD8 cells was not affected by GPR18 deficiency (Figure S5 in Supplementary Material).
Since KLRG1 + and KLRG1 − effector-memory cells differ in their distribution within the spleen, with KLRG1 + cells locating predominantly in the highly vascular red pulp (9,20), we tested for any effect of GPR18 deficiency on cell distribution using in vivo CD8α-PE labeling. This technique labels cells that are in blood-exposed compartments such as the red pulp while leaving cells in the lymphoid-rich white pulp unlabeled (18,21). As expected, KLRG1 − cells were predominantly protected from labeling, being enriched in the white pulp, while KLRG1 + cells were highly labeled ( Figure 4F). GPR18-deficiency  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.
did not affect the fraction of KLRG1 − or KLRG1 + cells that were labeled, indicating that the cells were located in their correct compartments.
Finally, to confirm that the phenotype observed was solely due to GPR18 deficiency, we restored GPR18 expression by transduction of GPR18 KO BM cells with a GPR18 and GFP encoding retrovirus, versus a GFP control retrovirus (empty vector). Irradiated mice were reconstituted with the transduced BM cells and then analyzed for the frequency of each CD8 cell type that was transduced (GFP + ) versus untransduced (GFP − ) to test for enrichment or depletion of the transduced cells. Compared to mice reconstituted with GPR18 KO BM transduced with the empty vector, mice receiving GPR18 KO BM transduced with GPR18 showed a selective increase in transduced CD8 EM and KLRG1 + cells in blood and spleen (Figures 5A,B; Figures  S6A,B in Supplementary Material). These data provide strong support for the conclusion that the altered CD8 T cell compartments in the GPR18-deficient mice reflects a direct requirement for GPR18.

DiscUssiOn
Our findings establish a cell intrinsic role for GPR18 in the normal accumulation of CD8 effector T cells, in particular, the KLRG1 + EM cell population. The CD8 effector cells studied here are those arising endogenously over time in mice housed in a specific pathogen-free colony. In experiments where we infected mice with the pathogen LCMV (Armstrong strain), we did not detect clear differences in the induced EM cell populations between GPR18 KO and littermate control mice. Further studies will be needed to define the types of response where GPR18 contributes to KLRG1 + CD8 EM cell development or maintenance, but at this time, we suggest the receptor is influencing their development in response to commensal microorganisms. Two ligands have been proposed for GPR18, N-arachidonyl glycine (NAGly), and resolvin-D2 (22)(23)(24). In our previous work and studies by others, NAGly has not been confirmed to be a functional GPR18 agonist (14,25). Future studies will be needed to determine whether resolvin-D2 or an as yet unidentified GPR18 ligand acts to promote effector CD8 T cell homeostasis. Our studies suggest that small molecule agonists of GPR18 might augment the size of the KLRG1 + effector CD8 T cell compartment, an effect that might be beneficial, for example, during viral responses or in the context of tumor immunotherapy. Our findings may also prove significant for understanding the GPR18 SNPs detected as being enriched in inflammatory bowel disease patients in genome-wide association studies (26,27