Figure 1.

Elevated expression of transcripts encoding GPR174 and other LysoPS receptors in T reg cells. (A) Diagram of the construct used to target the Gpr174 locus by homologous recombination. For more details, see Materials and methods. (B and C) Measurement of dTomato-GPR174 reporter allele expression in thymocytes (left) or splenocytes (right) by flow cytometry from 8-wk-old male Gpr174^−/Y mice. (B) Thymocyte populations are as follows: gray shaded, CD4⁺CD8⁺ DP; blue dashed, CD8⁺ SP; purple dotted, CD25⁻ CD4 SP; and red, CD25⁺ CD4 SP T reg cells. Splenocyte populations are as follows: gray shaded, background (splenocytes from wild-type mice); blue dashed, naive CD8⁺ T cells; purple dotted, CD25⁻ naive CD4⁺ T cells; and red, CD25⁺CD4⁺ T reg cells. (C) The mean fluorescence intensity (MFI) of dTomato-GPR174 is shown for the indicated cell populations. B cells were identified as B220⁺IgD^high splenocytes. Each dot represents a measurement from a separate mouse; n = 4. (D) Expression of dTomato-GPR174 was measured in naive CD4⁺ T cells cultured under Th0, Th1, or Th17 polarizing conditions for 5 d; representative flow cytometry data are shown. (E) The mRNA expression levels of the LysoPS receptors Gpr174, Gpr34, P2ry10, and P2ry10-L were measured by RT-PCR in the indicated sorted thymocyte and splenocyte populations from 8-wk-old wild-type mice. Populations were gated as described in Materials and methods. Cells were sorted in triplicate, and each dot represents the relative expression in a separate sorted cell population from a distinct mouse; n = 3; error bars show SD. All data in B–E are representative of at least three independent assays.