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The lysophosphatidylserine receptor GPR174 constrains regulatory T cell development and function.

Barnes MJ, Li CM, Xu Y, An J, Huang Y, Cyster JG - J. Exp. Med. (2015)

Bottom Line: In vivo, LysoPS was detected in lymphoid organ and spinal cord tissues and was abundant in the colon.This study provides evidence that a bioactive lipid, LysoPS, negatively influences T reg cell accumulation and activity through GPR174.As such, GPR174 antagonists might have therapeutic potential for promoting immune regulation in the context of autoimmune disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, and Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143 Howard Hughes Medical Institute, Department of Microbiology and Immunology, and Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143.

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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+IgDhigh 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.
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fig1: 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+IgDhigh 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.

Mentions: Our initial interest in GPR174 stemmed from an effort to identify GPCRs involved in regulating lymphocyte transit through lymphoid organs (Pham et al., 2008). Quantitative PCR analysis of the mRNA expression levels of 353 nonodorant GPCRs (Regard et al., 2008) in naive T and B cells identified Gpr174 (previously known as Fksg79) as one of the most abundantly expressed transcripts (not depicted). To characterize the role of this X-linked gene in the immune system, we replaced the single coding exon with a cassette encoding an “in-frame” dTomato allele (Fig. 1 A). Analysis of dTomato expression in Gpr174−/Y male mice (Fig. 1, B–D) confirmed high levels of GPR174 expression in naive T and B cells (Fig. 1, B and C), and dTomato expression patterns were similar to Gpr174 mRNA expression levels (Fig. 1, C and E). Naive T and B cell numbers and lymphoid tissue organization were normal in Gpr174−/Y mice (not depicted). In LN transit assays (Pham et al., 2008), no differences in trafficking between wild-type and Gpr174−/Y T or B cells were detected (not depicted). Further characterization of dTomato expression showed abundant GPR174 expression in CD25+ CD4 single-positive (SP) thymocytes and CD25+CD4+ T cells, populations that are highly enriched in T reg cells, compared with naive CD4+ T cells (Fig. 1, B and C). These Gpr174 gene expression patterns were confirmed by quantitative RT-PCR analysis of sorted cell populations from wild-type mice (Fig. 1 E). We observed that elevated Gpr174 expression correlated with Foxp3 expression in both immature T reg (Foxp3+CD25−) and mature T reg (Foxp3+CD25+) CD4 SP thymocytes, whereas levels were not elevated in Foxp3−CD25+ thymic T reg cell precursors (Fig. 1 E). In contrast, when we differentiated naive CD4+ T cells in Th1 or Th17 polarizing conditions, reduced GPR174-dTomato expression was observed compared with cells cultured in Th0 conditions (Fig. 1 D).


The lysophosphatidylserine receptor GPR174 constrains regulatory T cell development and function.

Barnes MJ, Li CM, Xu Y, An J, Huang Y, Cyster JG - J. Exp. Med. (2015)

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+IgDhigh 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.
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fig1: 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+IgDhigh 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.
Mentions: Our initial interest in GPR174 stemmed from an effort to identify GPCRs involved in regulating lymphocyte transit through lymphoid organs (Pham et al., 2008). Quantitative PCR analysis of the mRNA expression levels of 353 nonodorant GPCRs (Regard et al., 2008) in naive T and B cells identified Gpr174 (previously known as Fksg79) as one of the most abundantly expressed transcripts (not depicted). To characterize the role of this X-linked gene in the immune system, we replaced the single coding exon with a cassette encoding an “in-frame” dTomato allele (Fig. 1 A). Analysis of dTomato expression in Gpr174−/Y male mice (Fig. 1, B–D) confirmed high levels of GPR174 expression in naive T and B cells (Fig. 1, B and C), and dTomato expression patterns were similar to Gpr174 mRNA expression levels (Fig. 1, C and E). Naive T and B cell numbers and lymphoid tissue organization were normal in Gpr174−/Y mice (not depicted). In LN transit assays (Pham et al., 2008), no differences in trafficking between wild-type and Gpr174−/Y T or B cells were detected (not depicted). Further characterization of dTomato expression showed abundant GPR174 expression in CD25+ CD4 single-positive (SP) thymocytes and CD25+CD4+ T cells, populations that are highly enriched in T reg cells, compared with naive CD4+ T cells (Fig. 1, B and C). These Gpr174 gene expression patterns were confirmed by quantitative RT-PCR analysis of sorted cell populations from wild-type mice (Fig. 1 E). We observed that elevated Gpr174 expression correlated with Foxp3 expression in both immature T reg (Foxp3+CD25−) and mature T reg (Foxp3+CD25+) CD4 SP thymocytes, whereas levels were not elevated in Foxp3−CD25+ thymic T reg cell precursors (Fig. 1 E). In contrast, when we differentiated naive CD4+ T cells in Th1 or Th17 polarizing conditions, reduced GPR174-dTomato expression was observed compared with cells cultured in Th0 conditions (Fig. 1 D).

Bottom Line: In vivo, LysoPS was detected in lymphoid organ and spinal cord tissues and was abundant in the colon.This study provides evidence that a bioactive lipid, LysoPS, negatively influences T reg cell accumulation and activity through GPR174.As such, GPR174 antagonists might have therapeutic potential for promoting immune regulation in the context of autoimmune disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Microbiology and Immunology, and Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143 Howard Hughes Medical Institute, Department of Microbiology and Immunology, and Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94143.

Show MeSH
Related in: MedlinePlus