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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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CD4+ T cells show diminished Nur77 levels and enhanced proliferation in Gpr174−/Y mice. (A) Flow cytometry analysis of the mean fluorescence intensity (MFI) of Nur77-GFP expression in thymocytes from 6-wk-old wild-type and Gpr174−/Y Nur77-GFP+ littermate male mice. Each dot represents an individual mouse; n = 4. (B) Intracellular levels of Ki-67 expression in the indicated thymocyte populations from 6-wk-old wild-type and Gpr174−/Y littermate male mice were determined using flow cytometry. The percentage of Ki-67+ cells among Foxp3+ T reg and Foxp3− CD4 SP thymocytes is shown; n = 9 or 10. (A and B) Horizontal lines indicate the mean. (C) The effects of LysoPS on T cell proliferation under neutral conditions. CFSE-labeled LN cells from wild-type or Gpr174−/Y mice were cultured in round-bottom plates with 0.25 µg/ml soluble anti-CD3 and the indicated amounts of LysoPS. Cell proliferation was assessed 3 d later based on CFSE dilution that was measured by flow cytometry; live CD4+TCR-β+ T cells are shown. Gates indicate the percentage of cells that divided at least three times, and means ± SD are shown; n = 4. (D and E) Direct effects of GPR174 and its ligand LysoPS on proliferation and Foxp3 induction. In flat 96-well plates coated with anti-CD3 and anti-CD28 mAbs (both 2 µg/ml), a mixture of either wild-type (Ly5-1+) or Gpr174−/Y (Ly5-1+) with congenic wild-type (Ly5-2+) naive CD4+ T cells (D) or either wild-type or Gpr174−/Y naive CD4+ T cells (E) were added. Cells were cultured in the presence of 200 U/ml IL-2 (D) or 1 ng/ml TGF-β (E) and the indicated concentrations of 18:0 LysoPS for 4 d. Expression of Ly5-1 and Ly5-2 (D) or Foxp3 (E) was measured by flow cytometry; n = 4; error bars indicate SD. Counts were quantified based on the number of events acquired on a flow cytometer run for 60 s per sample (E). Cells used in E were isolated from mixed bone marrow chimeric mice to minimize extrinsic effects on naive T cells. (F) In an in vitro T reg cell suppressor assay, CD4+CD25+CD45RBhighCD103− T reg cells were sorted from spleens of wild-type and Gpr174−/Y littermate mice. T reg cells were cultured at the indicated ratios with 105 CFSE-labeled naive CD45.1+CD4+ T cells and 2 × 104 CD11c-enriched DCs in the presence of 0.5 µg/ml soluble anti-CD3ε mAb for 3 d. The proliferation index of CFSE-labeled cells was determined in triplicate cultures, and representative CFSE flow cytometry plots are shown. (G) A total of 2 × 105 naive CD4+ T cells from CD45.2+ wild-type or Gpr174−/Y littermate mice were cultured along with an equivalent number of cells from CD45.1+ wild-type mice under Th1 or Th17 polarizing conditions for 5 d. After restimulation with PMA and ionomycin, the percentage of CD45.2+ cells that secreted IFN-γ or IL-17 was determined by intracellular cytokine staining and flow cytometry analysis. (F and G) Error bars show SD. Data were evaluated by unpaired Student’s t test: *, P < 0.05; **, P < 0.01. All data are representative of three or more independent experiments.
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fig3: CD4+ T cells show diminished Nur77 levels and enhanced proliferation in Gpr174−/Y mice. (A) Flow cytometry analysis of the mean fluorescence intensity (MFI) of Nur77-GFP expression in thymocytes from 6-wk-old wild-type and Gpr174−/Y Nur77-GFP+ littermate male mice. Each dot represents an individual mouse; n = 4. (B) Intracellular levels of Ki-67 expression in the indicated thymocyte populations from 6-wk-old wild-type and Gpr174−/Y littermate male mice were determined using flow cytometry. The percentage of Ki-67+ cells among Foxp3+ T reg and Foxp3− CD4 SP thymocytes is shown; n = 9 or 10. (A and B) Horizontal lines indicate the mean. (C) The effects of LysoPS on T cell proliferation under neutral conditions. CFSE-labeled LN cells from wild-type or Gpr174−/Y mice were cultured in round-bottom plates with 0.25 µg/ml soluble anti-CD3 and the indicated amounts of LysoPS. Cell proliferation was assessed 3 d later based on CFSE dilution that was measured by flow cytometry; live CD4+TCR-β+ T cells are shown. Gates indicate the percentage of cells that divided at least three times, and means ± SD are shown; n = 4. (D and E) Direct effects of GPR174 and its ligand LysoPS on proliferation and Foxp3 induction. In flat 96-well plates coated with anti-CD3 and anti-CD28 mAbs (both 2 µg/ml), a mixture of either wild-type (Ly5-1+) or Gpr174−/Y (Ly5-1+) with congenic wild-type (Ly5-2+) naive CD4+ T cells (D) or either wild-type or Gpr174−/Y naive CD4+ T cells (E) were added. Cells were cultured in the presence of 200 U/ml IL-2 (D) or 1 ng/ml TGF-β (E) and the indicated concentrations of 18:0 LysoPS for 4 d. Expression of Ly5-1 and Ly5-2 (D) or Foxp3 (E) was measured by flow cytometry; n = 4; error bars indicate SD. Counts were quantified based on the number of events acquired on a flow cytometer run for 60 s per sample (E). Cells used in E were isolated from mixed bone marrow chimeric mice to minimize extrinsic effects on naive T cells. (F) In an in vitro T reg cell suppressor assay, CD4+CD25+CD45RBhighCD103− T reg cells were sorted from spleens of wild-type and Gpr174−/Y littermate mice. T reg cells were cultured at the indicated ratios with 105 CFSE-labeled naive CD45.1+CD4+ T cells and 2 × 104 CD11c-enriched DCs in the presence of 0.5 µg/ml soluble anti-CD3ε mAb for 3 d. The proliferation index of CFSE-labeled cells was determined in triplicate cultures, and representative CFSE flow cytometry plots are shown. (G) A total of 2 × 105 naive CD4+ T cells from CD45.2+ wild-type or Gpr174−/Y littermate mice were cultured along with an equivalent number of cells from CD45.1+ wild-type mice under Th1 or Th17 polarizing conditions for 5 d. After restimulation with PMA and ionomycin, the percentage of CD45.2+ cells that secreted IFN-γ or IL-17 was determined by intracellular cytokine staining and flow cytometry analysis. (F and G) Error bars show SD. Data were evaluated by unpaired Student’s t test: *, P < 0.05; **, P < 0.01. All data are representative of three or more independent experiments.

Mentions: To better characterize the development of thymic Gpr174−/Y T reg cells, we crossed Gpr174−/Y mice to animals expressing a Nur77-GFP transgene (Moran et al., 2011). Levels of Nur77 increase in positively selected thymic T reg cells and CD25+Foxp3− T reg cell precursors in response to recent MHC class II–dependent signaling. Importantly, Nur77 levels are reduced in conditions that favor enhanced T reg cell development, such as in response to exogenous TNFR family–mediated co-stimulation (Mahmud et al., 2014). CD25+ CD4 SP thymocytes in Gpr174−/Y mice expressed lower levels of Nur77-GFP, consistent with a model in which GPR174 deficiency results in the exposure of developing thymocytes to T reg cell–favoring signals (Fig. 3 A). Additionally, we noted that CD25− CD4 SP thymocytes showed a very slight reduction in Nur77-GFP levels (Fig. 3 A). Because TNF receptor family members contribute to thymic T reg cell development and reduced Nur77-GFP abundance (Mahmud et al., 2014), we measured levels of glucocorticoid-induced TNFR family–related gene (GITR), OX40, and TNFR2 in the Foxp3+ and Foxp3− CD4 SP subsets and detected no differences between wild-type and Gpr174−/Y thymocytes (not depicted). In line with the accumulation of Gpr174−/Y thymic T reg cells, more Foxp3+, but not Foxp3− CD4 SP thymocytes expressed Ki-67, a marker of cells that have recently divided, compared with cells in wild-type littermates (Fig. 3 B). This effect was most obvious in the thymus of mice younger than 8 wk old (not depicted). Together, these data suggest that GPR174 signaling might intrinsically constrain thymic T reg cell proliferation.


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)

CD4+ T cells show diminished Nur77 levels and enhanced proliferation in Gpr174−/Y mice. (A) Flow cytometry analysis of the mean fluorescence intensity (MFI) of Nur77-GFP expression in thymocytes from 6-wk-old wild-type and Gpr174−/Y Nur77-GFP+ littermate male mice. Each dot represents an individual mouse; n = 4. (B) Intracellular levels of Ki-67 expression in the indicated thymocyte populations from 6-wk-old wild-type and Gpr174−/Y littermate male mice were determined using flow cytometry. The percentage of Ki-67+ cells among Foxp3+ T reg and Foxp3− CD4 SP thymocytes is shown; n = 9 or 10. (A and B) Horizontal lines indicate the mean. (C) The effects of LysoPS on T cell proliferation under neutral conditions. CFSE-labeled LN cells from wild-type or Gpr174−/Y mice were cultured in round-bottom plates with 0.25 µg/ml soluble anti-CD3 and the indicated amounts of LysoPS. Cell proliferation was assessed 3 d later based on CFSE dilution that was measured by flow cytometry; live CD4+TCR-β+ T cells are shown. Gates indicate the percentage of cells that divided at least three times, and means ± SD are shown; n = 4. (D and E) Direct effects of GPR174 and its ligand LysoPS on proliferation and Foxp3 induction. In flat 96-well plates coated with anti-CD3 and anti-CD28 mAbs (both 2 µg/ml), a mixture of either wild-type (Ly5-1+) or Gpr174−/Y (Ly5-1+) with congenic wild-type (Ly5-2+) naive CD4+ T cells (D) or either wild-type or Gpr174−/Y naive CD4+ T cells (E) were added. Cells were cultured in the presence of 200 U/ml IL-2 (D) or 1 ng/ml TGF-β (E) and the indicated concentrations of 18:0 LysoPS for 4 d. Expression of Ly5-1 and Ly5-2 (D) or Foxp3 (E) was measured by flow cytometry; n = 4; error bars indicate SD. Counts were quantified based on the number of events acquired on a flow cytometer run for 60 s per sample (E). Cells used in E were isolated from mixed bone marrow chimeric mice to minimize extrinsic effects on naive T cells. (F) In an in vitro T reg cell suppressor assay, CD4+CD25+CD45RBhighCD103− T reg cells were sorted from spleens of wild-type and Gpr174−/Y littermate mice. T reg cells were cultured at the indicated ratios with 105 CFSE-labeled naive CD45.1+CD4+ T cells and 2 × 104 CD11c-enriched DCs in the presence of 0.5 µg/ml soluble anti-CD3ε mAb for 3 d. The proliferation index of CFSE-labeled cells was determined in triplicate cultures, and representative CFSE flow cytometry plots are shown. (G) A total of 2 × 105 naive CD4+ T cells from CD45.2+ wild-type or Gpr174−/Y littermate mice were cultured along with an equivalent number of cells from CD45.1+ wild-type mice under Th1 or Th17 polarizing conditions for 5 d. After restimulation with PMA and ionomycin, the percentage of CD45.2+ cells that secreted IFN-γ or IL-17 was determined by intracellular cytokine staining and flow cytometry analysis. (F and G) Error bars show SD. Data were evaluated by unpaired Student’s t test: *, P < 0.05; **, P < 0.01. All data are representative of three or more independent experiments.
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fig3: CD4+ T cells show diminished Nur77 levels and enhanced proliferation in Gpr174−/Y mice. (A) Flow cytometry analysis of the mean fluorescence intensity (MFI) of Nur77-GFP expression in thymocytes from 6-wk-old wild-type and Gpr174−/Y Nur77-GFP+ littermate male mice. Each dot represents an individual mouse; n = 4. (B) Intracellular levels of Ki-67 expression in the indicated thymocyte populations from 6-wk-old wild-type and Gpr174−/Y littermate male mice were determined using flow cytometry. The percentage of Ki-67+ cells among Foxp3+ T reg and Foxp3− CD4 SP thymocytes is shown; n = 9 or 10. (A and B) Horizontal lines indicate the mean. (C) The effects of LysoPS on T cell proliferation under neutral conditions. CFSE-labeled LN cells from wild-type or Gpr174−/Y mice were cultured in round-bottom plates with 0.25 µg/ml soluble anti-CD3 and the indicated amounts of LysoPS. Cell proliferation was assessed 3 d later based on CFSE dilution that was measured by flow cytometry; live CD4+TCR-β+ T cells are shown. Gates indicate the percentage of cells that divided at least three times, and means ± SD are shown; n = 4. (D and E) Direct effects of GPR174 and its ligand LysoPS on proliferation and Foxp3 induction. In flat 96-well plates coated with anti-CD3 and anti-CD28 mAbs (both 2 µg/ml), a mixture of either wild-type (Ly5-1+) or Gpr174−/Y (Ly5-1+) with congenic wild-type (Ly5-2+) naive CD4+ T cells (D) or either wild-type or Gpr174−/Y naive CD4+ T cells (E) were added. Cells were cultured in the presence of 200 U/ml IL-2 (D) or 1 ng/ml TGF-β (E) and the indicated concentrations of 18:0 LysoPS for 4 d. Expression of Ly5-1 and Ly5-2 (D) or Foxp3 (E) was measured by flow cytometry; n = 4; error bars indicate SD. Counts were quantified based on the number of events acquired on a flow cytometer run for 60 s per sample (E). Cells used in E were isolated from mixed bone marrow chimeric mice to minimize extrinsic effects on naive T cells. (F) In an in vitro T reg cell suppressor assay, CD4+CD25+CD45RBhighCD103− T reg cells were sorted from spleens of wild-type and Gpr174−/Y littermate mice. T reg cells were cultured at the indicated ratios with 105 CFSE-labeled naive CD45.1+CD4+ T cells and 2 × 104 CD11c-enriched DCs in the presence of 0.5 µg/ml soluble anti-CD3ε mAb for 3 d. The proliferation index of CFSE-labeled cells was determined in triplicate cultures, and representative CFSE flow cytometry plots are shown. (G) A total of 2 × 105 naive CD4+ T cells from CD45.2+ wild-type or Gpr174−/Y littermate mice were cultured along with an equivalent number of cells from CD45.1+ wild-type mice under Th1 or Th17 polarizing conditions for 5 d. After restimulation with PMA and ionomycin, the percentage of CD45.2+ cells that secreted IFN-γ or IL-17 was determined by intracellular cytokine staining and flow cytometry analysis. (F and G) Error bars show SD. Data were evaluated by unpaired Student’s t test: *, P < 0.05; **, P < 0.01. All data are representative of three or more independent experiments.
Mentions: To better characterize the development of thymic Gpr174−/Y T reg cells, we crossed Gpr174−/Y mice to animals expressing a Nur77-GFP transgene (Moran et al., 2011). Levels of Nur77 increase in positively selected thymic T reg cells and CD25+Foxp3− T reg cell precursors in response to recent MHC class II–dependent signaling. Importantly, Nur77 levels are reduced in conditions that favor enhanced T reg cell development, such as in response to exogenous TNFR family–mediated co-stimulation (Mahmud et al., 2014). CD25+ CD4 SP thymocytes in Gpr174−/Y mice expressed lower levels of Nur77-GFP, consistent with a model in which GPR174 deficiency results in the exposure of developing thymocytes to T reg cell–favoring signals (Fig. 3 A). Additionally, we noted that CD25− CD4 SP thymocytes showed a very slight reduction in Nur77-GFP levels (Fig. 3 A). Because TNF receptor family members contribute to thymic T reg cell development and reduced Nur77-GFP abundance (Mahmud et al., 2014), we measured levels of glucocorticoid-induced TNFR family–related gene (GITR), OX40, and TNFR2 in the Foxp3+ and Foxp3− CD4 SP subsets and detected no differences between wild-type and Gpr174−/Y thymocytes (not depicted). In line with the accumulation of Gpr174−/Y thymic T reg cells, more Foxp3+, but not Foxp3− CD4 SP thymocytes expressed Ki-67, a marker of cells that have recently divided, compared with cells in wild-type littermates (Fig. 3 B). This effect was most obvious in the thymus of mice younger than 8 wk old (not depicted). Together, these data suggest that GPR174 signaling might intrinsically constrain thymic T reg cell proliferation.

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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