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FcRγ controls the fas-dependent regulatory function of lymphoproliferative double negative T cells.

Juvet SC, Thomson CW, Kim EY, Han M, Zhang L - PLoS ONE (2013)

Bottom Line: We have shown previously that a significant proportion of DN T cells express FcRγ, and that this molecule is required for TCR transgenic DN T cells to suppress allogeneic immune responses.Furthermore, FcRγ acted in a cell-intrinsic fashion to limit DN T cell accumulation by increasing the rate of apoptosis in proliferated cells.These results indicate that FcRγ can confer Fas-dependent regulatory properties on LPR DN T cells, and suggest that FcRγ may be a novel marker for functional DN Tregs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Patients with autoimmune lymphoproliferative syndrome (ALPS) and lymphoproliferation (LPR) mice are deficient in Fas, and accumulate large numbers of αβ-TCR(+), CD4(-), CD8(-) double negative (DN) T cells. The function of these DN T cells remains largely unknown. The common γ subunit of the activating Fc receptors, FcRγ, plays an important role in mediating innate immune responses. We have shown previously that a significant proportion of DN T cells express FcRγ, and that this molecule is required for TCR transgenic DN T cells to suppress allogeneic immune responses. Whether FcRγ plays a critical role in LPR DN T cell-mediated suppression of immune responses to auto and allo-antigens is not known. Here, we demonstrated that FcRγ(+), but not FcRγ(-) LPR DN T cells could suppress Fas(+) CD4(+) and CD8(+) T cell proliferation in vitro and attenuated CD4(+) T cell-mediated graft-versus host disease. Although FcRγ expression did not allow LPR DN T cells to inhibit the expansion of Fas-deficient cells within the LPR context, adoptive transfer of FcRγ(+), but not FcRγ(-), DN T cells inhibited lymphoproliferation in generalized lymphoproliferative disease (GLD) mice. Furthermore, FcRγ acted in a cell-intrinsic fashion to limit DN T cell accumulation by increasing the rate of apoptosis in proliferated cells. These results indicate that FcRγ can confer Fas-dependent regulatory properties on LPR DN T cells, and suggest that FcRγ may be a novel marker for functional DN Tregs.

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FcRγ expression by LPR DN T cells is required for their regulatory function toward Fas-expressing syngeneic T cells.A. B6 CD4+ T cells (104/well) were co-cultured with irradiated CB6F1 splenocytes (105/well) and IL-2. Purified B6.LPR.FcRγ+/+ or B6.LPR.FcRγ−/− DN T cells were added in varying ratios. After 4 days, 3H-thymidine (1 µCi/well) was added. After 18 h, 3H-thymidine uptake was determined, and percent suppression was calculated. Two-way ANOVA p<0.0001; Bonferroni post-tests **p<0.01. Data are from one of three experiments with similar results. B. B6 or B6.LPR.FcRγ+/+ CD8+ T cells (104/well) were cultured as in C. Percent suppression was calculated. Two-way ANOVA p<0.0001 for the effect of FcRγ on suppression of B6 CD8+ T cells; p = 0.0043 for the effect of FcRγ on suppression of B6.LPR CD8+ T cells. Bonferroni post-tests **p<0.01. Data are from one of three experiments with similar results. C. Male CB6F1 mice were lethally irradiated and reconstituted with 2×106 TCD BM alone (BM Only) or with 106 B6 CD4+ T cells (BM+CD4+), with or without 5×106 B6.LPR.FcRγ+/+ or B6.LPR.FcRγ−/− DN T cells. Mice losing >25% of their body weight were sacrificed. *Log rank test p = 0.01 for the comparison of mice treated with B6.LPR.FcRγ+/+ and B6.LPR.FcRγ−/− DN T cells. Data are derived from two independent experiments, each with 2–5 mice per group. D. Naive B6 CD8+ T cells were used as responders and stimulated by irradiated bm1 splenocytes. Varying numbers of DN T cells isolated from spleens of bm1 splenocyte-treated B6.FcRγ+/+ (squares) or B6.FcRγ−/− (triangles) mice were added to the MLR cultures as putative suppressor cells. Cell proliferation was measured by 3H-thymidine incorporation. The data are expressed as percent inhibition of proliferation as compared with the controls to which no putative suppressor cells were added. Data points are the mean +/− SD of triplicate wells and are derived from one of three independent experiments.
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pone-0065253-g005: FcRγ expression by LPR DN T cells is required for their regulatory function toward Fas-expressing syngeneic T cells.A. B6 CD4+ T cells (104/well) were co-cultured with irradiated CB6F1 splenocytes (105/well) and IL-2. Purified B6.LPR.FcRγ+/+ or B6.LPR.FcRγ−/− DN T cells were added in varying ratios. After 4 days, 3H-thymidine (1 µCi/well) was added. After 18 h, 3H-thymidine uptake was determined, and percent suppression was calculated. Two-way ANOVA p<0.0001; Bonferroni post-tests **p<0.01. Data are from one of three experiments with similar results. B. B6 or B6.LPR.FcRγ+/+ CD8+ T cells (104/well) were cultured as in C. Percent suppression was calculated. Two-way ANOVA p<0.0001 for the effect of FcRγ on suppression of B6 CD8+ T cells; p = 0.0043 for the effect of FcRγ on suppression of B6.LPR CD8+ T cells. Bonferroni post-tests **p<0.01. Data are from one of three experiments with similar results. C. Male CB6F1 mice were lethally irradiated and reconstituted with 2×106 TCD BM alone (BM Only) or with 106 B6 CD4+ T cells (BM+CD4+), with or without 5×106 B6.LPR.FcRγ+/+ or B6.LPR.FcRγ−/− DN T cells. Mice losing >25% of their body weight were sacrificed. *Log rank test p = 0.01 for the comparison of mice treated with B6.LPR.FcRγ+/+ and B6.LPR.FcRγ−/− DN T cells. Data are derived from two independent experiments, each with 2–5 mice per group. D. Naive B6 CD8+ T cells were used as responders and stimulated by irradiated bm1 splenocytes. Varying numbers of DN T cells isolated from spleens of bm1 splenocyte-treated B6.FcRγ+/+ (squares) or B6.FcRγ−/− (triangles) mice were added to the MLR cultures as putative suppressor cells. Cell proliferation was measured by 3H-thymidine incorporation. The data are expressed as percent inhibition of proliferation as compared with the controls to which no putative suppressor cells were added. Data points are the mean +/− SD of triplicate wells and are derived from one of three independent experiments.

Mentions: To determine further the importance of FcRγ expression in the regulatory function of LPR DN T cells in an allogeneic setting, B6 CD4+ T cells were co-cultured with allogeneic CB6F1 splenocytes in the presence of either LPR FcRγ+/+ or LPR FcRγ−/− DN T cells in varying ratios. After 3 days, proliferation of CD4+ T cells was determined and the percent suppression calculated. As shown in Fig. 5A, LPR FcRγ−/− DN T cells had a reduced ability to suppress the alloantigen-driven proliferation of B6 CD4+ T cells in comparison with LPR FcRγ+/+ DN T cells. We observed a similar phenomenon when B6 CD8+ T cells were used as responders (Fig. 5B, closed symbols; two way ANOVA p<0.0001 for the effect of FcRγ; Bonferroni post test p<0.01 at all ratios). Thus, LPR DN T cells can inhibit syngeneic Fas+ T cells responding to alloantigens in an FcRγ-dependent manner.


FcRγ controls the fas-dependent regulatory function of lymphoproliferative double negative T cells.

Juvet SC, Thomson CW, Kim EY, Han M, Zhang L - PLoS ONE (2013)

FcRγ expression by LPR DN T cells is required for their regulatory function toward Fas-expressing syngeneic T cells.A. B6 CD4+ T cells (104/well) were co-cultured with irradiated CB6F1 splenocytes (105/well) and IL-2. Purified B6.LPR.FcRγ+/+ or B6.LPR.FcRγ−/− DN T cells were added in varying ratios. After 4 days, 3H-thymidine (1 µCi/well) was added. After 18 h, 3H-thymidine uptake was determined, and percent suppression was calculated. Two-way ANOVA p<0.0001; Bonferroni post-tests **p<0.01. Data are from one of three experiments with similar results. B. B6 or B6.LPR.FcRγ+/+ CD8+ T cells (104/well) were cultured as in C. Percent suppression was calculated. Two-way ANOVA p<0.0001 for the effect of FcRγ on suppression of B6 CD8+ T cells; p = 0.0043 for the effect of FcRγ on suppression of B6.LPR CD8+ T cells. Bonferroni post-tests **p<0.01. Data are from one of three experiments with similar results. C. Male CB6F1 mice were lethally irradiated and reconstituted with 2×106 TCD BM alone (BM Only) or with 106 B6 CD4+ T cells (BM+CD4+), with or without 5×106 B6.LPR.FcRγ+/+ or B6.LPR.FcRγ−/− DN T cells. Mice losing >25% of their body weight were sacrificed. *Log rank test p = 0.01 for the comparison of mice treated with B6.LPR.FcRγ+/+ and B6.LPR.FcRγ−/− DN T cells. Data are derived from two independent experiments, each with 2–5 mice per group. D. Naive B6 CD8+ T cells were used as responders and stimulated by irradiated bm1 splenocytes. Varying numbers of DN T cells isolated from spleens of bm1 splenocyte-treated B6.FcRγ+/+ (squares) or B6.FcRγ−/− (triangles) mice were added to the MLR cultures as putative suppressor cells. Cell proliferation was measured by 3H-thymidine incorporation. The data are expressed as percent inhibition of proliferation as compared with the controls to which no putative suppressor cells were added. Data points are the mean +/− SD of triplicate wells and are derived from one of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675138&req=5

pone-0065253-g005: FcRγ expression by LPR DN T cells is required for their regulatory function toward Fas-expressing syngeneic T cells.A. B6 CD4+ T cells (104/well) were co-cultured with irradiated CB6F1 splenocytes (105/well) and IL-2. Purified B6.LPR.FcRγ+/+ or B6.LPR.FcRγ−/− DN T cells were added in varying ratios. After 4 days, 3H-thymidine (1 µCi/well) was added. After 18 h, 3H-thymidine uptake was determined, and percent suppression was calculated. Two-way ANOVA p<0.0001; Bonferroni post-tests **p<0.01. Data are from one of three experiments with similar results. B. B6 or B6.LPR.FcRγ+/+ CD8+ T cells (104/well) were cultured as in C. Percent suppression was calculated. Two-way ANOVA p<0.0001 for the effect of FcRγ on suppression of B6 CD8+ T cells; p = 0.0043 for the effect of FcRγ on suppression of B6.LPR CD8+ T cells. Bonferroni post-tests **p<0.01. Data are from one of three experiments with similar results. C. Male CB6F1 mice were lethally irradiated and reconstituted with 2×106 TCD BM alone (BM Only) or with 106 B6 CD4+ T cells (BM+CD4+), with or without 5×106 B6.LPR.FcRγ+/+ or B6.LPR.FcRγ−/− DN T cells. Mice losing >25% of their body weight were sacrificed. *Log rank test p = 0.01 for the comparison of mice treated with B6.LPR.FcRγ+/+ and B6.LPR.FcRγ−/− DN T cells. Data are derived from two independent experiments, each with 2–5 mice per group. D. Naive B6 CD8+ T cells were used as responders and stimulated by irradiated bm1 splenocytes. Varying numbers of DN T cells isolated from spleens of bm1 splenocyte-treated B6.FcRγ+/+ (squares) or B6.FcRγ−/− (triangles) mice were added to the MLR cultures as putative suppressor cells. Cell proliferation was measured by 3H-thymidine incorporation. The data are expressed as percent inhibition of proliferation as compared with the controls to which no putative suppressor cells were added. Data points are the mean +/− SD of triplicate wells and are derived from one of three independent experiments.
Mentions: To determine further the importance of FcRγ expression in the regulatory function of LPR DN T cells in an allogeneic setting, B6 CD4+ T cells were co-cultured with allogeneic CB6F1 splenocytes in the presence of either LPR FcRγ+/+ or LPR FcRγ−/− DN T cells in varying ratios. After 3 days, proliferation of CD4+ T cells was determined and the percent suppression calculated. As shown in Fig. 5A, LPR FcRγ−/− DN T cells had a reduced ability to suppress the alloantigen-driven proliferation of B6 CD4+ T cells in comparison with LPR FcRγ+/+ DN T cells. We observed a similar phenomenon when B6 CD8+ T cells were used as responders (Fig. 5B, closed symbols; two way ANOVA p<0.0001 for the effect of FcRγ; Bonferroni post test p<0.01 at all ratios). Thus, LPR DN T cells can inhibit syngeneic Fas+ T cells responding to alloantigens in an FcRγ-dependent manner.

Bottom Line: We have shown previously that a significant proportion of DN T cells express FcRγ, and that this molecule is required for TCR transgenic DN T cells to suppress allogeneic immune responses.Furthermore, FcRγ acted in a cell-intrinsic fashion to limit DN T cell accumulation by increasing the rate of apoptosis in proliferated cells.These results indicate that FcRγ can confer Fas-dependent regulatory properties on LPR DN T cells, and suggest that FcRγ may be a novel marker for functional DN Tregs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Patients with autoimmune lymphoproliferative syndrome (ALPS) and lymphoproliferation (LPR) mice are deficient in Fas, and accumulate large numbers of αβ-TCR(+), CD4(-), CD8(-) double negative (DN) T cells. The function of these DN T cells remains largely unknown. The common γ subunit of the activating Fc receptors, FcRγ, plays an important role in mediating innate immune responses. We have shown previously that a significant proportion of DN T cells express FcRγ, and that this molecule is required for TCR transgenic DN T cells to suppress allogeneic immune responses. Whether FcRγ plays a critical role in LPR DN T cell-mediated suppression of immune responses to auto and allo-antigens is not known. Here, we demonstrated that FcRγ(+), but not FcRγ(-) LPR DN T cells could suppress Fas(+) CD4(+) and CD8(+) T cell proliferation in vitro and attenuated CD4(+) T cell-mediated graft-versus host disease. Although FcRγ expression did not allow LPR DN T cells to inhibit the expansion of Fas-deficient cells within the LPR context, adoptive transfer of FcRγ(+), but not FcRγ(-), DN T cells inhibited lymphoproliferation in generalized lymphoproliferative disease (GLD) mice. Furthermore, FcRγ acted in a cell-intrinsic fashion to limit DN T cell accumulation by increasing the rate of apoptosis in proliferated cells. These results indicate that FcRγ can confer Fas-dependent regulatory properties on LPR DN T cells, and suggest that FcRγ may be a novel marker for functional DN Tregs.

Show MeSH
Related in: MedlinePlus