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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 deficiency results in an increased accumulation of DN T cells.A. B6.LPR.FcRγ+/+ and B6.LPR.FcRγ−/− mice were given 4×107 CB6F1 splenocytes intravenously. After 7 days, varying numbers of DN T cells were purified and incubated for a further 3 days with irradiated CB6F1 splenocytes (105/well), after which 1 µCi 3H-thymidine was added to each culture. Thymidine uptake, reflecting live proliferated cell number, was determined by scintillation counting. Two-way ANOVA p<0.0001; **Bonferroni post tests p<0.001. B. B6.SCID mice (FcRγ+/+) received 107 B6.LPR.FcRγ+/+ (n = 3, upright triangles) or B6.LPR.FcRγ−/− (n = 3, inverted triangles) DN T cells. On days 1, 5, 7, 10, and 14 blood samples were obtained and peripheral blood mononuclear cells (PBMCs) were stained for TCRβ, CD4, CD8, and NK1.1 expression. The percentage of DN T cells in the PBMC compartment was then determined by flow cytometry. Two-way repeated measures ANOVA p = 0.0279 for the effect of FcRγ genotype; Bonferroni post test p<0.001 at day 14. C. At day 7 and day 14, the splenocytes of the DN T cell recipients were counted and stained for TCRβ, CD4, CD8, and NK1.1 and examined by flow cytometry. The number of splenic DN T cells in each type of recipient was then determined. Two-way ANOVA p = 0.0005 for the effect of FcRγ genotype; Bonferroni post test p<0.001 at day 14.
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pone-0065253-g002: FcRγ deficiency results in an increased accumulation of DN T cells.A. B6.LPR.FcRγ+/+ and B6.LPR.FcRγ−/− mice were given 4×107 CB6F1 splenocytes intravenously. After 7 days, varying numbers of DN T cells were purified and incubated for a further 3 days with irradiated CB6F1 splenocytes (105/well), after which 1 µCi 3H-thymidine was added to each culture. Thymidine uptake, reflecting live proliferated cell number, was determined by scintillation counting. Two-way ANOVA p<0.0001; **Bonferroni post tests p<0.001. B. B6.SCID mice (FcRγ+/+) received 107 B6.LPR.FcRγ+/+ (n = 3, upright triangles) or B6.LPR.FcRγ−/− (n = 3, inverted triangles) DN T cells. On days 1, 5, 7, 10, and 14 blood samples were obtained and peripheral blood mononuclear cells (PBMCs) were stained for TCRβ, CD4, CD8, and NK1.1 expression. The percentage of DN T cells in the PBMC compartment was then determined by flow cytometry. Two-way repeated measures ANOVA p = 0.0279 for the effect of FcRγ genotype; Bonferroni post test p<0.001 at day 14. C. At day 7 and day 14, the splenocytes of the DN T cell recipients were counted and stained for TCRβ, CD4, CD8, and NK1.1 and examined by flow cytometry. The number of splenic DN T cells in each type of recipient was then determined. Two-way ANOVA p = 0.0005 for the effect of FcRγ genotype; Bonferroni post test p<0.001 at day 14.

Mentions: Since LPR FcRγ−/− mice exhibit a much greater accumulation of DN T cells than LPR FcRγ+/+ mice [26], we hypothesized that FcRγ expression might inhibit DN T cell proliferation. To test this hypothesis, LPR FcRγ+/+ or LPR FcRγ−/− DN T cells were preactivated in vivo by infusing LPR FcRγ+/+ and LPR FcRγ−/− mice with allogeneic CB6F1 splenocytes. The purified DN T cells were then cultured in varying ratios with irradiated CB6F1 splenocytes and IL-2. Proliferation of DN T cells was measured by 3H-thymidine incorporation. As shown in Fig.2A, LPR FcRγ−/− DN T cells proliferated significantly greater than that of LPR FcRγ+/+ DN T cells. These data suggest that FcRγ expression in LPR DN T cells decreases their propensity to proliferate in response to alloantigen-stimulation in vitro.


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 deficiency results in an increased accumulation of DN T cells.A. B6.LPR.FcRγ+/+ and B6.LPR.FcRγ−/− mice were given 4×107 CB6F1 splenocytes intravenously. After 7 days, varying numbers of DN T cells were purified and incubated for a further 3 days with irradiated CB6F1 splenocytes (105/well), after which 1 µCi 3H-thymidine was added to each culture. Thymidine uptake, reflecting live proliferated cell number, was determined by scintillation counting. Two-way ANOVA p<0.0001; **Bonferroni post tests p<0.001. B. B6.SCID mice (FcRγ+/+) received 107 B6.LPR.FcRγ+/+ (n = 3, upright triangles) or B6.LPR.FcRγ−/− (n = 3, inverted triangles) DN T cells. On days 1, 5, 7, 10, and 14 blood samples were obtained and peripheral blood mononuclear cells (PBMCs) were stained for TCRβ, CD4, CD8, and NK1.1 expression. The percentage of DN T cells in the PBMC compartment was then determined by flow cytometry. Two-way repeated measures ANOVA p = 0.0279 for the effect of FcRγ genotype; Bonferroni post test p<0.001 at day 14. C. At day 7 and day 14, the splenocytes of the DN T cell recipients were counted and stained for TCRβ, CD4, CD8, and NK1.1 and examined by flow cytometry. The number of splenic DN T cells in each type of recipient was then determined. Two-way ANOVA p = 0.0005 for the effect of FcRγ genotype; Bonferroni post test p<0.001 at day 14.
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pone-0065253-g002: FcRγ deficiency results in an increased accumulation of DN T cells.A. B6.LPR.FcRγ+/+ and B6.LPR.FcRγ−/− mice were given 4×107 CB6F1 splenocytes intravenously. After 7 days, varying numbers of DN T cells were purified and incubated for a further 3 days with irradiated CB6F1 splenocytes (105/well), after which 1 µCi 3H-thymidine was added to each culture. Thymidine uptake, reflecting live proliferated cell number, was determined by scintillation counting. Two-way ANOVA p<0.0001; **Bonferroni post tests p<0.001. B. B6.SCID mice (FcRγ+/+) received 107 B6.LPR.FcRγ+/+ (n = 3, upright triangles) or B6.LPR.FcRγ−/− (n = 3, inverted triangles) DN T cells. On days 1, 5, 7, 10, and 14 blood samples were obtained and peripheral blood mononuclear cells (PBMCs) were stained for TCRβ, CD4, CD8, and NK1.1 expression. The percentage of DN T cells in the PBMC compartment was then determined by flow cytometry. Two-way repeated measures ANOVA p = 0.0279 for the effect of FcRγ genotype; Bonferroni post test p<0.001 at day 14. C. At day 7 and day 14, the splenocytes of the DN T cell recipients were counted and stained for TCRβ, CD4, CD8, and NK1.1 and examined by flow cytometry. The number of splenic DN T cells in each type of recipient was then determined. Two-way ANOVA p = 0.0005 for the effect of FcRγ genotype; Bonferroni post test p<0.001 at day 14.
Mentions: Since LPR FcRγ−/− mice exhibit a much greater accumulation of DN T cells than LPR FcRγ+/+ mice [26], we hypothesized that FcRγ expression might inhibit DN T cell proliferation. To test this hypothesis, LPR FcRγ+/+ or LPR FcRγ−/− DN T cells were preactivated in vivo by infusing LPR FcRγ+/+ and LPR FcRγ−/− mice with allogeneic CB6F1 splenocytes. The purified DN T cells were then cultured in varying ratios with irradiated CB6F1 splenocytes and IL-2. Proliferation of DN T cells was measured by 3H-thymidine incorporation. As shown in Fig.2A, LPR FcRγ−/− DN T cells proliferated significantly greater than that of LPR FcRγ+/+ DN T cells. These data suggest that FcRγ expression in LPR DN T cells decreases their propensity to proliferate in response to alloantigen-stimulation in vitro.

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