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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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CD8+ T cells proliferating in response to alloantigen are selectively killed by LPR DN T cells via the Fas pathway.CD8+ T cells (105/well) from B6 (Fas+/+) or B6.LPR (FasLPR/LPR) were labelled with CFSE and cocultured with irradiated CB6F1 splenocytes and IL-2 for 5 days without or with LPR DN T cells in the indicated ratios. After 5 days, the cultures were stained with anti-CD8-APC and 7-AAD and analyzed by flow cytometry. A. The percentage of undivided cells (CFSEhi) was used to determine the percent suppression at each DN:CD8+ T cell ratio. Two-way ANOVA p<0.0001 for the effect of CD8+ T cell Fas expression. B. Representative histograms of 7-AAD staining gated on proliferated (CFSE-diluted) Fas+/+ (top row) and FasLPR/LPR (bottom row) CD8+ T cells at the indicated DN:CD8+ ratios. Numbers inside histograms are the percentages of cells falling in the 7-AAD+ gate. C. The fold increase in cell death for proliferated Fas+/+ (white bars), proliferated FasLPR/LPR (black bars), unproliferated Fas+/+ (light grey bars), proliferated FasLPR/LPR (dark grey bars) CD8+ T cells is shown. Two-way ANOVA p<0.0001; Bonferrroni post-tests ***p<0.001 and *p<0.05. Data are derived from two independent experiments each with duplicate wells.
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pone-0065253-g006: CD8+ T cells proliferating in response to alloantigen are selectively killed by LPR DN T cells via the Fas pathway.CD8+ T cells (105/well) from B6 (Fas+/+) or B6.LPR (FasLPR/LPR) were labelled with CFSE and cocultured with irradiated CB6F1 splenocytes and IL-2 for 5 days without or with LPR DN T cells in the indicated ratios. After 5 days, the cultures were stained with anti-CD8-APC and 7-AAD and analyzed by flow cytometry. A. The percentage of undivided cells (CFSEhi) was used to determine the percent suppression at each DN:CD8+ T cell ratio. Two-way ANOVA p<0.0001 for the effect of CD8+ T cell Fas expression. B. Representative histograms of 7-AAD staining gated on proliferated (CFSE-diluted) Fas+/+ (top row) and FasLPR/LPR (bottom row) CD8+ T cells at the indicated DN:CD8+ ratios. Numbers inside histograms are the percentages of cells falling in the 7-AAD+ gate. C. The fold increase in cell death for proliferated Fas+/+ (white bars), proliferated FasLPR/LPR (black bars), unproliferated Fas+/+ (light grey bars), proliferated FasLPR/LPR (dark grey bars) CD8+ T cells is shown. Two-way ANOVA p<0.0001; Bonferrroni post-tests ***p<0.001 and *p<0.05. Data are derived from two independent experiments each with duplicate wells.

Mentions: Much of the literature on DN Tregs demonstrates that these cells inhibit T cell responses via the Fas pathway (reviewed in [30], [31]), and this is true for LPR DN T cells as well [4], [29]. While Fas-mediated cytolysis of activated T cells is well described, there are also data to show that Fas ligation on naïve T cells can inhibit their initial activation without causing apoptosis [32], [33]. We recently demonstrated that proliferating, alloreactive Fas+ CD4+ T cells were selectively killed by LPR DN T cells during a CFSE suppression assay [29]. To determine whether this is also true for CD8+ T cells, we co-cultured CFSE-labelled B6 or LPR CD8+ T cells with irradiated CB6F1 splenocytes and IL-2. LPR DN T cells were added to the cultures in varying ratios and after 5 days, CFSE dilution and 7-AAD staining were jointly examined in CD8+ T cells by flow cytometry. As shown in Fig. 6A, the proliferation of Fas-expressing, but not Fas-deficient, CD8+ T cells was suppressed by LPR DN T cells (two-way ANOVA p<0.0001).


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)

CD8+ T cells proliferating in response to alloantigen are selectively killed by LPR DN T cells via the Fas pathway.CD8+ T cells (105/well) from B6 (Fas+/+) or B6.LPR (FasLPR/LPR) were labelled with CFSE and cocultured with irradiated CB6F1 splenocytes and IL-2 for 5 days without or with LPR DN T cells in the indicated ratios. After 5 days, the cultures were stained with anti-CD8-APC and 7-AAD and analyzed by flow cytometry. A. The percentage of undivided cells (CFSEhi) was used to determine the percent suppression at each DN:CD8+ T cell ratio. Two-way ANOVA p<0.0001 for the effect of CD8+ T cell Fas expression. B. Representative histograms of 7-AAD staining gated on proliferated (CFSE-diluted) Fas+/+ (top row) and FasLPR/LPR (bottom row) CD8+ T cells at the indicated DN:CD8+ ratios. Numbers inside histograms are the percentages of cells falling in the 7-AAD+ gate. C. The fold increase in cell death for proliferated Fas+/+ (white bars), proliferated FasLPR/LPR (black bars), unproliferated Fas+/+ (light grey bars), proliferated FasLPR/LPR (dark grey bars) CD8+ T cells is shown. Two-way ANOVA p<0.0001; Bonferrroni post-tests ***p<0.001 and *p<0.05. Data are derived from two independent experiments each with duplicate wells.
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pone-0065253-g006: CD8+ T cells proliferating in response to alloantigen are selectively killed by LPR DN T cells via the Fas pathway.CD8+ T cells (105/well) from B6 (Fas+/+) or B6.LPR (FasLPR/LPR) were labelled with CFSE and cocultured with irradiated CB6F1 splenocytes and IL-2 for 5 days without or with LPR DN T cells in the indicated ratios. After 5 days, the cultures were stained with anti-CD8-APC and 7-AAD and analyzed by flow cytometry. A. The percentage of undivided cells (CFSEhi) was used to determine the percent suppression at each DN:CD8+ T cell ratio. Two-way ANOVA p<0.0001 for the effect of CD8+ T cell Fas expression. B. Representative histograms of 7-AAD staining gated on proliferated (CFSE-diluted) Fas+/+ (top row) and FasLPR/LPR (bottom row) CD8+ T cells at the indicated DN:CD8+ ratios. Numbers inside histograms are the percentages of cells falling in the 7-AAD+ gate. C. The fold increase in cell death for proliferated Fas+/+ (white bars), proliferated FasLPR/LPR (black bars), unproliferated Fas+/+ (light grey bars), proliferated FasLPR/LPR (dark grey bars) CD8+ T cells is shown. Two-way ANOVA p<0.0001; Bonferrroni post-tests ***p<0.001 and *p<0.05. Data are derived from two independent experiments each with duplicate wells.
Mentions: Much of the literature on DN Tregs demonstrates that these cells inhibit T cell responses via the Fas pathway (reviewed in [30], [31]), and this is true for LPR DN T cells as well [4], [29]. While Fas-mediated cytolysis of activated T cells is well described, there are also data to show that Fas ligation on naïve T cells can inhibit their initial activation without causing apoptosis [32], [33]. We recently demonstrated that proliferating, alloreactive Fas+ CD4+ T cells were selectively killed by LPR DN T cells during a CFSE suppression assay [29]. To determine whether this is also true for CD8+ T cells, we co-cultured CFSE-labelled B6 or LPR CD8+ T cells with irradiated CB6F1 splenocytes and IL-2. LPR DN T cells were added to the cultures in varying ratios and after 5 days, CFSE dilution and 7-AAD staining were jointly examined in CD8+ T cells by flow cytometry. As shown in Fig. 6A, the proliferation of Fas-expressing, but not Fas-deficient, CD8+ T cells was suppressed by LPR DN T cells (two-way ANOVA p<0.0001).

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