IL-6 receptor blockade corrects defects of XIAP-deficient regulatory T cells - PubMed (original) (raw)

IL-6 receptor blockade corrects defects of XIAP-deficient regulatory T cells

Wan-Chen Hsieh et al. Nat Commun. 2018.

Abstract

X-linked lymphoproliferative syndrome type-2 (XLP-2) is a primary immunodeficiency disease attributed to XIAP mutation and is triggered by infection. Here, we show that mouse Xiap-/- regulatory T (Treg) cells and human XIAP-deficient Treg cells are defective in suppressive function. The Xiap-/- Treg cell defect is linked partly to decreased SOCS1 expression. XIAP binds SOCS1 and promotes SOCS1 stabilization. Foxp3 stability is reduced in Xiap-/- Treg cells. In addition, Xiap-/- Treg cells are prone to IFN-γ secretion. Transfer of wild-type Treg cells partly rescues infection-induced inflammation in Xiap-/- mice. Notably, inflammation-induced reprogramming of Xiap-/- Treg cells can be prevented by blockade of the IL-6 receptor (IL-6R), and a combination of anti-IL-6R and Xiap-/- Treg cells confers survival to inflammatory infection in Xiap-/- mice. Our results suggest that XLP-2 can be corrected by combination treatment with autologous iTreg (induced Treg) cells and anti-IL-6R antibody, bypassing the necessity to transduce Treg cells with XIAP.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1

XIAP-deficiency impairs Treg cell suppressive function. a Comparable control and _Xiap_−/− tTreg cells and iTreg cells development. The fractions of splenic CD4+Foxp3+ population from control (WT) and _Xiap_−/− mice were determined (tTreg). WT and _Xiap_−/− CD4+CD25− T cells were treated with anti-CD3/CD28 plus TGF-β and IL-2, and Foxp3 expression at day 5 was assessed (iTreg). Experiments were independently repeated six times. b XIAP-deficiency does not affect the Treg cells phenotype. Expressions of CTLA-4, GITR, LAG3 and FR4 in WT and _Xiap_−/− tTreg cells were determined. Numbers indicate mean fluorescence intensities. c Normal IL-10 and TGF-β production in _Xiap_−/− tTreg cells. CD4+CD25+ cells were stimulated with anti-CD3/CD28 and IL-2 for 96 h, before generation of IL-10 and TGF-β was determined. d Impaired in vitro suppressive activity of _Xiap_−/− tTreg cells. CD4+CD25- cells were incubated with anti-CD3, antigen-presenting cells, and the indicated ratios of WT and _Xiap_−/− CD4+CD25+ tTreg cells. [3H]thymidine incorporation was determined at 80 h. Values are mean ± SD of triplicate samples in an experiment. *P < 0.05, **P < 0.01 for unpaired _t_-test. Experiments were reproduced independently three times with similar outcomes. e Knockdown of XIAP in human Treg cells. The levels of XIAP in control (pLKO.3) and XIAP-knockdown human iTreg cells were determined by immunoblots. f Impaired suppressive activity in XIAP-deficient human tTreg cells. Human effector T cells (Teff) were labeled with 2 μM CFSE, activated by anti-CD3/CD28 in the presence of the indicated ratio of human control and XIAP-knockdown tTreg cells, and collected at 72 h. Proliferation was determined by gating CD4+ T cells for their CFSE intensity in flow cytometry. g, h Diminished inhibitory activity of _Xiap_−/− tTreg cells in vivo. CD4+CD25− effector T cells were administered intraperitoneally into _Rag1_−/− mice with WT or _Xiap_−/− tTreg cells. Body weight was determined at the indicated time-points (g). Data are the mean ± SD of six mice in each group. ***P < 0.001 for two-way ANOVA. Mice were killed at day 27 and colons were removed, fixed in paraformaldehyde, sectioned, and stained with H&E. Micrographs are representative of the six mice in each group. Bar indicates 100 μm

Fig. 2

Foxp3 instability in activated _Xiap_−/− Treg cells. a, b Reduced Foxp3+ population in activated _Xiap_−/− tTreg cells. WT and _Xiap_−/− tTreg cells were stimulated with anti-CD3/CD28 in the presence of IL-2 for 4 days. Foxp3 expression of activated tTreg cells was determined by intracellular staining and analyzed by flow cytometry. The gated section represents the Foxp3+ population and the number indicates the percentage of each population (a). The Foxp3+ fractions in WT and _Xiap_−/− tTreg cells were quantitated (b), n = 5. c, d Diminished Foxp3high population in adoptively transferred _Xiap_−/− tTreg cells. CD45.2+ tTreg cells were recovered from CD45.1+ _Rag1_−/− mice into which CD45.2+ tTreg cells and CD45.1+ CD4+CD25− T cells had been transferred a month earlier. The isolated CD45.2+ CD4+ T cells were reactivated with TPA/A23187 and Foxp3 expression was determined by intracellular staining (c). The Foxp3+ fractions in the transferred WT and _Xiap_−/− tTreg cells re-isolated from _Rag1_−/− mice were quantitated (d), n = 9. *P < 0.05 for unpaired t-test

Fig. 3

XIAP interacts with SOCS1 and enhances SOCS1 expression. a XIAP deficiency impairs IL-2-induced SOCS1 expression. T cells were collected at 0, 10, 20, 40, 60, and 120 min after IL-2 treatment. SOCS1 expression of lysate was detected by anti-SOCS1. Protein levels were quantitated by densitometry and normalized by actin control. The level of SOCS1 in WT T cells was used as 1 for comparison. b XIAP-deficiency decreases SOCS1 expression in human iTreg cells. Control and XIAP-knockdown human iTreg cells were treated with IL-2 and the levels of SOCS1 were determined at the indicated time-points. c XIAP enhances SOCS1 expression. XIAP-FLAG and SOCS1-HA were co-transfected into HEK293T cells. After 24 h of transfection, cell lysates were prepared and SOCS1 and XIAP expression was determined with anti-FLAG and anti-HA. d XIAP interacts with SOCS1. XIAP-FLAG and SOCS1-HA were co-transfected into HEK293T cells as indicated. Total cell lysates were immunoprecipitated by anti-HA and the presence of SOCS1 and XIAP-FLAG in the precipitates and lysates was determined. * indicates immunoglobulin heavy chain. e Endogenous XIAP interacts with SOCS1. Mouse peripheral T cells from spleen and lymph nodes were treated with IL-2 as indicated and then 600 μg of cell lysates were immunoprecipitated with anti-SOCS1 or control goat IgG. The contents of endogenous XIAP were determined. * indicates immunoglobulin heavy chain. f The BIR1 domain of XIAP interacts with SOCS1. Full-length (FL), RING domain-deleted (ΔR), N-terminus (N), C-terminus (C), BIR1, BIR2 or BIR3 of XIAP-FLAG were co-transfected with SOCS1-HA into HEK293T cells. Total cell lysates were immunoprecipitated with anti-HA and the presence of XIAP variants and SOCS1 in the pull-down complex and cell lysates was determined. g The SH2 domain of SOCS1 binds XIAP. Full-length (FL), SOSC box-deleted (ΔSB), N-terminal-deleted (ΔN), N-terminal (N), SH2 domain, or SOCS box (SB) of SOCS1 were transfected with XIAP-FLAG into HEK293T cells as indicated. Total cell lysates were immunoprecipitated by anti-FLAG and the presence of SOCS1 variants and XIAP in the precipitates and cell lysates was determined. Each experiment (a, c–g) was independently repeated three times with similar results

Fig. 4

XIAP enhances the association of Elongin B/C with SOCS1 and promotes SOCS1 K63 ubiquitination. a XIAP increases the association of Elongin B/C with SOCS1. Full-length, ΔRING (ΔR) or C-terminal (C) XIAP-FLAG was co-transfected with SOCS1-HA and Elongin B/C-Myc into HEK293T cells. Elongin B/C-Myc in cell lysates was pulled down by anti-Myc and the presence of SOCS1 and XIAP in the precipitates and lysates was determined by the respective antibodies. b XIAP enhances the addition of K63 ubiquitin to SOCS1 in vivo. XIAP-FLAG was co-transfected with SOCS1-myc and Elongin B/C-Myc into HEK293T cells, with WT Ub, K48 Ub or K63 Ub, as indicated. SOCS1 was immune-precipitated, and the association of ubiquitin type determined. c XIAP promotes SOCS1 K63 poly-ubiquitination in vitro. Recombinant K63 ubiquitin-HA, E1, E2 (UBC13), SOCS1-HA (on Mag beads), Elongin B/C-Myc, or XIAP-FLAG was added as indicated to a ubiquitination reaction conducted at 30 °C for 1 h. Around 10% of the reaction mixture was used for Western blotting. SOCS1-HA was pulled down from the rest of the reaction mixture and ubiquitination of the SOCS1 complex was determined. Experiments were independently repeated three times (a, c) or twice (b)

Fig. 5

Enhanced conversion of XIAP-deficient Treg cells into Foxp3+IFN-γ+ cells. a, b Increased transition of _Xiap_−/− tTreg cells into IFN-γ-producing cells. WT and _Xiap_−/− tTreg cells (Foxp3-GFP tagged) were isolated by GFP-expression and re-stimulated with plate-bound anti-CD3/CD28 and IL-2 (CD3/CD28), with additional IL-12 ( + IL-12, 50 ng ml−1), for 4 days. tTreg cells were then reactivated with TPA/A23187 for 6 h, and the expressions of IFN-γ in the gated GFP+ (representing Foxp3+) fraction were determined by intracellular staining (a). Quantitation of Foxp3+IFN-γ+ cells from tTreg cells isolated by either Foxp3-GFP or CD4+CD25+ expression (b), n = 10. ***P < 0.001 for unpaired _t_-test. c Increased secretion of IFN-γ by _Xiap_−/− tTreg cells. Purified WT and _Xiap_−/− tTreg cells were stimulated as in (a), or with additional IL-6 plus IL-1α + IL-1β ( + IL-6/IL-1), and re-activated with TPA/A23187 for 24 h and the levels of IFN-γ in supernatants was analyzed by ELISA. d, e Enhanced expression and secretion of IFN-γ by human XIAP-knockdown iTreg cells. Human control and XIAP-knockdown iTreg cells were activated by anti-CD3/CD28 and IL-2, with the addition of IL-12 or IL-6, as indicated, for 4 days. Secretion of IFN-γ was quantitated (d) and intracellular expressions of Foxp3 and IFN-γ were determined (e). f Elevated secretion of IL-17A by _Xiap_−/− tTreg cells. WT and _Xiap_−/− tTreg cells were stimulated as in (c) and the generated IL-17 was quantitated by ELISA. g, h Increased expression of IFN-γ and IL-17 in the adoptively transferred _Xiap_−/− tTreg cells. WT or _Xiap_−/− CD45.2+ tTreg cells were administrated into _Rag1_−/− CD45.1+ mice together with WT CD45.1+ CD4+CD25− effector T cells. Mice were killed after 27 days and CD4+ T cells from spleen and lymph nodes were isolated. CD4+ T cells were activated by TPA/A23187 and the expressions of Foxp3, IFN-γ (g) and IL-17A (h) in CD45.2+ cells were determined by intracellular staining. Values (c, e, f) are mean ± SD of triplicate samples in an experiment. *P < 0.05, **P < 0.01, ***P < 0.001 for unpaired t-test. All experiments (a–h) were independently repeated three times with similar results

Fig. 6

Transfer of Treg cells rescues _Xiap_−/− mice from lethal Candida albicans infection. a Increased expression of IFN-γ+ and IL-17+ cells in _Xiap_−/− Treg cells upon Candida albicans infection. WT and _Xiap_−/− female mice were intravenously infected with C. albicans (1 × 105), and mice were killed at day 10. T cells were isolated from spleen, and the frequencies of CD4+Foxp3+, Foxp3+IFN-γ+ and Foxp3+IL-17+ cells were quantitated by intracellular staining. b Transfer of WT iTreg cells partially rescues _Xiap_−/− mice from _C. albicans_-induced lethality. WT and _Xiap_−/− female mice were intravenously administered with C. albicans (1 × 105) and a group of _Xiap_−/− mice also received WT iTreg cells (1 × 106) at day 2. Survival of mice was monitored and is presented as a Kaplan–Meier survival curve (n = 7 for each group). **P < 0.01. c Reduced kidney inflammation in _C. albicans_-infected _Xiap_−/− mice with WT iTreg cells transfer. Kidney was isolated from _Xiap_−/− mice 10 days after C. albicans injection and infiltrated neutrophil contents were determined. d Reduced inflammatory cytokine production in _C. albicans_-infected _Xiap_−/− mice into which WT iTreg cells had been transferred. Serum from mice was collected at the indicated time-points after C. albicans injection, and the levels of IL-6, TNF, MCP-1, and IL-12 were determined, n = 6. Values (a, d) are mean ± SD of samples. *P < 0.05, **P < 0.01, ***P < 0.001 for unpaired _t_-test

Fig. 7

Anti-IL-6R reduces the expression of IFN-γ in activated _Xiap_−/− Treg cells. a, b Anti-IL-6R decreases IFN-γ expression in re-stimulated _Xiap_−/− Treg cells. WT and _Xiap_−/− iTreg cells were stimulated with anti-CD3/CD28 and IL-2, or with additional IL-12 as indicated, in the absence or presence of anti-IL-6R (50 μg ml−1) for 4 days. iTreg cells were re-stimulated with TPA/A23187 for 24 h and IFN-γ production was determined by ELISA (a), or reactivated with TPA/A23187 for 5 h and the expressions of Foxp3 and IFN-γ were determined by intracellular staining (b). c, d Anti-IL-6R inhibits IFN-γ expression in human Treg cells. Control and human XIAP-knockdown iTreg cells were stimulated as in (a, b), with additional IL-6 as indicated, and secretion (c) or intracellular expression (d) of IFN-γ was determined. e Inability of anti-TNF or anti-IL-1R to inhibit the production of IFN-γ in activated _Xiap_−/− Treg cells. WT and _Xiap_−/− iTreg cells were stimulated, as described in (a), in the presence or absence of anti-TNF or anti-IL-1R (50 μg ml−1 each) for 4 days. Treg cells were re-stimulated with TPA/A23187 for 24 h and the secreted IFN-γ was determined by ELISA. f Anti-IL-6R rescues the impaired suppressive activity of _Xiap_−/− tTreg cells in vivo. CD45.2+ WT or _Xiap_−/− tTreg cells were co-transferred with CD45.1+ CD4+CD25- effector T cells into male CD45.1+ _RagI_−/− mice. Anti-IL-6R antibody (500 μg per mouse) was intraperitoneally administrated at day 0, followed by weekly dosing of 500 μg. The body weights of mice were monitored at the indicated time-points. ***P < 0.001 by two-way ANOVA. g, h Anti-IL-6R restores Foxp3 stability and reduces IFN-γ expression in _Xiap_−/− tTreg cells transferred in vivo. Lymphocytes were isolated from spleen and mesenteric lymph nodes of mice in (f) and reactivated with TPA/A23187. The expressions of Foxp3 (g) and IFN-γ (h) in CD45.2+ T cells were determined by intracellular staining. Values are mean ± SD of triplicate samples in an experiment. *P < 0.05, **P < 0.01, ***P < 0.001 for unpaired _t_-test (a, c, g, h). Experiments (a–e) were independently repeated three times with similar results

Fig. 8

Combination anti-IL-6R and _Xiap_−/− Treg cells rescue mice from infection-induced inflammation. a, b _Xiap_−/− iTreg cells plus anti-IL-6R rescue _Xiap_−/− mice from C. albicans infection. Male _Xiap_−/− mice (KO) were intravenously administered with C. albicans and _Xiap_−/− iTreg cells (1 × 106) or anti-IL-6R antibody (500 μg) at day 2, or both, as indicated. Survival of mice is presented as a Kaplan–Meier survival curve (a). n = 11 for KO, n = 10 for KO + KO iTreg, n = 6 for KO + Ab, n = 9 for KO + KO iTreg + Ab. ***P < 0.001 for Log-rank (Mantel–Cox) Test (a). n.s., not significant. The serum level of IL-6 was determined on day 14 after infection (b). *P < 0.05 for unpaired _t_-test (b). c, d Anti-IL-6R prevents the conversion of the transferred _Xiap_−/− Treg cells. CD45.1+ _Xiap_−/− iTreg cells were transferred into CD45.2+ _Xiap_−/− mice infected with C. albicans as in (a). CD45.1+ T cells were recovered at day 10 post-infection, and the expression of Foxp3 and IFN-γ was determined (c). The Foxp3+IFN-γ+ fractions in the CD45.1+ Treg cells were quantitated (d), n = 5. **P < 0.01 for unpaired _t_-test (d). e, f Anti-IL-6R reduces kidney neutrophil infiltration and fungal load in _C. albicans_-infected _Xiap_−/− mice with KO iTreg cells transfer. Male _Xiap_−/− mice were infected with C. albicans, as described in (a). Kidneys were isolated 14 days post-infection and neutrophils (e) and C. albicans titres (f) were quantitated. *P < 0.05, **P < 0.01 for unpaired _t_-test. n.s., not significant. g Anti-IL-6R does not affect IL-17 production. Male CD45.1+ _Xiap_−/− mice were infected with C. albicans and treated with anti-IL-6R and anti-IL-6R plus CD45.2+ KO iTreg cells. Spleen T cells and B cells were isolated 7 days after treatments. CD45.2+ iTreg cells were depleted. T cells were incubated with B cells and heat-killed C. albicans (HKCA) for 4 days, and the production of IL-17A after stimulation with TPA/A23187 (10/100 ng ml−1) was determined by ELISA, . Values are mean ± SD of triplicate samples in an experiment. Experiments were independently repeated three times with similar results

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IL-6 receptor blockade corrects defects of XIAP-deficient regulatory T cells - PubMed (original) (raw)