TRAF3 regulates the effector function of regulatory T cells and humoral immune responses - PubMed (original) (raw)

TRAF3 regulates the effector function of regulatory T cells and humoral immune responses

Jae-Hoon Chang et al. J Exp Med. 2014.

Abstract

Regulatory T cells (Treg cells) control different aspects of immune responses, but how the effector functions of Treg cells are regulated is incompletely understood. Here we identified TNF receptor-associated factor 3 (TRAF3) as a regulator of Treg cell function. Treg cell-specific ablation of TRAF3 impaired CD4 T cell homeostasis, characterized by an increase in the Th1 type of effector/memory T cells. Moreover, the ablation of TRAF3 in Treg cells resulted in increased antigen-stimulated activation of follicular T helper cells (TFH cells), coupled with heightened formation of germinal centers and production of high-affinity IgG antibodies. Although the loss of TRAF3 did not reduce the overall frequency of Treg cells, it attenuated the antigen-stimulated production of follicular Treg cells (TFR cells). TRAF3 signaling in Treg cells was required to maintain high level expression of inducible co-stimulator (ICOS), which in turn was required for TFR cell generation and inhibition of antibody responses. These findings establish TRAF3 as a mediator of Treg cell function in the regulation of antibody responses and suggest a role for TRAF3 in mediating ICOS expression in Treg cells.

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Figures

Figure 1.

Figure 1.

Treg cell–specific TRAF3 ablation does not reduce Treg cell frequency but partially impairs the in vivo function of Treg cells. (A) Treg (CD4+CD25+YFP+) and non-Treg (CD4+CD25−YFP−) T cells were sorted from the WT-R26YFP (WT) and _Traf3Treg-KO_R26YFP (KO) mice, and TRAF3 expression was assessed by immunoblot. β-Actin served as a loading control. (B) The frequency of Foxp3+ Treg cells (among CD3+CD4+ cells) in the spleen of WT and Traf3Treg-KO (KO) mice (6 wk old) was assessed by flow cytometry; data are presented as mean ± SD of five mice per group. Data are representative of five independent experiments. (C) Naive CD4+ T cells (Teff) were labeled with CFSE and activated by anti-CD3 plus antigen-presenting cells (irradiated splenocytes from WT mice, depleted of CD3+ T cells) in the presence of the indicated ratios of WT or KO Treg cells. Naive CD4+ T cell proliferation was measured as dilution of the cytosolic dye CFSE. Data are presented as a representative plot (left) and a summary graph (right) of six mice per genotype in three independent experiments. (D) _Rag1_-KO mice were adoptively transferred with WT B6.SJL CD45RBhi naive CD4+ T cells (CD45.1+) along with either PBS buffer or sorted Treg cells (CD45.1−), and body weight was monitored. (E and F) CD45RBhi CD4+ T cells (CD45.1+) were transferred into recipient mice as in D (on week 5), and the total number (E) and frequency (F) of Th1 and Th17 cells in the spleen of the recipient mice were assessed by ICS. Eight mice in two different experiments were used for each group. (G and H) The frequency of adoptively transferred Foxp3+ Treg cells (G) and IL-10–producing Treg cells (gated on Foxp3+ cells) in the spleen of the recipient mice described in D was determined by flow cytometry (5 wk after adoptive transfer; H). (C–E, G, and H) Mean ± SD is shown. Data are representative of two independent experiments. *, P < 0.05; and **, P < 0.01.

Figure 2.

Figure 2.

Altered CD4+ T cell homeostasis in Traf3Treg-KO mice. (A–D) Spleens (A–C; n = 4–5) or small and large intestine lamina propria (D; n = 6) were harvested from 8-wk-old WT or Traf3Treg-KO (KO) mice and analyzed by flow cytometry for the frequency (A) and absolute number (B) of memory-like (CD44hiCD62Llo) CD4+ T cells, and the frequency of IL-17– and IFN-γ–producing CD4+ T cells (C and D) was assessed using ICS. Mean ± SD is shown. Data are representative of five independent experiments. *, P < 0.05.

Figure 3.

Figure 3.

Treg cell–specific TRAF3 controls antigen-stimulated humoral immune responses. (A) WT and Traf3Treg-KO mice (8 wk old) were immunized i.p. with SRBCs, and serum SRBC–specific Ig was measured 14 d later by ELISA (n = 5). (B and C) WT and Traf3Treg-KO mice (6–8 wk old) were immunized i.p. with KLH-NP26 (B) or Ficoll-NP26 for 12 d (C), and NP9 (high affinity) Ig was measured 12 d later by ELISA (B: n = 4; C: n = 9). (D) Basal concentrations of Ig isotypes and IgG subclasses in the sera of 18–20-wk-old WT and Traf3Treg-KO mice were measured by ELISA (n = 7). (E and F) WT and Traf3Treg-KO mice (8 wk old; n = 10) were intranasally infected with influenza virus H1N1 and monitored for bodyweight loss (E) and serum anti-H1N1 IgG antibody titer on day 13 (F). (A–F) Mean ± SD is shown. Data are representative of four (A–D) or two (E and F) independent experiments. *, P < 0.05; and **, P < 0.01.

Figure 4.

Figure 4.

Treg cell–specific TRAF3 regulates TFH cell function and GC formation. (A and B) WT and Traf3Treg-KO (KO) mice (8 wk old) were immunized i.p. with SRBCs, and the frequency of GC B cells (CD95+GL7+) among B220+ cells was assessed by flow cytometry. Data are presented as a representative plot (A) and mean ± SD based on five mice for each group (B). (C) Mice were immunized as in A and B, and GCs were analyzed by immunofluorescence detection of PNA+ cells. Graph shows average GC areas as mean ± SD based on multiple slides. Data are representative of four WT and three Traf3Treg-KO (KO) mice. Bars, 500 µm. (D) Frequency of W33 to L mutation in the GC B cells of SRBC-immunized WT and Traf3Treg-KO mice (10 d after immunization), determined by sequencing the cDNA clones constructed using pooled RNA from six WT and six KO mice. (E and F) WT-R26YFP (WT) or _Traf3Treg-KO_R26YFP (KO) mice were immunized as in A, and 10 d later, the frequency of TFH cells (CXCR5+PD-1+) among CD4+YFP− T cells was assessed by flow cytometry. Data are presented as a representative plot (E) and mean ± SD (F) based on six mice for each group. (G) TFH cells were sorted from the spleens of the WT and KO mice in E and F, and expression of the indicated genes was assessed by RT-PCR. Mean ± SEM is shown. Data in A–G are representative of three to four independent experiments. *, P < 0.05; and **, P < 0.01.

Figure 5.

Figure 5.

TRAF3 is required for TFR cell induction. (A and B) WT-R26YFP and _Traf3Treg-KO_-R26YFP (KO) mice (8 wk old) were immunized with SRBCs, and 10 d later, the frequency of TFR cells (CXCR5+PD-1+YFP+CD4+) in the spleen was assessed by flow cytometry. Data are presented as a representative plot (A) and mean ± SD (B) based on eight WT and nine KO mice. (C and D) Mice were immunized as in A and B, and the frequency and absolute number of TFR cells was assessed by flow cytometry based on surface staining of CXCR5 and intracellular staining of Bcl-6 within Foxp3+CD4+ Treg cells. Data are presented as a representative plot (C) and mean ± SD (D) based on eight WT and nine KO mice. (A–D) Data are representative of three to four independent experiments.*, P < 0.05; and **, P < 0.01.

Figure 6.

Figure 6.

TRAF3 mediates ICOS gene expression via a MAP kinase pathway in Treg cells. (A) Expression of the indicated surface markers on Foxp3+CD4+ Treg cells from the spleens of WT and Traf3Treg-KO mice (6 wk old) was assessed by flow cytometry. (B) Expression of the indicated genes in Treg cells from 6-wk-old WT or Traf3Treg-KO (KO) mice was assessed by RT-PCR. Mean ± SEM is shown. (C) The frequency of IL-10–producing cells among YFP+CD4+ Treg cells from the spleen of WT-R26YFP (WT) and _Traf3Treg-KO_-R26YFP (KO) mice was assessed by flow cytometry. Data are presented as a representative plot (left) and the mean ± SD of six mice for each group. The left panel of C shows the isotype control for IL-10 ICS. (D) GFP+CD25+CD4+ Treg cells were sorted from the spleens of three WT and three KO mice and stimulated for 24 h with PMA and ionomycin. IL-10 secretion was measured by ELISA. (E and F) WT or Traf3Treg-KO (KO) mice (6 wk old; n = 5) were immunized with SRBCs, and 10 d later, ICOS expression in TFR (E) or TFH (F) cells was assessed by flow cytometry. Graph in E shows the mean fluorescence intensity (MFI) of ICOS based on five mice for each group. (D–F) Mean ± SD is shown. (G) _Rag1_-KO recipient mice were adoptively transferred with 1:1 ratio of mixed bone marrows of WT (B6.SJL, CD45.1+) and Traf3Treg-KO (KO, CD45.2+) mice. 6 wk later, mice were immunized with SRBCs, and ICOS expression on gated WT (CD4+CD45.1+Foxp3+) and KO (CD4+CD45.2+Foxp3+) Treg cells was assessed by flow cytometry at day 10 after immunization. Graph shows MFI as mean ± SD from four mice for each group. Data are representative of three to four (A–F) or two (G) independent experiments. *, P < 0.05; and **, P < 0.01.

Figure 7.

Figure 7.

NIK overexpression in Treg cells does not inhibit the expression of ICOS and IL-10 or perturb antibody production. (A) The frequency of memory (CD44hiCD62Llo) and naive (CD44loCD62Lhi) CD4+ T cells in WT and NIKΔT3Treg-Tg mice (12 wk old; n = 4) was analyzed by flow cytometry. (B) Serum IgM and IgG in unmanipulated WT and NIKΔT3Treg-Tg mice (12–16 wk old; n = 4) was measured by ELISA. (C) Expression of ICOS in CD4+Foxp3+ T cells from WT and NIKΔT3Treg-Tg mice (6 wk old; n = 3) was assessed by flow cytometry. (D) Frequency of IL-10–producing cells among GFP+CD4+ Treg cells from the spleen of WT and NIKΔT3Treg-Tg mice (6 wk old; n = 3) was measured by ICS. (B and D) Mean ± SD is shown. (E) Expression of the indicated surface markers on Foxp3+CD4+ Treg cells from the spleens of WT and NIKΔT3Treg-Tg mice (8 wk old) was assessed by flow cytometry. Data are representative of four independent experiments.

Figure 8.

Figure 8.

TRAF3 mediates TCR/CD28-stimulated activation of ERK and AP1. (A) Treg cells (CD4+YFP+CD25+) were sorted from WT-R26YFP and Traf3Treg-KO-R26YFP mice and stimulated for the indicated time periods with anti-CD3 plus anti-CD28 using a cross-linking method. Levels of total and phosphorylated ERK1,2 were assessed by immunoblot. Data are presented as a representative blot (left) and a summary graph based on quantification of three independent blots (right). Mean ± SD is shown. (B) ICOS expression on sorted WT Treg cells that were either not treated (NT) or treated with anti-CD3 plus anti-CD28 in the absence (T) or presence (T + U0126) of an ERK inhibitor, 50 µM U0126, for 24 h was assessed by flow cytometry. (C) Nuclear extracts were prepared from WT or TRAF3-deficient CD4+ T cells stimulated with anti-CD3 plus anti-CD28 for the indicated time periods, and EMSA was performed using ICOS AP1 or the control NF-Y oligonucleotide probes. Data are representative of three independent experiments. **, P < 0.01.

Figure 9.

Figure 9.

ICOS is essential for controlling TFH cell function and GC formation. Sorted naive CD4+ T cells (CD4+CD62LhiCD44loCD25−) from WT B6.SJL mice (5 wk old; CD45.1+) and sorted Treg T cells (CD4+YFP+CD25+) from WT, Traf3Treg-KO (_Traf3_-KO), or _Icos_-KO mice (6 wk old; CD45.2+) were mixed in a 5:1 ratio and intravenously transferred into Tcrb/_Tcrd_-dKO mice. 2 d later, recipient mice were immunized with SRBCs and sacrificed for experiments 14 d after immunization. (A) Frequency of Treg cells (CD45.1−) and naive CD4+ T cells (CD45.1+) in the spleen of Tcrb/_Tcrd_-dKO recipients was assessed by flow cytometry. (B) Frequency of GC B cells (GL7+CD95+) among B220+ cells was assessed by flow cytometry. Data are presented as a representative plot (left) and mean ± SD (right) based on three mice for each group. (C) SRBC-specific antibody levels in the sera of Tcrb/_Tcrd_-dKO recipient mice transferred with WT, _Traf3_-KO, and _Icos_-KO Treg cells was assessed by ELISA. Mean ± SD is shown. (D) Frequency of plasma cells (B220−CD138+) in the spleens of the Tcrb/_Tcrd_-dKO recipients was assessed by flow cytometry. (E and F) Frequency of the TFH (CXCR5+PD-1+Foxp3−CD4+) cells among CD4+Foxp3− T cells (E) and the frequency of TFR (CXCR5+PD-1+GFP+CD4+) cells among CD4+Foxp3+ Treg cells (F) were assessed by flow cytometry. Data are presented as mean ± SD based on three mice for each group and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

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