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

The induction of follicular regulatory T cells and control of germinal center reactions require the adaptor protein Traf3, in part because of Traf3-induced expression of ICOS.

Foxp3 + regulatory T cells (T reg cells) form a major population of CD4 + immunosuppressive T cells that plays a pivotal role in maintaining peripheral immune tolerance and preventing autoimmune diseases (Sakaguchi et al., 2008). In addition, T reg cells also restrain the immunity against foreign antigens and cancer. The development, maintenance, and function of T reg cells are dependent on the master transcription factor Foxp3 and factors that regulate Foxp3 expression and function (Josefowicz et al., 2012). Genetic deficiencies in these core regulatory factors typically lead to impaired self-tolerance and homeostasis of T cells, coupled with severe autoimmune disorders. Strong evidence suggests that T reg cells represent a diverse cell population, comprising functionally distinct subsets that control different types of immune responses (Campbell and Koch, 2011;Josefowicz et al., 2012). The molecular mechanism that regulates the differentiation and function of the diverse T reg cells subsets remains poorly understood.
Recent studies have identified a specific subset of T reg cells, the follicular T reg cells (T FR cells), which are localized in the B cell follicles and specialized for the control of germinal center (GC) reactions (Chung et al., 2011;Linterman et al., 2011;Wollenberg et al., 2011). Formation of GCs is essential for various events of a T-dependent humoral immune response, such as antibody class switching, somatic hypermutation, and affinity maturation (Ramiscal and Vinuesa, 2013). The GC reactions depend on follicular T helper cells (T FH cells), a subset of CD4 + T effector cells which provide essential help to cognate B cells for their activation and differentiation in GCs (Linterman et al., 2012).

TRAF3 deficiency partially impaired the in vivo function of T reg cells
To study the function of TRAF3 in T reg cells, we generated T reg -conditional TRAF3 KO mice by crossing the Traf3-floxed mice with Foxp3-Cre mice (Fig. 1 A). Ablation of TRAF3 did not reduce, but even moderately increased, the frequency of T reg cells in the spleen (Fig. 1 B). To examine the effect of TRAF3 deficiency on the immunosuppressive function of T reg cells, we performed in vitro suppression assays using T reg cells purified from the spleen of WT and Traf3 Treg-KO mice. The WT and TRAF3-deficient T reg cells had comparable efficiencies in suppressing the proliferation of naive T cells ( Fig. 1 C), suggesting a dispensable role for TRAF3 in regulating the in vitro function of T reg cells.
We next examined the in vivo function of T reg cells using a well-defined adoptive transfer model, which involved cotransfer of CD45RB hi naive CD4 + T cells with T reg cells purified from the WT and Traf3 Treg-KO mice. As expected, adoptive transfer of CD45RB hi naive T cells into Rag1-KO mice induced severe body weight loss of the recipient mice, coupled with massive expansion of the transferred T cells, and these pathological phenotypes were efficiently blocked when the naive T cells were cotransferred with WT T reg cells (Fig. 1 D). Compared with the WT T reg cells, the TRAF3-deficient T reg cells exhibited a reduced ability to suppress the expansion and pathological activity of the naive T cells (Fig. 1, E and F). Although the mutant T reg cells remained fully functional in inhibiting the production of Th17 cells from the cotransferred naive CD4 + T cells, their ability to suppress the generation of Th1 cells was partially impaired (Fig. 1 F). After the adoptive transfer, a lower number of TRAF3-deficient T reg cells was recovered compared with that of the WT T reg cells ( Fig. 1 G), suggesting reduced expansion or survival of the mutant T reg cells. Furthermore, they showed a reduced level of IL-10 production ( Fig. 1 H). Collectively, these data suggest that T reg cell-specific TRAF3 deficiency does not compromise the homeostasis or in vitro function of T reg cells but partially impairs the in vivo functions of T reg cells.

Altered CD4 T cell homeostasis in Traf3 Treg-KO mice
To assess the function of the TRAF3-deficient T reg cells under nontransferred conditions, we examined the homeostasis of CD4 + T cells in peripheral lymphoid organs. The spleens and lymph nodes of the Traf3 Treg-KO mice contained an enhanced frequency of CD4 + T cells with memory-like markers (CD44 hi CD62L lo ; Fig. 2, A and B). Among the memory-like CD4 + T cells in the peripheral lymphoid organs, there was a significant increase in the frequency of IFN--producing Th1 cells in the Traf3 Treg-KO mice, although the frequency of Th17 cells was generally low and not significantly different between the WT and mutant mice (Fig. 2 C).
T reg cells play an important role in maintaining T cell homeostasis in the intestine, an organ with dynamic interactions between the immune system and microbiota (MacDonald et al., 2011). The T reg cell-specific TRAF3 deficiency resulted The T FR cells resemble the T FH cells in that they express high levels of the chemokine receptor CXCR5, the inducible co-stimulator (ICOS), and the inhibitory receptor PD-1 (Linterman et al., 2012). However, in contrast to T FH cells, the T FR cells express Foxp3 and possess immunosuppressive function. It appears that the T FR cells are generated from CXCR5  T reg cells, instead of naive T cells, in response to T cell-dependent antigens. The T FR cell production requires the transcription factor Bcl-6 and is negatively regulated by the inhibitory receptor PD-1 (Chung et al., 2011;Linterman et al., 2011;Sage et al., 2013). However, the intracellular signaling events involved in the induction of T FR cells are largely unknown.
The TNF receptor (TNFR)-associated factors (TRAFs) form a family of signaling adaptors that mediate signal transduction from both the TNFRs and various other immune receptors (Ha et al., 2009). Among the TRAF family members, TRAF3 is particularly complex in signaling functions, which differ in the context of different receptors in different cell types (Hildebrand et al., 2011). In B cells, TRAF3 functions as a negative regulator of the noncanonical NF-B signaling pathway and B cell survival. TRAF3 physically interacts with the NF-B-inducing kinase (NIK) and mediates continuous degradation of this central component of the noncanonical NF-B pathway (Liao et al., 2004). Thus, loss of TRAF3 causes constitutive activation of noncanonical NF-B, coupled with B cell hyperplasia and aberrant production of antibodies (He et al., 2006;Xie et al., 2007;Gardam et al., 2008). Similar abnormalities have been observed in B cell conditional transgenic mice expressing a stable form of NIK that lacks the TRAF3-binding motif (NIKT3; Sasaki et al., 2008). In contrast to its negative role in B cell homeostasis, TRAF3 has a positive role in mediating TCR signaling and T celldependent immune responses (Xie et al., 2011). Interestingly, the TRAF3 deficiency is associated with an increase in the frequency of T reg cells, although the role of TRAF3 in regulating the homeostasis and function of T reg cells remains unknown (Hildebrand et al., 2011;Xie et al., 2011).
In the present study, we directly examined the function of TRAF3 in T reg cells by generating T reg cell-specific TRAF3 KO (Traf3 Treg-KO ) mice. We show that TRAF3 is dispensable for the homeostasis of the general population of T reg cells. However, TRAF3 has a particularly important role in the regulation of T FR cell generation and GC reactions. The T reg cell-specific TRAF3 deficiency severely inhibited the antigen-stimulated production of T FR cells, coupled with deregulated activation of T FH cells and heightened GC reactions. Consequently, the Traf3 Treg-KO mice have elevated production of high-affinity antibodies of the IgG subtypes. Mechanistically, TRAF3 is required for maintaining the high-level expression of ICOS in T reg cells. We provide evidence that ICOS deficiency compromises the induction of T FR cells and the control of GC reactions. These findings shed light on the signaling mechanism that mediates T FR cell induction and identify TRAF3 as a crucial factor involved in this specific process. (A) T reg (CD4 + CD25 + YFP + ) and non-T reg (CD4 + CD25  YFP  ) T cells were sorted from the WT-R26 YFP (WT) and Traf3 Treg-KO R26 YFP (KO) mice, and TRAF3 expression was assessed by immunoblot. -Actin served as a loading control. (B) The frequency of Foxp3 + T reg cells (among CD3 + CD4 + cells) in the spleen of WT and Traf3 Treg-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 (T eff ) 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 T reg 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 CD45RB hi naive CD4 + T cells (CD45.1 + ) along with either PBS buffer or sorted T reg cells (CD45.1  ), and body weight was monitored. (E and F) CD45RB hi 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 + T reg cells (G) and IL-10-producing T reg 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. obtained when the mice were immunized with another protein antigen composed of the hapten 4-hydroxy-3-nitrophenylacetyl (NP) conjugated to the carrier protein KLH (NP-KLH; Fig. 3 B). ELISA assays using NP9-BSA (for high affinity) and NP26-BSA (for global affinity) suggested that the T reg cellspecific TRAF3 ablation particularly enhanced the production of high-affinity antibodies ( Fig. 3 B and not depicted). In response to immunization by a nonprotein antigen, NP-Ficoll, the production of high-affinity IgG, mainly IgG2b, was only moderately enhanced in the Traf3 Treg-KO mice (Fig. 3 C).
We also examined the effect of TRAF3 deficiency on the basal level of antibodies in different ages of mice. At a young age, the WT and Traf3 Treg-KO mice had comparable serum concentrations of antibodies (not depicted). However, older Traf3 Treg-KO mice (18-20 wk old) had an elevated basal level of serum IgG, particularly IgG1 and IgG2b (Fig. 3 D). These results suggest that expression of TRAF3 in T reg cells is important for controlling the magnitude of humoral immune responses, especially the production of high-affinity IgG antibodies.
To further assess the role of T reg cell-specific TRAF3 in mediating humoral immune responses, we used a model of influenza infection, which is known to trigger strong T celldependent antibody responses (Elsner et al., 2012). As expected, the WT mice experienced bodyweight loss after the intranasal infection with the A/PR8 (H1N1) strain of influenza virus (Fig. 3 E). Importantly, under the same infection conditions, the Traf3 Treg-KO mice had much less bodyweight reduction compared with the WT mice ( Fig. 3 E). Moreover, the Traf3 Treg-KO mice also produced significantly higher levels of antiviral IgG antibodies (Fig. 3 F). These results emphasize the biological significance of the TRAF3-mediated T reg cell regulation.
in a moderate increase in the frequency of CD4 + T cells, but not the CD8 + T cells, in the lamina propria of the small and large intestines (not depicted). Among the CD4 + T cells, there was a significantly increased frequency of Th1 cells in the lamina propria of Traf3 Treg-KO mice (Fig. 2 D). Thus, the loss of TRAF3 in T reg cells altered the homeostasis of T cells, a finding which was consistent with the partially compromised function of the TRAF3-deficient T reg cells (Fig. 1, D-F). It is important to note, however, that the overall phenotype of the Traf3 Treg-KO mice in T cell homeostasis was considerably milder compared with that observed in mutant mice lacking Foxp3 or its major regulators that mediate the development and core functions of T reg cells (Josefowicz et al., 2012). Moreover, the Traf3 Treg-KO mice only displayed relatively mild tissue inflammation at older ages, mostly seen in the lung and the liver (not depicted). These results suggest that TRAF3 may regulate a specific function, instead of mediating the core suppressive activity, of T reg cells.

T reg cell-specific TRAF3 controls antigen-stimulated humoral immune responses
In addition to maintaining T cell homeostasis and preventing autoimmune diseases, T reg cells play an important role in modulating immune responses to foreign antigens. To examine the role of TRAF3 in mediating this latter function of T reg cells, we analyzed the effect of T reg cell-specific TRAF3 deficiency on the induction of antibodies by well-characterized protein and nonprotein antigens. Upon immunization of a protein antigen, sheep RBCs (SRBCs), the young Traf3 Treg-KO and WT control mice produced comparable levels of IgM (not depicted). However, the Traf3 Treg-KO mice produced significantly more IgG and its subtypes (Fig. 3 A). Similar results were  deficiency preferentially promoted the production of highaffinity antibodies of a major secondary isotype, IgG, we examined the role of TRAF3 in regulating GC formation by flow cytometry, based on the typical surface markers of GC B cells. As expected, immunization of the WT animals with SRBCs induced production of GC B cells, which peaked around 1 wk and declined around 2 wk after the immunization T reg cell-specific TRAF3 regulates T FH cell function and GC formation A hallmark of T cell-dependent antibody responses is the formation of GCs, which are required for various events of the humoral immune response, including antibody class switching, somatic hypermutation, and affinity maturation (Ramiscal and Vinuesa, 2013). Because the T reg cell-specific TRAF3

Figure 4. T reg cell-specific TRAF3 regulates T FH cell function and GC formation. (A and B) WT and Traf3
Treg-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 Traf3 Treg-KO (KO) mice. Bars, 500 µm. (D) Frequency of W33 to L mutation in the GC B cells of SRBC-immunized WT and Traf3 Treg-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-R26 YFP (WT) or Traf3 Treg-KO R26 YFP (KO) mice were immunized as in A, and 10 d later, the frequency of T FH 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) T FH 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. Mitsdoerffer et al., 2010;Linterman et al., 2012). Thus, the perturbed T FH cell activation provided explanations for the aberrant formation of GCs and hyper production of IgG isotypes in Traf3 Treg-KO mice.

TRAF3 is important for T FR cell induction
Recent studies identified a subset of T reg cells, the T FR cells, which controls the function of T FH cells and GC reactions (Chung et al., 2011;Linterman et al., 2011;Wollenberg et al., 2011). T FR cells are characterized by expression of the chemokine receptor CXCR5, which mediates their migration into the lymphoid follicles (Linterman et al., 2012). In addition, the T FR cells also express high levels of the co-inhibitory molecule PD-1 and the transcription factor Bcl-6, respectively exerting negative and positive roles in T FR regulation (Chung et al., 2011;Linterman et al., 2011;Wollenberg et al., 2011;Sage et al., 2013).
The particularly prominent phenotype of the Traf3 Treg-KO mice in T FH cell activation and humoral immune responses prompted us to examine the induction and function of T FR cells. We analyzed the T FR cells based on their surface expression of CXCR5 and PD-1 within the population of GFP + (expressed from the Foxp3-GFP-hCre transgene) T reg cells. As expected, the spleens of the WT mice contained a small population of CXCR5 + PD-1 + GFP + T FR cells, and this population was significantly increased in response to SRBC immunization (Fig. 5, A and B). Under unimmunized conditions, the T FR frequency of the Traf3 Treg-KO mice was only moderately lower than that of the WT mice (Fig. 5, A and B). However, these mutant animals were attenuated in antigen-stimulated production of T FR cells after SRBC immunization (Fig. 5,  A and B). To further confirm this remarkable result, we stained the T FR cells based on their surface expression of CXCR5 ( Fig. 4 A). Importantly, the Traf3 Treg-KO mice produced a markedly higher frequency of GC B cells than the WT mice (Fig. 4, A and B). Parallel immunofluorescence assays also revealed the formation of enlarged GCs in the spleen of the Traf3 Treg-KO mice after SRBC immunization (Fig. 4 C).
Formation of GCs is associated with antibody somatic hypermutations, a mechanism which generates high-affinity antibodies (Allen et al., 1987;Weiss et al., 1992). Given the enhanced GC formation in Traf3 Treg-KO mice, we analyzed the antibody somatic hypermutation frequency in the immunized WT and Traf3 Treg-KO mice based on the tryptophan (W) 33 to leucine (L) hotspot mutation in the Ig heavy chain variable gene V H 186.2 (Allen et al., 1988). Consistent with the elevated GC formation in the Traf3 Treg-KO mice, these mutant animals had a profoundly higher frequency of W33 to L mutations (Fig. 4 D).
GC formation and antibody class switching are dependent on T FH subtype of CD4 + helper T cells. We found that in response to SRBC immunization, the WT and Traf3 Treg-KO mice produced a comparable frequency of T FH cells (Fig. 4, E and F). Interestingly, however, the T FH cells derived from the Traf3 Treg-KO mice displayed hyperexpression of genes encoding several cytokine genes, including IL-4, IL-10, IL-17, and IFN- (Fig. 4 G). Furthermore, the expression of several transcription factors, such as Bcl-6, Gata-3, and RORt, were also elevated in the T FH cells derived from the immunized Traf3 Treg-KO mice (Fig. 4 G). Of note, T FH cells are known to produce different effector cytokines depending on the nature of antigens, which appears to influence the antibody class switching in a humoral immune response (Bauquet et al., 2009;Linterman et al., 2012). Several of these cytokines, IL-4, IL-17, and IFN-, have been shown to mediate B cell help and induction of antibody class switching toward the generation of different IgG subtypes (Bauquet et al., 2009;

NIK overexpression does not inhibit ICOS expression
The signaling function of TRAF3 is complex and may vary among cell types (Hildebrand et al., 2011). TRAF3 is known to bind to NIK and mediate NIK degradation, thereby negatively regulating the noncanonical NF-B signaling (Sun, 2012). Loss of TRAF3 causes accumulation of NIK and constitutive activation of noncanonical NF-B in B cells and T cells (Liao et al., 2004;He et al., 2006;Xie et al., 2007;Gardam et al., 2008). To examine how TRAF3 regulates ICOS expression, we examined the potential involvement of noncanonical NF-B activation. We used a transgenic mouse expressing a stabilized form of NIK lacking its TRAF3-binding motif (NIKT3) under the control of a loxP-flanked stop cassette (Sasaki et al., 2008). By crossing these mice with the Foxp3-GFP-hCre mice, we generated T reg -conditional NIKT3 transgenic (NIKT3 Treg-Tg ) mice. As seen with the Traf3 Treg-KO mice, the NIKT3 Treg-Tg mice displayed a moderate increase in memory-like effector T cells (Fig. 7 A). This result suggested that deregulated noncanonical NF-B activation might partially impair the function of T reg cells. However, the NIKT3 Treg-Tg mice did not show obvious abnormalities in antibody responses (Fig. 7 B) or reduced expression of ICOS and IL-10 in T reg cells (Fig. 7, C and D). In fact, the level of ICOS was even slightly enhanced in these mutant T reg cells (Fig. 7 C), probably reflecting their abnormal activation. Consistent with this idea, another activation marker, CD103, was also enhanced on the NIKT3 T reg cells (Fig. 7 E).

TRAF3-mediated ICOS induction involves activation of ERK and AP1
In conventional T cells, TRAF3 has a role in mediating TCR-stimulated activation of the MAP kinase ERK (Xie et al., 2011). We found that loss of TRAF3 in T reg cells also inhibited the activation of ERK by the TCR and CD28 signals (Fig. 8 A). To test whether attenuated ERK activation contributed to the reduced expression of ICOS in T reg cells, we used a selective ERK inhibitor, U0126. Indeed, the ERK inhibitor severely inhibited the induction of ICOS expression (Fig. 8 B). ICOS promoter contains an AP1-binding site that is important for promoter activation by the TCR/CD28 signals (Watanabe et al., 2012). Consistently, we found that crosslinking the TCR and CD28 in WT T cells led to rapid activation of AP1 that bound to the ICOS AP1 probe (Fig. 8 C). Moreover, the TRAF3 deficiency attenuated the activation of AP1 (Fig. 8 C). These findings suggest that TRAF3 regulates ICOS gene induction via an ERK-AP1 signaling pathway. and intracellular expression of Bcl-6 and Foxp3. This method once again revealed a defect of the Traf3 Treg-KO mice in T FR cell production in response to immunization with SRBCs (Fig. 5, C and D). Thus, although TRAF3 is dispensable for the homeostasis of T reg cells, it is important for the induction of a specific subset of T reg cells, the T FR cells, involved in the control of GC formation and humoral immune responses. This finding explains the aberrant induction of GCs and highaffinity antibodies in the Traf3 Treg-KO mice.

TRAF3 mediates ICOS gene expression in T reg cells
To assess the molecular mechanism by which TRAF3 regulates T reg cell function, we examined the expression of signature molecules on WT and TRAF3-deficient T reg cells. Although the TRAF3 deficiency did not affect the expression of most of the T reg cell surface markers analyzed, it caused an increase in the frequency of CD103 hi T reg cell populations (Fig. 6 A). The increase in CD103 hi T reg cells might be caused by their elevated activation, as expression of this molecule is known to be associated with effector/memory type T reg cells (Huehn et al., 2004). Interestingly, the TRAF3-deficient T reg cells had a profound reduction in the expression level of ICOS ( Fig. 6 A), a co-stimulatory molecule thought to mediate specific T reg cell functions, such as IL-10 gene induction and T FR cell development (Ito et al., 2008;Busse et al., 2012;Redpath et al., 2013;Sage et al., 2013). QPCR analysis further confirmed the defect in Icos gene expression (Fig. 6 B). Moreover, the TRAF3-deficient T reg cells were also impaired in the induction of IL-10 expression (Fig. 6, B-D). These findings further suggest the involvement of TRAF3 in the regulation of specific aspects of T reg cell functions.
Because Traf3 Treg-KO mice had a defect in T FR cell induction, we tested whether TRAF3 was required for antigenstimulated ICOS expression on T FR cells. As seen with the total T reg cells, ICOS expression was reduced on T FR cells under unimmunized conditions (Fig. 6 E). Importantly, the defect of the TRAF3-deficient T FR cells in ICOS expression became much more drastic after immunization of the mice with the protein antigen SRBCs (Fig. 6 E). Furthermore, this defect appeared to be cell intrinsic because it was not detected in the Foxp3  non-T reg cells (Fig. 6 F). A mixed bone marrow adoptive transfer experiment revealed that the TRAF3-deficient T reg cells had reduced ICOS expression even when they were developed in the presence of WT T reg cells in the same recipient mice (Fig. 6 G), thus further suggesting a cell-intrinsic role for TRAF3 in the regulation of ICOS expression in T reg 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 Traf3 Treg-KO (KO) mice (6 wk old; n = 5) were immunized with SRBCs, and 10 d later, ICOS expression in T FR (E) or T FH (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 Traf3 Treg-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 + ) T reg 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.

Tcrb/Tcrd double KO (Tcrb/Tcrd-dKO) mice with WT naive CD4 + T cells together with T reg cells purified from WT,
Traf3 Treg-KO , or Icos-KO mice and then challenged the mice with SRBC antigen. The different T reg cells were highly similar in homeostasis after the adoptive transfer ( Fig. 9 A). In contrast, although the WT T reg cells efficiently suppressed the induction of GCs in the immunized recipient mice, the TRAF3-deficient T reg cells were largely defective in this function (Fig. 9 B). The

ICOS expression in T reg cells is required for the formation and function of T FR cells
A recent study suggests that Icos-KO mice have a defect in T FR cell induction, although it is unclear whether this function is cell intrinsic (Sage et al., 2013). We used a T reg cell adoptive transfer model to examine whether the expression of ICOS in T reg cells is required for the development and function of T FR cells. We adoptively transferred the T cell-deficient approach, it is thought that TRAF3 may regulate the development of T reg cells (Hildebrand et al., 2011). Our present study revealed that ablation of TRAF3 in committed T reg cells, using the Foxp3-Cre system, also caused a moderate, but significant, increase in the T reg cell frequency. Our finding is consistent with the previous observation and further suggests that TRAF3 may negatively regulate both the early development and the late-stage maturation or survival of T reg cells. Interestingly, despite their increased T reg cell frequency, the Traf3 Treg-KO mice had impaired conventional T cell homeostasis, characterized by an increase in the percentage of CD4 + T cells with memory/effector surface markers. This result suggests a positive role for TRAF3 in the regulation of T reg cell function. In contrast, the phenotype of the Traf3 Treg-KO mice in T cell homeostasis is considerably weaker than that observed in mice lacking Foxp3 or other factors required for the development and core functions of T reg cells. The Traf3 Treg-KO mice also had only moderate immune cell infiltrations in the nonlymphoid tissues, particularly the lung. Consistently, adoptive transfer experiments revealed that the TRAF3-deficient T reg cells had weakly reduced ability to suppress the expansion and pathogenesis of CD4 + T cells. We observed reduced expression of IL-10 in the TRAF3-deficient T reg cells, which may contribute to their partially impaired function in maintaining immune homeostasis.
Despite the relatively weak phenotype in immune homeostasis, the Traf3 Treg-KO mice had a prominent abnormality in antigen-stimulated GC reactions. In response to T-dependent antigens, these mutant animals formed enlarged GCs in the spleen and produced markedly more GC B cells, coupled with hyper production of high-affinity IgG subtypes. Gene expression analyses suggest the aberrant activation of T FH cells. Of particular interest is the elevated production of various effector cytokines, including IL-4, IL-10, IL-17, and IFN-, by the T FH cells derived from the immunized Traf3 Treg-KO mice. Production of these cytokines by normal T FH cells has been observed similar deficiency was observed with the ICOS-deficient T reg cells (Fig. 9 B).
We next examined the production of high-affinity IgG antibodies in the immunized recipient mice. Consistent with the deficiency of the Traf3-KO and Icos-KO T reg cells in suppressing the GC formation, they also displayed attenuated function in the control of IgG production compared with the WT T reg cells (Fig. 9 C). Moreover, the recipient mice of Traf3-KO and Icos-KO T reg cells contained substantially higher levels of plasma cells (Fig. 9 D) and T FH cells (Fig. 9 E), coupled with reduced frequency of T FR cells (Fig. 9 F). Collectively, these data suggest that TRAF3 and ICOS have a T reg cell-intrinsic role in mediating the generation and function of T FR cells.

DISCUSSION
The Foxp3 + T reg cells represent a functionally diverse population composed of subsets that regulate different immune functions (Campbell and Koch, 2011;Josefowicz et al., 2012). The signaling mechanism that regulates the functional diversity of T reg cells is still poorly understood. In the present study, we have identified TRAF3 as a signaling factor that mediates the effector function of T reg cells, particularly their control of GC formation and antibody responses. Our data suggest that TRAF3 is involved in the induction of the recently defined T FR cells, a subset of T reg cells specialized in the control of GC reactions (Chung et al., 2011;Linterman et al., 2011;Wollenberg et al., 2011). Consequently, the mice with T reg cell-specific TRAF3 deficiency display heightened responses to antigens in the formation of GCs and production of high-affinity antibodies.
A recent study suggests that TRAF3 ablation in total T cells, by crossing the Traf3-floxed mice with CD4-Cre mice, causes an increase in the percentage of Foxp3 + T reg cells (Xie et al., 2011). Because TRAF3 is knocked out during the double-positive stage of thymocyte development in this cells (CD4 + YFP + CD25 + ) were sorted from WT-R26 YFP and Traf3 Treg-KO -R26 YFP 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 T reg 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. attenuated in this response. In an adoptive transfer model, the TRAF3-deficient T reg cells also displayed a severe defect in generating T FR cells and suppressing GC responses. Under the transfer conditions, the TRAF3-deficient T reg cells also had significantly reduced ability to suppress the production of T FH cells. These results suggest that TRAF3 may regulate a signaling pathway in T reg cells that is particularly important for their conversion into T FR cells during an immune response.
The generation of T FR cells from Foxp3 + T reg cells is critically dependent on the transcription factor Bcl-6 (Chung et al., previously and thought to regulate B cell proliferation and antibody class switching (Rousset et al., 1992;King et al., 2008;Bauquet et al., 2009;Mitsdoerffer et al., 2010). Thus, deregulated production of these effector cytokines by T FH cells may contribute to the heightened GC formation and antibody responses in the Traf3 Treg-KO mice. The aberrant T FH cell activation was apparently caused by the defect of these mutant animals in producing T FR cells. Upon immunization with a T-dependent antigen, the WT mice had a substantial increase in the T FR cells, whereas the Traf3 Treg-KO mice were severely Figure 9. ICOS is essential for controlling T FH cell function and GC formation. Sorted naive CD4 + T cells (CD4 + CD62L hi CD44 lo CD25  ) from WT B6. SJL mice (5 wk old; CD45.1 + ) and sorted T reg T cells (CD4 + YFP + CD25 + ) from WT, Traf3 Treg-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 T reg 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 T reg 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 T FH (CXCR5 + PD-1 + Foxp3  CD4 + ) cells among CD4 + Foxp3  T cells (E) and the frequency of T FR (CXCR5 + PD-1 + GFP + CD4 + ) cells among CD4 + Foxp3 + T reg 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.
T reg cell function and humoral immune responses, but also shed new light onto the signaling mechanism regulating the specific functions of T reg cells.

MATERIALS AND METHODS
Mice. Foxp3-GFP-hCre BAC transgenic mice (Zhou et al., 2008; The Jackson Laboratory) were backcrossed for nine generations to the C57BL/6 background and then crossed with Traf3-floxed mice (C57BL/6 background; Gardam et al., 2008) to produce age-matched Traf3 +/+ Foxp3 GFP-hCre (termed WT) and Traf3 fl/fl Foxp3 GFP-hCre (termed Traf3 Treg-KO ) mice. In some experiments, these mice were further crossed with the Rosa26-YFP Cre reporter (termed R26 YFP ; C57BL/6J background; The Jackson Laboratory) to generate WT and Traf3 Treg-KO mice that express YFP in their T reg cells (termed WT-R26 YFP and Traf3 Treg-KO R26 YFP mice, respectively). The Traf3-floxed mice were also crossed with CD4-Cre (C57BL/6 background; The Jackson Laboratory) mice to produce mice harboring TRAF3 deletion in total T cells (called Traf3 TKO mice). NIKT3 mice (C57BL/6 background; The Jackson Laboratory) express a stabilized form of NIK lacking the TRAF3-binding motif (NIKT3) from the Rosa26 locus under the control of a loxP-flanked stop cassette (Sasaki et al., 2008). These mice were crossed with the Foxp3-GFP-hCre mice (backcrossed to C57BL/6 background) to generate T regconditional NIKT3 transgenic (NIKT3 Treg-Tg ) and internal WT control mice. B6.SJL (expressing the CD45.1 congenic marker), Tcrb/Tcrd-dKO, Icos-KO, and Rag1-KO mice (all in C57BL/6 background) were obtained from the Jackson Laboratory. For all experiments, WT littermates were used as controls. Mice were maintained in a specific pathogen-free facility of the University of Texas MD Anderson Cancer Center, and all animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Texas MD Anderson Cancer Center.

Influenza viral infection. WT and Traf3
Treg-KO mice were subjected to intranasal infection with a nonlethal dose (25 tissue culture ID 50 units in 25 µl PBS per mouse) of influenza virus A/PR8 (H1N1) under isoflurane anesthesia. Body weight change was monitored every another day for 13 d. On day 13, influenza-specific antibodies in the serum were detected by ELSIA as described previously (Elsner et al., 2012). In brief, ELISA plates were coated with influenza virus overnight at 4°C, blocked with 1% BSA in PBS, and then loaded with serum samples prediluted in 1% BSA. After 3 h of incubation, the plates were washed three times with PBST buffer, and the bound antibody was detected by with biotinylated goat anti-mouse antibody and streptavidin-HRP using TMB as substrate. 2011; Linterman et al., 2011;Wollenberg et al., 2011). Our data suggested that TRAF3 is not required for expression of Bcl-6 or the various T reg cell signature molecules. Instead, TRAF3 appeared to be required for maintaining the highlevel expression of ICOS in T reg cells because the TRAF3 deficiency greatly reduced, although did not completely block, the expression of ICOS at both RNA and protein levels. By using an adoptive transfer approach, we obtained in vivo evidence that ICOS expression in T reg cells is crucial for T reg cell-mediated suppression of GC formation and antibody responses. Like the TRAF3-deficient T reg cells, the ICOS-deficient T reg cells were severely attenuated in the generation of T FR cells and suppression of GC induction in response to immunization. These findings provide a mechanistic insight into the function of TRAF3 in regulating T FR cell production and antibody responses. We believe that ICOS induction is an important, but probably not the only, mechanism by which TRAF3 regulates T FR cell induction and antibody responses.
The signaling function of TRAF3 is complex and differs in different cell types and in the context of different receptors (Hildebrand et al., 2011). One important function of TRAF3 is to mediate degradation of NIK and, thereby, negatively regulate the noncanonical NF-B signaling (Liao et al., 2004). This negative signaling function of TRAF3 is particularly important for maintaining normal homeostasis and function of B cells, although its role in other cell types remains unclear (He et al., 2006;Xie et al., 2007;Gardam et al., 2008). Our data suggest that the activation of noncanonical NF-B might contribute to the moderate defect of the TRAF3-deficient T reg cells in maintaining CD4 + T cell homeostasis because perturbed CD4 + T cell homeostasis was also detected in mice with conditional expression of stable form of NIK (NIKT3 Treg-Tg mice). However, the noncanonical NF-B activation is unlikely responsible for the functional defect of the TRAF3deficient T reg cells in the regulation of GC reactions. Unlike the TRAF3 deficiency, conditional expression of NIKT3 in T reg cells did not cause hyperproduction of IgG or other antibody isotypes. The NIKT3-expressing T reg cells did not show any defect in ICOS expression either. Instead, our data suggest a positive role for TRAF3 in mediating the TCR signaling in T reg cells. As seen in conventional T cells (Xie et al., 2011), the loss of TRAF3 in T reg cells resulted in attenuated activation of the MAP kinase ERK. We obtained evidence that ERK is important for TCR-stimulated ICOS expression in T reg cells.
In summary, we have identified TRAF3 as a signaling factor that mediates the effector function of T reg cells, particularly in the control of humoral immune responses. Although TRAF3 is dispensable for the homeostasis of T reg cells, it is crucial for antigen-stimulated production of T FR cells. Our data suggest that the T reg cell regulatory function of TRAF3 involves induction of the co-stimulatory molecule ICOS, which is the result of impaired ERK activation. Our study has also revealed the requirement of T reg cell-specific ICOS in the induction of T FR cells and the control GC responses. These findings not only establish a novel role of TRAF3 in the regulation of by flow cytometric cell sorting based on the B220 + CD95 + GL7 + surface markers. Somatic hypermutation was analyzed based on the W33 to L hotspot mutation, essentially as previously described (Muramatsu et al., 2000). In brief, V H 186.2 transcripts were amplified by PCR using the V H 186.2 (5-GCT-GTATCATGCTCTCTTG-3) and CH-1 (5-GGTGCTATCCGGTC-CCTGAGT-3) primer pairs, and the PCR products were inserted into the pGEM-T vector by TA cloning. Randomly picked cDNA clones were sequenced for identification of the W33 to L mutations.
Statistical analysis. Prism software (GraphPad Software) was used for twotailed unpaired Student's t tests. P-values of <0.05 or <0.01 were considered significant and very significant, respectively.
Online supplemental material. Table S1 lists the gene-specific primers used for real-time RT-PCR analyses. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20131019/DC1.
Histology and GC analysis. Organs were removed from sacrificed mice, fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned for hematoxylin-eosin staining. For GC formation analysis, SRBC-immunized mice were sacrificed after 10 d of immunization. Frozen spleen sections were fixed in methanol/acetone (2:1) for 10 min, followed by blocking for 30 min in a biotin-and serum-free SuperBlock buffer (Thermo Fisher Scientific). The GCs were detected using biotinylated peanut agglutinin (Vector Laboratories) followed by Alexa Fluor 594-conjugated streptavidin (Molecular Probes). The sections were mounted using an antiquenching agent (Vectashield; Vector Laboratories) and analyzed under a fluorescent microscope (DP70; Olympus). Panoramic pictures were taken and the GCs counted. The area and perimeter of each GC from each mouse (n = 3) were obtained using ImageJ software (National Institutes of Health). The area and perimeter are expressed in micrometers. GC B cells were analyzed by flow cytometry after staining with FITC-conjugated anti-CD95, PE-conjugated anti-B220, and APC-conjugated anti-GL7, and plasma B cells were analyzed similarly by staining with FITC-conjugated anti-B220 and PE-conjugated anti-CD138. For detecting T FH cells, the cells were stained with FITC-conjugated anti-CD4, PE-conjugated PD-1, and APC-conjugated CXCR5, followed by permeabilization with a Foxp3 staining kit (eBioscience) and intracellular staining with PE-conjugated anti-Bcl-6. T FH cells were detected similarly except the inclusion of intracellular staining with FITC-conjugated anti-Foxp3 or the GFP marker.

ELISA.
For SRBC or NP-specific antibody detection, Nunc Immuno Plate MaxiSorp plates (Thermo Fisher Scientific) were coated with SRBC lysate, NP9-BSA, or NP26-BSA and incubated overnight at 4°C. After blocking with 1% BSA in PBS, the plates were incubated with serially diluted standard or samples and then with HRP-conjugated detection antibodies. After colorimetric reaction (tetramethylbenzidine; Moss), the absorbance at 450 nm was measured using an ELISA plate reader. For IL-10 detection, Nunc plates were coated with 1 µg/ml anti-IL-10 capture antibody in 50 mM sodium bicarbonate and incubated overnight at 4°C. The coated plates were incubated with supernatant of the T reg cell culture and subjected to colorimetric detection as described above.
Immunoblot and real-time quantitative RT-PCR. Purified T reg and CD4 + T cells were lysed in RIPA buffer supplemented with inhibitors of phosphatases and the proteases. For analyzing ERK phosphorylation, the cells were stimulated with agonistic anti-CD3 and anti-CD28 antibodies using a crosslinking method (Reiley et al., 2007) before lysis. The cell lysates were fractionated by SDS-PAGE and subjected to immunoblot assays with the indicated antibodies. For analyzing RNA expression, total cellular RNA was isolated and subjected to real-time quantitative RT-PCR assays as previously described (Chang et al., 2011). The gene-specific primer sets are listed in Table S1.
In vitro and in vivo T reg cell suppression assays. The in vitro T reg cell suppression assay was performed as previously described (Chang et al., 2012). In vivo T reg cell assays were performed essentially as described previously (Chang et al., 2012). For examining the in vivo roles of T reg cells in the regulation of GC reactions, sorted CD4 + CD62L hi CD44 lo CD25  naive T cells from B6.SJL mice (5-6 wk old) and sorted CD4 + GFP + CD25 + T reg cells from WT, Traf3 Treg-KO , and Icos-KO mice (5-6 wk old) were intravenously transferred into Tcrb/Tcrd-dKO mice. Recipient mice were immunized with SRBC 2 d after adoptive transfer and sacrificed 14 d later for analyzing serum antibodies and lymphocyte populations.
Antibody somatic hypermutation analyses. WT and Traf3 Treg-KO mice were immunized with SRBCs and sacrificed after 10 d for isolating GC B cells