Tespa1 Deficiency Dampens Thymus-Dependent B-Cell Activation and Attenuates Collagen-Induced Arthritis in Mice

Thymocyte-expressed, positive selection-associated 1 (Tespa1) plays an important role in both T cell receptor (TCR)-driven thymocyte development and in the FcεRI-mediated activation of mast cells. Herein, we show that lack of Tespa1 does not impair B cell development but dampens the in vitro activation and proliferation of B cells induced by T cell-dependent (TD) antigens, significantly reduces serum antibody concentrations in vivo, and impairs germinal center formation in both aged and TD antigen-immunized mice. We also provide evidence that dysregulated signaling in Tespa1-deficient B cells may be linked to CD40-induced TRAF6 degradation, and subsequent effects on 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2) phosphorylation, MAPK activation, and calcium influx. Furthermore, we demonstrate that Tespa1 plays a critical role in pathogenic B cells, since Tespa1-deficient chimeric mice showed a lower incidence and clinical disease severity of collagen-induced arthritis. Overall, our study demonstrates that Tespa1 is essential for TD B cell responses, and suggests an important role for Tespa1 during the development of autoimmune arthritis.

Thymocyte-expressed, positive selection-associated 1 (Tespa1) plays an important role in both T cell receptor (TCR)-driven thymocyte development and in the FcεRI-mediated activation of mast cells. Herein, we show that lack of Tespa1 does not impair B cell development but dampens the in vitro activation and proliferation of B cells induced by T cell-dependent (TD) antigens, significantly reduces serum antibody concentrations in vivo, and impairs germinal center formation in both aged and TD antigen-immunized mice. We also provide evidence that dysregulated signaling in Tespa1-deficient B cells may be linked to CD40-induced TRAF6 degradation, and subsequent effects on 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2 (PLCγ2) phosphorylation, MAPK activation, and calcium influx. Furthermore, we demonstrate that Tespa1 plays a critical role in pathogenic B cells, since Tespa1-deficient chimeric mice showed a lower incidence and clinical disease severity of collagen-induced arthritis. Overall, our study demonstrates that Tespa1 is essential for TD B cell responses, and suggests an important role for Tespa1 during the development of autoimmune arthritis.
Keywords: tespa1, Plcg2, B-cell, collagen-induced arthritis, cD40 inTrODUcTiOn B lymphocytes play crucial roles in adaptive immunity by recognizing foreign antigens and eliciting appropriate host protective responses. The developmental fate of B cells, as well as their function during immune responses, is critically regulated by the B-cell receptor (BCR). BCR signaling is initiated by antigen ligation, which triggers the formation of the BCR signalosome, a multifunctional protein complex that includes the protein tyrosine kinase Lyn, spleen tyrosine kinase (Syk), B cell linker protein (BLNK), Bruton agammaglobulinemia tyrosine kinase (Btk), phospholipase Cγ2 (PLCγ2), and inositol 1,4,5-trisphosphate receptor type 2 (IP3R2), ultimately leading to calcium mobilization and the activation of several downstream pathways (1)(2)(3)(4)(5). In addition, during thymusdependent B-cell activation, several coreceptors such as CD40, CD19, CD22, CD21, and FcγRIIB, act to quantitatively modify BCR signaling. The signals generated by the costimulatory receptors are the means through which T cell help modulates BCR signaling (6)(7)(8). CD40 is one of the most important coreceptors, and plays a crucial role during T cell-dependent (TD) B cell activation, immunoglobulin class switching, and the development of humoral memory. The signaling pathways emanating from the BCR and CD40 can cooperate in a synergistic or additive manner, but the molecular mechanisms underlying these interactions are not completely understood. CD40 is a member of the TNFR family, and several members of the TNFR-associated factor (TRAF) family have been shown to bind to CD40 and serve as adaptor proteins in the CD40 signaling pathway. TRAFs clearly have important roles in B cell regulation, but the nature of these roles is still unresolved (9)(10)(11)(12)(13)(14).
Thymocyte-expressed, positive selection-associated 1 (Tespa1) was originally identified as a critical signaling molecule involved in T cell selection and maturation. Tespa1 regulates T cell receptor (TCR) signaling through direct binding to PLCγ1 and IP3R1, thereby facilitating TCR-induced calcium signals and thymocyte development. It also participates in mitochondrial Ca 2+ uptake via mitochondria-associated ER membranes in T cells (15)(16)(17)(18). Furthermore, Tespa1 was also found to negatively regulate FcεRImediated signaling and mast cell-mediated allergic responses (18). Besides T cells and mast cells, Tespa1 is also highly expressed in most subsets of peripheral B cells, suggesting a potential role in B cell function (19,20).
In this study, we show that the absence of Tespa1 does not impair B cell development, but significantly reduces the activation and proliferation of B cells induced by TD antigens, both in vitro and in bone marrow chimeras with Tespa1-deficient B cells (Tespa1 B−/− ). We also found a potential role for Tespa1 in the stabilization of TRAF 6 and the phosphorylation of PLCγ2 induced by CD40.
Finally, since B cells or some B cell subpopulations play crucial roles in the development of rheumatoid arthritis (RA) in humans and of collagen-induced arthritis (CIA) in mice (21)(22)(23)(24)(25), we employed CIA as a model to evaluate the role of Tespa1 in B cell-associated autoimmune diseases, and found that Tespa1deficient chimeras showed a lower incidence and clinical disease severity index of autoimmune arthritis. This suggests that Tespa1 is a potential therapeutic target in human RA.

MaTerials anD MeThODs ethics statement
This investigation was conducted in accordance with the ethical standards of the Declaration of Helsinki, followed national and international guidelines and was approved by the review board of the School of Medicine, Huzhou University.

animals and immunization
Tespa1 −/− mice were generated by homologous recombinationmediated gene targeting at the Shanghai Research Center for Model Organisms, as previously described in Ref. (16). Mice on a mixed 129 × C57BL/6 background were backcrossed onto the C57BL/6 background for 6-8 generations. B6.129S2-Igh-6tm1Cgn (μMT) mice were a kind gift from Qi Hai (Tsinghua University, Beijing, China). For the experiments with mixed bone marrow chimeras, C57BL/6 mice were lethally irradiated (8.5 Gy) and reconstituted with a mixed suspension of μMT (80%) and Tespa1 +/+ or Tespa1 −/− (20%) bone marrow cells at least 8 weeks before immunization. All mice were housed at the Zhejiang University Laboratory Animal Center. Animal experiment protocols were approved by the Review Committee of Zhejiang University School of Medicine and were in compliance with institutional guidelines. For TD responses, mice were immunized with 100 µg of NP-KLH (Biosearch Technologies, Novato, CA, USA) mixed with Imject alum (Thermo Fisher Scientific, MA, USA) on days 0 (primary immunization) and 21 (secondary immunization). For TI responses, 20 ng of NP-LPS or 20 µg of NP-Ficoll (Biosearch Technologies, Novato, CA, USA) were injected intraperitoneally.

Flow cytometry analysis
Single-cell suspensions were prepared from bone marrow isolated from the tibia and femur of one leg, and from the spleen and peritoneal cavity using standard procedures. Following red blood cell lysis, Fc receptors were blocked with anti-CD16/32 Ab (2.4G2), and cells were stained with the antibodies mentioned above in 0.5% FBS in PBS. For the proliferation assays, lymphocytes were loaded with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, Carlsbad, CA, USA), stimulated as described, and cell divisions were assessed after 3 days by Flow cytometry. Data were collected on a FACSCanto™ II (BD Biosciences San Jose, CA, USA) instrument and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

ca 2+ Flux
Splenocyte suspensions at a density of 5 × 10 6 cells/ml were loaded with 4 mg/ml of Fluo4 (Invitrogen, Carlsbad, CA, USA) in RPMI without sera for 1 h at 37°C and washed twice with calcium-free Hank's buffered saline (HBSS; pH 7.4). Cells were labeled with PE/ Cy5-conjugated anti-B220 for identification of B cells. After washing, cells were suspended at 2 × 10 6 cells/ml in calcium-free HBSS, and prewarmed at 37°C for 10 min. Next, 10 µg/ml of anti-mouse CD40 antibody was added to induce calcium flux during the first 60 s, followed by CaCl2 at a final concentration of 2 mM for the next 180 s and 1 µM ionomycin (Sigma-Aldrich, St. Louis, MO, USA) for an additional 60 s. Mean fluorescence ratios were plotted after analysis with FlowJo software (Tree Star, Ashland, OR, USA). elisa Total serum Ig levels were quantified by ELISA using the Mouse Ig Isotyping ELISA Ready-Set-Go! kit (ebioscience, San Diego, CA, USA), according to the manufacturer's instructions. The concentrations were calculated based on the OD450 values obtained with serial dilutions, using values in the linear portion of the response curve to calculate the serum Ab levels. NP-specific and CII-specific ELISAs were performed following similar methods, but plates were coated with 10 μg/ml of NP-BSA (Biosearch Technologies, Novato, CA, USA) or 5 µg/ml of bovine collagen type II (Sigma-Aldrich, St. Louis, MO, USA).

immunohistochemistry and histological analysis
Germinal centers (GCs) were stained in splenic paraffin sections using biotinylated peanut agglutinin (PNA), streptavidin-HRP, and the diaminobenzidine method. For histopathologic analysis of joint tissues, whole ankles, and ankle joints were fixed in 10% formalin for 3 days. After decalcification for 18 days in Cal-Ex II (Fisher Scientific, Springfield, NJ, USA), the specimens were processed for paraffin embedding. Tissue sections (5 µm) were stained with H&E for microscopic evaluation.
immunoprecipitation and immunoblot analysis Cells stimulated as described were lysed with NP-40 lysis buffer (Beyotime Biotechnology; Shanghai, China), containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 10 mM PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Cwbiotech, Beijing, China). The lysates were incubated on ice for 20-30 min, vortexed extensively, and centrifuged (13,000 rpm × 10 min, 4°C). Samples were then boiled for 5 min, subjected to 10% SDS-PAGE, blotted, probed with commercially available antibodies, and bound antibodies detected using the Tanon 5500 enhanced chemiluminescence detection system (Tanon, Shanghai, China).  To investigate whether Tespa1 is required for B cell development, we used flow cytometric analysis to quantify the number of developing and mature B cells in lymphoid tissues of Tespa1-deficient and wild-type (WT) mice. We found that the numbers of pro-B, pre-B, immature B, mature B, B1, and plasma cells were unaltered in the bone marrow of these mice (Figures 1A,D). Furthermore, there was also no change in the number of B1 cells in the peritoneal cavity (Figures 1B,D). In addition, we examined various B cell subsets in the spleen, and found that the lack of Tespa1 did not alter the numbers of mature B cells, immature B cells, T1, T2, T3 B cells, age-associated B cells (24), follicular B cells, marginal zone B cells, switched memory B cells, unswitched memory B cells, plasma cells, or B1 cells (Figures 1C,D).

induction of cia
aged Tespa1-Deficient Mice have lower Baseline concentrations of serum immunoglobulins  Figure 3A). We found no significant alteration in the percentages of T and B cells in the peripheral blood of reconstituted chimeric mice, further confirming that Tespa1 deficiency does not affect the development of B cells (Figure 3B). After reconstitution, mice were immunized with the following NP-conjugated antigens: NP-KLH (TD antigen), NP-LPS (TI-1 antigen) and NP-Ficoll (TI-2 antigen), and hapten-specific responses were measured. After immunization with NP-KLH, we detected a reduction of all types of NP-specific Ig responses in Tespa1 B−/− mice when compared with the Tespa1 B+/+ controls, especially following the secondary immunization. By contrast, no significant differences were seen with TI-antigens ( Figure 3C).
Germinal centers are the sites where T-B cell interactions occur during TD B cell responses, and these structures are a hallmark of Th activity (27,28). To determine whether Tespa1 deficiency affected GC formation, the spleens of Tespa1 B−/− and Tespa1 B+/+ mice immunized with NP-KLH were removed at day 21 postimmunization and analyzed by flow cytometry and immunohistochemistry. We found that the percentage of B220 + Fas + GL-7 + GC-B cells was significantly higher in immunized Tespa1 B+/+ mice. Staining with PNA also showed enhanced GC formation in Tespa1 B+/+ mice when compared with Tespa1 B−/− (Figure 3D).

Tespa1 Deficiency impairs Thymus-Dependent B-cell activation and Proliferation
To characterize the effect of Tespa1 on B cell activation at the cellular level, B cells from Tespa1 +/+ and Tespa1 −/− mice were enriched and stimulated in vitro with anti-mouse CD40 antibody (TD response), LPS (TI-1 response) and anti-IgM F(ab′)2 (TI-2 response) as described in Section "Materials and Methods. " The surface expression of antigen-presenting molecules (MHC II), costimulatory molecules (CD80 and CD86), and activation markers (CD21, CD23, CD25, CD44, and CD69) was analyzed by flow cytometry (Figure 4). We found that Tespa1-deficient B cells expressed lower levels of activation markers (CD25, CD69, CD80, CD86, and MHC-II) following stimulation with anti-CD40 mAb (Figures 4A,B). By contrast, no significant differences were observed after stimulation with anti-IgM F(ab′)2 or LPS. In addition, the in vitro proliferation, measured with CFSE, was significantly reduced in Tespa1-deficient splenic B cells following anti-CD40 mAb stimulation (Figures 4C,D).

altered cD40 signaling events in Tespa1-Deficient B cells
The above results indicate that Tespa1 positively regulates thymus-dependent B-cell activation, both in vitro and in vivo. Next, we examined the activation of intracellular signaling pathways in Tespa1 wild type (WT) and knockout (KO) B cells. Since we detected differences in B cell activation only for thymus-dependent responses, B cells were stimulated with anti-mouse CD40 for different time periods and analyzed by Western blot. The levels of total (t) and phosphorylated (p) BCR-proximal tyrosine kinases Lyn and Syk were unchanged in B cells derived from Tespa1 KO mice when compared with WT controls (Figure 5A). In addition, we investigated CD40 signaling mechanisms by examining TRAFs and found that Tespa1 deficiency impaired the stabilization of (D) (Left) Splenocytes from Tespa1-deficient or WT chimeras immunized with NP-KLH were stained with anti-B220, anti-Fas (CD95), and anti-GL-7 antibodies. Cells were gated on the B220 + population. (Right) Immunohistochemical staining with peanut agglutinin (brown) of spleens from control and Tespa1-deficient chimeras collected on day 21 after immunization. Scale bar, 1,000 µm. Summarized data shows the mean ± SEM from three separate experiments (n = 10 mice per group), *p < 0.05, **p < 0.01. TRAF6 but not TRAF2 or TRAF3 following CD40 stimulation (Figures 5B,E). We also examined the levels of phosphorylation of other components of the BCR signalosome, including PLCγ2, BLNK, Btk, Grb2, and LAB, and only found significantly reduced phosphorylation of PLCγ2 in Tespa1 KO B cells after stimulation (Figures 5C,E). In addition, Tespa1 deficiency resulted in the attenuated activation of distal signaling mitogen-activated protein kinases ERK (Figures 5D,E), which are widely reported to be critical for B cell activation. These data suggest that the absence of Tespa1 perturbs a principal signaling axis (CD40/TRAF6/PLCγ2/

MAPK) in B cells. A transient increase of intracellular calcium
is essential for the activation, proliferation, and differentiation of B cells (29), and the phosphorylation of PLCγ2 could influence calcium influx. As expected, we found that B cells from Tespa1 WT mice had much higher levels of calcium flux than Tespa1 KO B cells (Figure 5F). In summary, these data suggest that the positive regulation of B cell activation by Tespa1 involves modulation of BCR signaling events.

Decreased severity of cia in Tespa1-Deficient chimeras
Because B cells are known to play an essential role during the effector phase of autoimmune arthritis, we examined the progression of CIA, a murine model of human RA, in Tespa1 B−/− and Tespa1 B+/+ chimeras. The incidence and clinical disease severity index of CIA were significantly reduced in Tespa1 B−/− chimeric mice when compared with Tespa1 B+/+ chimeric controls ( Figure 6A). In the radiological and histologic analysis, we observed more radiological abnormalities and more leukocytic infiltration in the ankle joints of Tespa1 B+/+ than in Tespa1 B−/− chimeras (Figures 6B,C).
Finally, anti-CII IgM, IgG1, IgG2a IgG2b, and IgG3 antibody levels were measured in the chimeric mice by ELISA. Anticollagen IgG, IgG1, and IgG2a antibody levels were significantly decreased in Tespa1 B−/− mice, whereas the IgG2b and IgG3 levels did not differ in Tespa1 B−/− mice ( Figure 6D). Taken together, our experiments suggest that Tespa1 modulates the onset and severity of CIA.

DiscUssiOn
In this study, we found an unexpected role for Tespa1 in CD40mediated B cell activation and proliferation. The absence of Tespa1 did not affect the development of B cells but did impair thymus-dependent B-cell activation, both in vitro and in vivo.
Optimal B cell activation depends on signals generated by Ag recognition through the BCR, as well as on additional signals provided by cognate interactions with T helper cells, including those triggered by CD40-CD40L interactions (30). The cross talk between CD40 and the BCR modulates the activation of multiple signaling molecules such as PI3K, NF-κB, PLCγ2, and MAPK; and regulates the development, survival, differentiation, and immune responsiveness of B cells (9,10,(31)(32)(33). CD40 is a member of the TNFR family and possesses neither intrinsic kinase activity nor conserved tyrosine residues in its cytoplasmic (CY) tail. Several TRAF members bind to CD40 and appear to function as adaptor proteins (6,11). TRAF6 is one of the most important TRAFs and contributes to the CD40-mediated activation of B cell. TRAF6 may function as an adapter molecule, an activator of mitogenactivated protein kinases, or act as a repressor of certain signaling circuits (34). In different immune cells, proteasome-dependent TRAF6 degradation has been observed and was reported to impair the downstream signaling pathways leading to cell activation (35)(36)(37). However, the molecular mechanisms underlying the stabilization of TRAF6 are not well understood.
Phosphoinositide-specific C phospholipases (PLC) are a critical group of cell-signaling molecular switches that regulate the formation of the second messengers inositol 1,4,5-trisphospate (IP3) and diacylglycerol. There are six families of PLC enzymes, differing in their structural organization and amino acid sequence (38). PLCγ2 is highly expressed in cells of hematopoietic origin and plays a critical role in B cell function (39)(40)(41). In some previous reports, PLCγ2 phosphorylation was detected following CD40 ligation, and a complex between TRAF6 and PLCγ2 was also identified in B-cells (9,10,42). However, the signaling pathways regulating these activation processes remain poorly understood.
In this study, we found that the absence of Tespa1 decreased the stabilization of TRAF6, attenuated the phosphorylation of PLCγ2 in anti-CD40-stimulated B cells, and impaired specific distal signaling events, such as the activation of ERK mitogenactivated protein kinases and calcium flux. By contrast, it had no effect on other adaptor proteins, such as BLNK, Btk, and Grb2.
These findings imply that Tespa1 may be involved in the formation of TRAF6-PLCγ2 complexes as reported previously (42), and suggest there is a CD40/TRAF6/Tespa1/PLCγ2/MAPK signaling axis in involved in CD40-induced B cell activation.
To elucidate the possible role of Tespa1 in B cell-related autoimmune diseases, we induced CIA in bone marrow chimeric mice and showed that the development of CIA was clearly attenuated in Tespa1-deficient chimeras, suggesting that Tespa1 may be a potential therapeutic target in human RA.
Our study has limitations and there are still many unanswered questions. We cannot exclude the possibility that Tespa1 may be involved in the regulation of other B cell signaling pathways since we detected no significant changes in NK-κB, which is the classic protein that is activated downstream of CD40 and TRAF 6. We also did not find the target protein that interacts directly with Tespa1 in B cells.
More importantly, it has been reported that decreased expression of Tespa1 in peripheral blood mononuclear cell is partially associated with a reduced susceptibility to human RA, but not with disease severity. Since many different types of immune cells are involved in the development of RA (43), and Tespa1 may also modulate the functions of these immune cells, this may mask its role in B cell thymus-dependent humoral responses.
Taken together, our results suggest that Tespa1 may be a previously unsuspected missing component of the CD40-proximal signaling machinery in B cells, placing Tespa1 in a key position to regulate B cell-mediated autoimmune diseases and suggests possible new signaling pathways in B-lymphocytes. Further research along these lines may increase our understanding of the mechanistic relation between human autoimmune diseaseassociated alleles and B-cell physiology.

eThics sTaTeMenT
This investigation was conducted in accordance with the ethical standards of the Declaration of Helsinki, followed national and international guidelines and was approved by the review board of the School of Medicine, Huzhou University.
aUThOr cOnTriBUTiOns YY and WH were involved in design, performing and analysis of experiments, and contributed to drafting of the manuscript. XiaoL, XiawL, JQ, and XA were involved in performing of experiments. HH and HS provided resources and were involved in editing the manuscript. LL was involved in the conception of the study. HZ was involved in design of the study.