Gm614 Protects Germinal Center B Cells From Death by Suppressing Caspase-1 Transcription in Lupus-Prone Mice

Only a few signaling pathways have been reported in germinal center (GC) B-cell proliferation and death. In this study, we showed that a novel uncharacterized Gm614 protein is highly expressed in GC B cells from lupus-prone mice. Critically, ablation of this GC B-cell-specific Gm614 promoted GC B-cell death and mitigation of autoimmune symptoms, whereas overexpression protected GC B cells from death and exacerbated autoimmune symptoms. We demonstrated that mechanistically, nuclear-localized Gm614 reduced caspase-1 expression in GC B cells by binding with caspase-1 promoter to suppress its activation. Our results suggest that Gm614 protects GC B cells from death by suppressing caspase-1 transcription in autoimmune diseases. This may provide some hints for targeting the cell proliferation involved in autoimmune diseases.


INTRODUCTION
B cells play an important role in autoimmune diseases. They can present antigen (Ag) to T cells, secrete inflammatory cytokines, and differentiate into autoantibody-producing plasma cells (PCs) (1). B cells are regarded as a valid target for autoimmune diseases, and depletion of B cells with rituximab has proven to be an effective treatment of autoimmune diseases (2). Further exploration of potential B-cell targets may open up new therapeutic opportunities.
Most autoimmune diseases involve an imbalance of B-cell proliferation and death. Specifically, lymphoproliferative syndrome illustrates the role of cell death in autoimmune diseases with natural mutants such as a homozygous null FAS ligand (FASLG) leading to uncontrolled proliferation of lymphocytes accompanied by autoimmune cytopenia (3). Lupus-prone MRL/lpr mice demonstrate similar autoimmune symptoms as systemic lupus erythematosus (SLE) due to the lymphoproliferation-producing spontaneous mutation of Fas (4). Although the parental strains display limited autoimmunity, NZB/W F1 mice develop inflammation-driven lupus-like features similar to that of human lupus patients (5). Therefore, exploring the mechanisms underlying disregulation involved in B-cell death and proliferation will be important in finding effective B-cell-targeting agents for autoimmune diseases.
Germinal centers (GC) may be important sites of immune dis-regulation in autoimmune diseases (6). Spontaneous GC formation in the spleens of several autoimmune mouse strains promote the development of a lupus-like disease (6), and ectopic GC-like structures are also involved in autoimmune diseases such as SLE (7). B cells with a GC phenotype (B220 + PNA + ) have been found in the kidneys of an SLE mouse model (8). In GC, Ag-activated B cells undergo a proliferation phase in the GC dark zone (DZ), followed by a non-proliferative selection stage in the GC light zone (LZ) where B cells are selected to differentiate into memory B cells or PCs or die (9).
The signaling pathways involved in germinal center (GC) Bcell proliferation and death are largely unknown. In the present study, Gm614 was shown to be highly expressed in GC B cells from lupus-prone mice. Importantly, our results suggest that Gm614 protects GC B cells from death by suppressing caspase-1 transcription in autoimmune diseases.

Ethics Committee Approval
Care, use, and treatment of mice in this study were in strict agreement with international guidelines for the care and use of laboratory animals. This study was approved by the Animal Ethics Committee of Beijing Institute of Brain Disorders.

Mice and Immunization
All mice used in the study are on the C57BL/6 background. Seven-week-old female C57BL/6 mice were purchased from the Chinese Academy of Medical Sciences (Beijing, China). Lupusprone MRL/MpJ/lpr/lpr (MRL/lpr) mice and age-matched MRL/ MpJ/+/+ (MRL/+) mice (Nanjing Biomedical Research Institute of Nanjing University, Nanjing, China) have been previously described (10). Other lupus-prone NZB/WF1 mice (New Zealand Black/New Zealand White F1 mice) and their control NZW/LacJ mice were purchased from Chinese Academy of Medical Sciences, Beijing, China, and have been previously described (11). Gm614 fl/fl (Gm614 F/F ) mice were developed by Cyagen Biosciences Inc., Guangzhou, China. Cg1-cre (Cg1 cre ) (B6.129P2[Cg]-Ighg1tm1[IRES-cre]Cgn/J) mice as a tool to mediate GC-specific knocked-out of a gene (12,13) were purchased from Shanghai Biomodel Organism Science & Technology Development Co., Ltd. (Shanghai, China). Gm614 F/F mice and Cg1 cre mice were crossed to obtain GCspecific Gm614-null (Cg1 cre Gm614 F/F , Gm614 cKO) animals. Gm614 transgenic (Tg) mice were developed by Cyagen Biosciences Inc., Guangzhou, China. BM Chimeras have been used to develop B-specific depletion/expression of a gene (14,15). mMT mice (B6.129S2-Ighmtm1Cgn/J, Stock No: 002288, The Jackson Lab) were irradiated (1150 cGy) and reconstituted with BM from mMT mice (80%) and Gm614 Tg mice (20%). Thus, 80% of the hematopoietic cells (except B cells) in the chimeric mice will be wild-type in gene expression whereas the B cells can only be derived from Gm614 Tg precursors. The reconstituted mice were named as B Gm 614 Tg mice. B non Tg mice were used as the control. To induce lupus, about 9-week-old mice were injected intraperitoneally (i.p.) with 0.5 ml pristane (2,6,10,14-Tetramethylpentadecane, Cat no. 138462500, Fisher Scientific Corp., USA). In some experiments, mice were injected i.p. with 100 mg of the protein antigen KLH (Keyhole limpet haemocyanin; Sigma, St Louis, MO) in a volume of 0.2 mL of saline.

iTraq Quantitative Proteomics
Each sample of 200 mg total protein in 200 mL denaturing buffer was reduced with 2 mL 1 M DTT at 60°C for 1 h, and then the cysteine residues were blocked by adding 10 mL 1 M IAA for 40 min at room temperate under dark conditions. The reduced and alkylated protein mixtures were subjected to the FASP protocol with spin ultra-filtration units containing a nominal molecular weight cutoff of 10,000 Da (Sartorius, Gottingen, Germany) and centrifuged at 12,000 rpm for 20 min. The bottom solution was then discarded. After washing with 100 mL dissolution buffer three times, the protein digestions were conducted by incubating the proteins and the Sequencing Grade Modified Trypsin (Promega Corporation, WI, USA) in a 1:50 ratio (trypsin-to-protein mass) at 37°C overnight. After digestion, the liberated peptides were collected by centrifugation at 12,000 rpm for 20 min and the filtration units were washed with 50 mL of UA buffer. The resultant peptide mixture samples were labeled using an 8

Quantitative PCR Analysis
Total RNA was extracted from cells with Trizol (Invitrogen Life Technologies). The final RNA pellets were dissolved in 0.1 mM EDTA (2 ml/mg original wet weight). Reverse transcription reactions were carried out on 22 ml of sample using superscript II RNA H-Reverse Transcriptase (Invitrogen Life Technologies) in a reaction volume of 40 ml. All samples were diluted in 160 ml nuclease-free water. qPCR was employed to quantify mouse gene expression from the cDNA samples. Mouse gene expression was normalized to the levels of the b-actin gene.

Production of Anti-Gm614 Antibody
The production of the polyclonal antibody in Rabbit has been previously described (19,23). Briefly, The C-terminal end of mouse Gm614 (sequence: SPKGGGAQQTTST SSQLKSKGGR) corresponding to amino acids 144-166 was chosen for peptide synthesis. The peptide was coupled to keyhole limpet and used for the immunization of rabbits and production of anti-Gm614. The polyclonal antibody was purified by protein A chromatograph, and its reactivity with recombinant mouse Gm614 was confirmed by ELISA.

Western Blot Analysis
On a 10% SDS-polyacrylamide gel, 25 µg of cell protein from whole-cell lysates was electrophoretically separated and then transferred to a PVDF membrane. This membrane was blocked for 1 hour in the solution with 5% fat-free dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) at room temperature. The blots were then incubated overnight at 4°C with rabbit antibodies against caspase-1 (#24232, Cell Signaling Tech), Gm614 (described above), and b-actin (#4970, Cell Signaling Tech) antibodies diluted 1:1000 in TBS-T containing 5% bovine serum albumin. The membrane was washed for 5 min every time and totally for 4 times with TBS-T, and incubated for 45 min at room temperature with HRP (horseradish peroxidase)conjugated secondary antibody F(ab')2 (Zymed Laboratories, San Francisco, CA) (1:20 000 in TBS-T containing 5% bovine serum albumin). Finally, the ECL detection system (Amersham, Arlington Heights, IL) was used to show the protein band.
Hematoxylin and Eosin (H&E) Staining and Histopathology Scoring H&E staining has been described previously (24,25). Kidney specimens were fixed in formalin for 24 h, dehydrated by successive incubation in 70%, 80%, and 90% ethanol for 3 h, and then 100% ethanol I for 2 h, 100% ethanol II for 2 h, and vitrified with xylene I and xylene II for 20 min. After immersing in paraffin I and II for 40 min, the specimens were embedded and sliced. Staining was performed as follows: hematoxylin staining for 15 min, followed by hydrochloric acid alcohol solution for 35 s, eosin staining for 10 min, followed by 90% ethanol for 40 s. Neutral balsam was then used for mounting and the section was observed and photographed by light microscope.

ELISA Assay
Blood was taken from vena cava with syringe and carefully put in a 1.5 mL E-tube. The tube was placed for 30 min at room temperature (RT) for clotting and then centrifuged for 10 min at 10000 rpm in RT. Sera in the supernatant were collected and tested by ELISA against the following recombinant proteins: Ro60, La, and Sm/RNP (Euroimmun AG, Lübeck, Germany, and Orgentec Diagnostics GmbH, Mainz, Germany), dsDNA (Sigma) in the presence of histone (Sigma), or KLH (Sigma). Goat anti-rat polyvalent HRP-labeled anti-IgG and -IgM antibodies were used as the secondary antibodies (Sigma, SAB3700666-2MG).

Propidium Iodide (PI)/FACS Analysis
Cell cycle and death analysis has been described previously (19,23). Briefly, cells were collected and washed 1 time with 5-10 ml of 1×PBS. Cells were suspended in 500 ml 1× PBS containing + 0.1% glucose (at 4°C), and 5 ml of cold 70% EtOH (kept at −20°C) was immediately added, mixed, and kept at 4°C for 1 h. Cell were then spun down and washed once with 1× PBS (10 ml). Without adding more PBS, cells were then spun again for 2 min so that the residual PBS could be removed and cells were then suspended in 300 ml 69 mM PI (propidium iodide, Cat# 537059, Calbiochem, San Diego, CA) solution with 38 mM Na Citrate (Cat# C7254 Sigma, St. Louis, MO) and 20 ml of 10 mg/ml RNase (Cat# R4875, Calbiochem, San Diego, CA). Cells were mixed, incubated at 37°C for 30-45 min, and analyzed by FACS.

Immunofluorescence and Confocal Microscopy
Cells were seeded onto glass coverslips in 24-well plates, washed with PBS, fixed in 4% formaldehyde solution for 30 min, and permeabilized for 5 min with 0.5% Triton X-100 in TBS. After extensive wash, cells were blocked with 2% BSA in PBS for 30 min. Coverslips were incubated with DAPI and observed under a confocal laser scanning microscope (Leica TCS-SP2). Images were directly captured, saved, and transferred to Adobe Photoshop 5.5.

Chip-Sequencing Assay
Chromatin immunoprecipitation (Chip) analysis has been described previously (27). Briefly, cells were cross-linked with 1% formaldehyde for 10 min at RT, quenched by the addition of glycine to a final concentration of 0.125 M, and frozen. Chip was performed on two independent pools of cells, each comprising 3 samples. Cell lysis and nuclei isolation was performed using the TruChIP High Cell Chromatin Shearing Kit with SDS (Covaris). Nuclei were sonicated in the S220 Ultrasonicator (Covaris) in order to obtain chromatin fragments ranging from 200 to 500 bp. Sheared chromatin from about 5-10 ×10 6 cells was incubated overnight with 4 µg of anti-EGFP (Abcam). Protein A magnetic beads were then added for 4 hr incubation followed by sequential washes at increasing stringency and reverse cross-linking. Upon RNAse and proteinase K treatment, Chip DNA was purified using the MiniElute Reaction Cleanup Kit (Qiagen) and quantified using the Quant-iT PicoGreen dsDNA Reagent (Life Technologies). Chip-seq library preparation, sequencing, and analysis were done by IGenecode.

Statistics
Statistics were analyzed by using GraphPad Prism (version 5.0, GraphPad Software Inc., USA). The data were shown as mean ± standard error of the mean (SEM). Student's t test was employed to determine significance between two groups (paired or unpaired) and One-Way or Two-Way ANOVA analysis was used to determine significance among several groups. Differences were considered statistically significant when p < 0.05.

Gm614 Expression Was Increased in Germinal Center (GC) B Cells of Lupus-Prone Mice
Our previous studies have shown that Atacicept (TACI-IgG) achieves similar clinical results to belimumab by reducing the levels of circulating mature and activated B cells in the lupusprone mice (11,28). To explore the molecular mechanisms, the transcripts in B220 + B cells from TACI-IgG-treated lupus-prone mice were assessed using Affymetrix Microarrays described in our previous study (20). We determined gene Gm614 resulted in 0.16, 0.12, and 0.14 fold down-regulation in three independent experiments shown in Supplementary Tables 1, 2, and 3, respectively. These results suggest that Gm614 may be associated with lupus progression.
To determine Gm614 expression in B-cell subpopulations in lupus-prone mice, flow cytometry (FACS) was used to sort immature B cells, mature B cells, GC B cells, PBs, and PCs from splenocytes of lupus-prone MRL/lpr (Figures 1A, B) and NZB/WF1 (Figures 1C, D) mice. qPCR (Figures 1A, C) and western blot (Figures 1B, D) analysis showed that both MRL/lpr and NZB/WF1 mice have higher Gm614 expression in GC B cells compared to the control. These results indicate that lupus-upregulated Gm614 expression in GC B cells is a more universal inflammation-driven phenomenon.

Gm614 Knocked-Out (KO) in GC B Cells Mitigated Autoimmune Symptoms
To explore the effect of Gm614 KO on lupus symptoms, we developed Cg1 cre Gm614 fl/fl (Gm614 cKO) mice (Supplementary Figure 1). Our data showed that Gm614 expression was knocked-out in GC B cells from Gm614 cKO mice (Supplementary Figure 1). However, Gm614 cKO mice did not demonstrated any open autoimmune symptoms.
The hydrocarbon oil 2, 6, 10, 14-tetramethylpentadecane (TMPD; also known as pristane)-induced experimental lupus mice displayed some important immunologic and clinical features that are similar to those in human SLE (29)(30)(31). To explore the effect of Gm614 cKO on lupus progression, we injected pristane into WT and Gm614 cKO mice. H&Estaining of the mouse kidney sections at 6 months showed reduced inflammatory cell infiltration and overall normal structure of the glomerular region in Gm614 cKO mice (Figure 2A). To evaluate the effect of Gm614 cKO on pristane-induced renal injury, histological scoring based on the typical microscopic features of chronic tubular damage, including extensive tubular necrosis and dilatation, as well as cast formation and loss of brush border was adopted. The results suggest that Gm614 cKO significantly protected the kidney from pristane-induced damage ( Figure 2B). Critically, Gm614 cKO reduced anti-nucleic antibodies (ANA) IgM and IgG, and antinucleosome IgG but not total IgG in pristane-induced lupus ( Figure 2C). Furthermore, we showed that Gm614 was depleted in the GC B cells of Gm614 cKO mice ( Figure 2D). This results in reduction in GC B cells (Figures 2E, F), PBs, and PCs ( Figures  2G, H) in Gm614 cKO mice. Taken together, these results suggest that Gm614 KO in GC B cells mitigates autoimmune symptoms by reducing GC B cells and subsequent PBs and PCs.

Gm614 Transgene (Tg) in B Cells (BGm614 Tg) Exacerbated Autoimmune Symptoms
To further explore the effect of Gm614 overexpression on lupus, BM Chimeras were used to develop B-specific overexpression of Gm614 in B Gm614 Tg mice (Supplementary Figure 2). Figure 2); however, these mice did not demonstrate any open autoimmune symptoms.

Overexpression of Gm614 was showed in B cells but not T cells (Supplementary
To explore the effect of Gm614 overexpression on lupus progression, we injected pristane into B non Tg and B Gm614 Tg mice. H&E-stained sections of the kidney were analyzed 6 months after pristane injection. The results showed that Gm614 Tg promoted inflammatory cell infiltration into the kidney in pristane-induced lupus ( Figure 3A). To evaluate the effect of Gm614 Tg on pristane-induced renal injury, histological scoring based on the typical microscopic features of chronic tubular damage, including extensive tubular necrosis and dilatation, as well as cast formation and loss of brush border was adopted. The results suggest that Gm614 Tg significantly promotes pristane-induced damage in the kidney ( Figure 3B). In addition, anti-nucleic antibodies (ANA) IgM and IgG, and antinucleosome IgG but not total IgG were elevated in B Gm614 Tg mice ( Figure 3C). This may result from Gm614 overexpression in GC B cells of these mice ( Figure 3D). Gm614 overexpression in GC B cells up-regulated GC B cells (Figures 3E, F), PBs, and PCs ( Figures 3G, H) in pristane-induced lupus. These results suggest that Gm614 overexpression in GC B cells exacerbated autoimmune symptoms by up-regulating GC B cells and subsequent PBs and PCs.

Gm614 Protected GC B Cells From Death
To explore the role of Gm614 in GC B cells, we first determined the effect of Gm614 on GC. We found that the percentages of CD86 lo CXCR4 hi dark zone (DZ) and CD86 hi CXCR4 lo light zone (LZ) B cells were unchanged between B Gm614 Tg mice and B non Tg mice ( Figures 4A, B). These results suggest that Gm614 Tg does not affect the ratio of B cells in DZ to LZ. Furthermore, we found that Gm614 Tg did not affect the GC B cell cycle but suppressed GC B cell death (Figures 4C, D). On the other hand, we also used Gm614 cKO mice to detect the effect of Gm614 cKO on GC B cells. We found that Gm614 cKO did not affect the ratio of GC B cells in DZ to LZ (Figures 4E, F) and the cell cycle but promoted GC B cell death (Figures 4G, H). These results demonstrate that Gm614 up-regulates GC B cells by protecting GC B cells from death.

Gm614 Suppressed Caspase-1 Expression in GC B Cells
To explore the mechanisms by which Gm614 protects GC B cells from death, we used RNA-sequencing and iTRAQ quantitative proteomics to determine the transcripts and proteomics in GC B cells, respectively. We found caspase-1 mRNA ( Figure 5A) and protein ( Figure 5B) were obviously increased in Gm614 -/-GC B cells. These results were further proven by qPCR ( Figure 5C) and western blot ( Figure 5D) assays. Our data suggest that Gm614 cKO up-regulated caspase-1 expression in GC B cells. On the other hand, we also used RNA-sequencing ( Figure 5E) and iTRAQ quantitative proteomics to determine the effect of Gm614 Tg on the transcripts and proteomics in GC B cells. Gm614 Tg obviously reduced caspase-1 mRNA ( Figure 5E) and protein ( Figure 5F) in GC B cells. This was subsequently proven by qPCR ( Figure 5G) and western blot ( Figure 5H) assays. Our results suggest that Gm614 suppressed caspase-1 expression in GC B cells.

Gm614 Suppressed Caspase-1 Transcription by Binding With the Caspase-1 Promoter
To explore the mechanisms by which Gm614 suppressed caspase-1 expression in GC B cells, we first determined the subcellular location of Gm614. We found that Gm614 was expressed in the nucleus ( Figure 6A). Further, we found that nuclear localization sequence (NLS) was located in the C-terminus (172∼191) of Gm614 (Figures 6B, C). We proposed that Gm614 could bind with DNA. Genome-wide mapping of Gm614 binding in GC B cells using ChIP-seq showed that Gm614 could bind Up2k, which is often contained within the promoter ( Figure 6D). De novo motif prediction by DNA sequences enriched in Gm614 binding regions demonstrated 6 Gm614 binding motifs ( Figure 6E). Genomic snapshots depicting the ChIP-seq results showed two Gm614 binding sites (-1612 ∼ -1601, -1273 ∼ -1262) at the caspase-1 genomic loci promoter regions ( Figure 6F, lower panel). Critically, these two sites were the same sequences (TTTTTTTTTCTT) as the second motif suggested by de novo motif prediction ( Figure 6F, upper panel). These results indicate that Gm614 could bind with the promoter of caspase-1. Dual luciferase reporter gene expression was analyzed to examine the effect of Gm614 on the caspase-1 promoter and we found that Gm614 could effectively suppress its activation ( Figure 6G). However, Gm614 did not suppress the activation of caspase-1 promoters with deletions at the -1612 ∼ -1601 or -1273 ∼ -1262 sites that binds Gm614 ( Figure 6G). These results suggest that Gm614 suppressed caspase-1 transcription by binding with the caspase-1 promoter.

Gm614 Promoted KLH-Induced GC B-Cell Responses
To study whether a foreign antigen promoted GC B cells to express Gm614, we determined the expression of Gm614 in spontaneous GCs of WT mice and KLH-immunized WT mice. We found that Gm614 expression was up-regulated in GC B cells by foreign antigen KLH (Figures 7A, B). To further explore whether Gm614 plays an important role in an optimal GC responses induced by an foreign antigen, we examined splenic CD19 + B220 + CD38 lo GL7 hi GC B cells, CD138 + B220 + PBs, and CD138 -B220 + PCs cells and anti-KLH IgM, IgG, and IgG1 in the sera from KLH-immunized WT, Cg1 cre , Gm614 F/F , and Cg1 cre Gm614 F/F mice. We found that Gm614 cKO reduced the absolute number of GC B cells (Figure 7C), PBs and PCs ( Figure 7D), anti-KLH IgM, IgG, and IgG1 antibodies ( Figure  7E) induced by KLH. These results suggest that Gm614 cKO suppressed KLH-induced GC B-cell responses. In addition, we also determined splenic CD19 + B220 + CD38 lo GL7 hi GC B cells, CD138 + B220 + PBs, and CD138 -B220 + PCs cells and anti-KLH IgM, IgG, and IgG1 in the sera from KLH-immunized B non Tg and B Gm614 Tg mice. Our data demonstrated that Gm614 Tg upregulated the absolute number of GC B cells (Figure 7F), PBs and PCs ( Figure 7G), anti-KLH IgM, IgG, and IgG1 antibodies

DISCUSSION
Germinal center (GC) B-cell proliferation, differentiation, survival, and death are strictly regulated by many molecules including Bcl6 and Aid. The transcriptional repressor Bcl6 directly controls many GC B cell functions including activation, survival, DNA damage response, cell cycle arrest, cytokines, toll-like receptors, TGF-b, WNT signaling, and so on (32). In GC, immunoglobulin (Ig) genes in Ag-activated B cells undergo somatic hypermutation (SHM) and class switching recombination (CSR) initiated by Aid (33,34). Apart from Bcl6 and Aicda (Aid), Gm614 is highly expressed in GC B cells from lupus-prone mice (Figure 1). In addition, Gm614 protects GC B cells from death (Figure 4), and the expression of Gm614 in GC B cells of lupus-prone mice plays an important role in the balance between GC B-cell survival and death.
Cell-intrinsic induction of Bcl-6 has been shown to promote autoimmune germinal centers (35). In addition, increased Aid expression is also associated with some autoimmune diseases, such as SLE (36). We demonstrated here that lupus could upregulate Gm614 expression in GC B cells (Figure 1). This increased Gm614 expression could effectively up-regulate GC B cells, resulting in increased PBs and PCs, and finally exacerbated autoimmune symptoms (Figure 3), whereas Gm614 cKO effectively reduced GC B cells, resulted in decreased PBs and PCs and mitigation of autoimmune symptoms (Figure 2). These results suggest that similar to Bcl-6 and Aid, Gm614 also has a critical role in autoimmune diseases.
Dark zones in mature GC contain mainly proliferating cells, and some apoptotic B cells and non-selected B cells also undergo apoptosis in light zones of GC (37). The up-regulation of B7-H1 on activated B cells suggests the possibility that PD-1/B7-H1 interactions may regulate survival of self-reactive B cells during GC reactions (38-40). We showed here that up-regulation of Gm614 protected GC B cells from apoptosis or death (Figure 4). Because apoptosis plays critical roles in the initiation, progression, and remission of autoimmune diseases, it is the major target of therapeutic interventions for autoimmune diseases (41). For example, induction of GC B-cell apoptosis by targeting Gm614 may be a potential way to control autoimmune diseases. Activated caspase-1 induces pyroptosis, inflammatory and necrosis-like programmed cell death (42). However, interleukin (IL)-1b and IL-18 secretion is rare in GC B cells. Thus, the main activity of caspase-1 is to induce B cell death (42). Critically, it has been shown that caspase-1 regulates splenic B-cell apoptosis independent of IL-1b and IL-18 (43). Our data here showed that GC B cell apoptosis or death (Figure 4) is positively associated with caspase-1 expression ( Figure 5). Thus, Gm614-mediated suppression of caspase-1 expression ( Figure 5) is at least partly involved in the imbalance between B-cell proliferation and death in autoimmune diseases.
Apart from the role of Gm614 in the autoimmune environment, we also explored the role of Gm614 in GC B cells in general. We found that foreign antigens (e.g., KLH) could up-regulate Gm614 expression in GC B cells (Figures 7A, B). Furthermore, Gm614 cKO suppressed KLH-induced GC B-cell responses ( Figure 7C), which resulted in reduced PBs and PCs ( Figure 7D) and anti-KLH IgM, IgG, and IgG1 antibodies ( Figure 7E). In addition, we showed that Gm614 Tg promoted KLH-induced GC B-cell responses ( Figure 7F), which resulted in increased PBs and PCs ( Figure 7G) and anti-KLH IgM, IgG, and IgG1 antibodies ( Figure 7H). These results define the role of Gm614 in GC B cells in general. Thus, Gm614 is required for GC B cell survival irrespective of an autoimmune or non-autoimmune environment. This will have implication in vaccine development.
In conclusion, we showed that a novel uncharacterized Gm614, highly expressed in GC B cells of lupus-prone mice, protected GC B cells from death and exacerbated autoimmune symptoms by binding with caspase-1 promoter to suppress its activation. Thus, our results provide a novel mechanism underlying the imbalance between B-cell proliferation and death in autoimmune diseases Splenocytes and sera were collected from KLH-immunized WT, Cg1 cre , Gm614 F/F , and Cg1 cre Gm614 F/F mice. Splenic CD19 + B220 + CD38 lo GL7 hi GC B cells, CD138 + B220 + PBs, and CD138 -B220 + PCs cells were analyzed and the absolute number of GC B cells (C), and PBs and PCs (D) was shown. Anti-KLH IgM, IgG, and IgG1 were determined in the sera using ELISA assay (E). (F-H) Gm614 Tg promoted GC B-cell responses induced by KLH. Splenocytes and sera were collected from KLH-immunized B non Tg and B Gm614 Tg mice. Splenic CD19 + B220 + CD38 lo GL7 hi GC B cells, CD138 + B220 + PBs, and CD138 -B220 + PCs cells were analyzed and the absolute number of GC B cells (F), and PBs and PCs (G) was shown. Anti-KLH IgM, IgG, and IgG1 were determined in the sera using ELISA assay (H). (A-H) Data represent three independent experiments, with six samples per group per experiment. Two-Way (D, E, G, H) and One-Way (C) ANOVA was followed by Bonferroni post-tests and two tailed Student's t-test (A, F), Error bars, s.e.m., *p < 0.05, **p < 0.01, ***p < 0.001. and provides some hints for treatment of autoimmune diseases by targeting Gm614.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal Ethics Committee of Beijing Institute of Brain Disorders.

AUTHOR CONTRIBUTIONS
YH, RX, BZ, and SZ performed the experiments, and XW contributed essential reagents and materials for the experiments.