Maintenance of Germinal Center B Cells by -9 through Promotion of and Inhibition of

This information is current as Jingting Zhang, Srikanth Kodali, Min Chen and Jin Wang of October 4, 2021. J Immunol published online 20 May 2020 http://www.jimmunol.org/content/early/2020/05/19/jimmun ol.2000359 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 20, 2020, doi:10.4049/jimmunol.2000359 The Journal of Immunology

Maintenance of Germinal Center B Cells by Caspase-9 through Promotion of Apoptosis and Inhibition of Necroptosis

Jingting Zhang,* Srikanth Kodali,*,† Min Chen,‡ and Jin Wang*,x

In response to T cell–dependent Ag encounter, naive B cells develop into germinal center (GC) B cells, which can further differentiate into Ab-secreting plasma cells or memory B cells. GC B cells are short lived and are prone to caspase-mediated apoptosis. However, how apoptotic regulate GC B cell fate has not been fully characterized. In this study, we show that mice with B cell–specific knockout of caspase-9 had decreases in GC B cells and Ab production after immunization. Caspase-9–deficient B cells displayed defects in caspase-dependent apoptosis but increases in necroptosis signaling. Additional deletion of Ripk3 restored GC B cells and Ab production in mice with B cell–specific knockout of caspase-9. Our results indicate that caspase-9 plays an important role in the maintenance of Ab responses by promoting apoptosis and inhibiting necroptosis in B cells. The Journal of Immunology, 2020, 205: 000–000. Downloaded from erminal centers (GCs) are special regions within sec- from Fas and BCR can mediate apoptosis during negative selec- ondary lymphoid organs that are seeded by B cells fol- tion of GC B cells (17, 18), caspase-9 is also involved in BCR- G lowing T cell–dependent Ag activation (1, 2). GCs are induced apoptosis in this process (18). segregated into dark zones and light zones (3–5). In the dark In addition to the induction of apoptosis, caspases have been zones, B cells undergo proliferation and somatic hypermutation of shown to regulate lymphocyte survival and development (19, 20).

their Ig , after which they migrate to the light zones for Ag Caspase-3 and caspase-6 can regulate cell cycling in B cells that is http://www.jimmunol.org/ affinity–based selection (3–5). The selected B cells can return to important for B cell activation and differentiation into plasma cells the dark zones for further proliferation and somatic hypermutation (21, 22). Caspase-8 is able to cleave and inactivate Ripk1 to in- or exit the GCs and differentiate into plasma cells or long-lived hibit necroptosis (23, 24), a form of programmed memory B cells. However, only a small fraction of B cells survive through necrosis (25). Necroptosis signaling involves the activa- the competition and selection in the GC to become plasma cells or tion of Ripk1, Ripk3, and its substrate, mixed lineage kinase do- memory B cells (1, 6, 7). Instead, a majority of GC B cells are main-like protein (MLKL) (26–32). PGAM5 can be activated by cleared by apoptosis (7). the Ripk1–Ripk3–MLKL complex to induce Drp1-mediated mi- Caspases are endoproteases that play essential roles in the ex- tochondrial fragmentation and necroptosis (33, 34). Necroptosis ecution of apoptosis (8, 9). Apoptotic caspases are subdivided into plays an essential role in immune cells in response to infection by guest on October 4, 2021 initiators and effectors based on their ordering in the apoptosis (35, 36). Cell membrane rupture may cause cell contents to spill signaling cascade (10). Caspase-8 is an initiator caspase of the into the organ, releasing damage-associated molecular patterns. extrinsic apoptosis pathway mediated by the TNFR family death Immune cells are then recruited into the damaged tissues (37). receptors (11, 12), whereas caspase-9 is the initiator caspase in the Caspase-8 deficiencies lead to decreased cell proliferation in intrinsic apoptosis pathway mediated through disruption of mi- T cells in humans and mice (38–40). Such defects in T cell pro- tochondria (13–15). The activation of the initiator caspases results liferation in caspase-8 knockout mice have been shown to be in the activation of downstream effector caspases, including rescued by additional deletion of an essential necroptosis , caspase-3, caspase-6, and caspase-7 (8, 16). Although signaling Ripk3 (40). This suggests that caspase-8 signaling is important for the protection of cell survival and proliferation by inhibiting necroptosis. *Immunobiology and Transplant Science Center, Houston Methodist Research Insti- Knockout studies suggest that caspase-9 is essential for † tute, Houston, TX 77030; Department of Pathology and Immunology, Baylor Col- mitochondrion-dependent apoptosis induced by various stimuli lege of Medicine, Houston, TX 77030; ‡Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, TX 77030; and (41, 42). Caspase-9 can also inhibit inflammatory responses by xDepartment of Surgery, Weill Cornell Medical College, Cornell University, New promoting immunologically silent apoptosis (43, 44), suggesting York, NY 10065 that caspase-9 may also contribute to the protection of cell sur- ORCID: 0000-0002-3116-0838 (J.W.). vival. In this study, we show that GC B cell numbers and primary Received for publication April 1, 2020. Accepted for publication April 28, 2020. Ab responses are decreased in mice with B cell–specific deletion 2 2 This work was supported by funding from the National Institutes of Health Na- of caspase-9 (B/Casp9 / ). B cells deficient in caspase-9 dis- tional Institute of Allergy and Infectious Diseases Grants R01AI116644 and R01AI123221 (to J.W.) and Cancer Prevention and Research Institute of Texas Grant played elevated levels of necroptosis signaling, although deletion 2/2 RP160384 (to J.W. and M.C.). of Ripk3 rescued GC B cells in B/Casp9 mice. Our results Address correspondence and reprint requests to Dr. Jin Wang, Houston Methodist suggest that caspase-9 protects GC B cells and Ab responses by Research Institute, 6670 Bertner Avenue, R7-214, Houston, TX 77030. E-mail inhibiting necroptosis. address: [email protected] Abbreviations used in this article: ASC, Ab-secreting cell; B/Casp92/2, B cell–specific deletion of caspase-9; DKO, double knockout; GC, germinal center; MLKL, mixed Materials and Methods lineage kinase domain-like protein; NP, 4-hydroxy-3-nitrophenylacetyl; NP-KLH, Mice NP–keyhole limpet hemocyanin; T1, transitional 1; T2, transitional 2; WT, wild-type. Two loxP sites were inserted into both sides of exon 6 of caspase-9 in a Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 9.3 kB HindIII genomic DNA from a bacterial artificial clone

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000359 2 INHIBITION OF NECROPTOSIS BY CASPASE-9 IN B CELLS

(BACPAC Resources). The construct was used for homologous recombi- flow cytometry on an LSRFortessa (BD Biosciences). To determine nation in embryonic stem cells to generate caspase-9-flox mice. CD19-cre necroptosis-related cell death, activated B cells were cultured in the mice (The Jackson Laboratory) were crossed with caspase-9-flox mice for presence of 20 mM Z-IETD and 20 ng/ml TNF-a (R&D Systems) with or B/Casp92/2. Ripk32/2 mice (45) were crossed with B/Casp92/2 mice to without 20 mg/ml Necrostatin-1 (Enzo Life Sciences, Farmingdale, NY) generate Ripk32/2B/Casp92/2 mice. Sex- and age-matched mice, 6–8 wk for 20 h. Propidium iodide was added, and the cells were analyzed by old and on the C57BL/6 background, were used for all experiments. Mice flow cytometry. Propidium iodide2 viable B cells were quantified, and were bred and maintained in a specific pathogen-free facility at Houston the percentage of cell death was calculated as described (48): % cell Methodist Research Institute. Experiments were performed according to death = (untreated 2 treated)/untreated 3 100%. federal and institutional guidelines and with the approval of the Institu- tional Animal Care and Use Committee. ELISA Immunization Clear polystyrene 96-well microplates (Corning, Corning, NY) were coated with 5 mg/ml NP5-BSA or NP25-BSA (Biosearch Technologies) overnight Mice were immunized with 100 mg of 4-hydroxy-3-nitrophenylacetyl at 4˚C. After blocking with PBS containing 0.05% Tween-20 at 37˚C for (NP)–keyhole limpet hemocyanin (NP-KLH) (Biosearch Technologies, 2 h, diluted serum samples were added and incubated at 4˚C overnight. Novato, CA) precipitated with 100 ml Imject Alum Adjuvant (Thermo The plates were incubated with HRP-conjugated goat anti-mouse IgG1 Fisher Scientific, Waltham, MA) by i.p. injection. Mice were used for (SouthernBiotech, Birmingham, AL) for 1 h, followed by development various analyses 2 wk after immunization (46). using TMB Peroxidase EIA Substrate Kit (Bio-Rad Laboratories, Hercu- les, CA). The reactions were stopped with 1 N H2SO4, and the absorbance Flow cytometric analysis of each well was measured using a Synergy H1 Hybrid Multi-Mode Spleen cells were isolated, and RBCs were lysed with ammonium chloride Microplate Reader (BioTek Instruments) at 450 nm. Ab titers were cal- culated as described (46). lysing buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA). Splenocytes were then used for various analyses. To detect NP-specific GC ELISPOT B cells, splenocytes were stained with V450–anti-IgM (BD Biosciences, Downloaded from San Jose, CA), Pacific Blue–anti-CD4, Pacific Blue–anti-CD8a, Pacific MultiScreen 96-Well Filtration Plates (MilliporeSigma, Burlington, MA) Blue–anti-CD11b, Pacific Blue–anti-CD11c, Pacific Blue–anti-Gr-1, Pa- were precoated with 20 mg/ml NP5-BSA or NP25-BSA at 4˚C overnight. cific Blue–anti-IgD, Brilliant Violet 421–anti-CD138, allophycocyanin/ After blocking with RPMI 1640 complete medium for 2 h at 37˚C, cells Fire-750–anti-B220, Alexa Fluor 647–anti-IgG1, PerCP/Cy5.5–anti-GL-7, (1–5 3 105/well) were then added to the plates and incubated at 37˚C PE/Cy7–anti-CD38 (BioLegend, San Diego, CA), and NP conjugated with overnight. The cells were lysed with H2O and then incubated in RPMI PE (Santa Cruz Biotechnology, Dallas, TX) for 15 min on ice. For intra- 1640 complete medium for 2 h at room temperature. The plates were in-

cellular staining of active caspases, splenocytes were stained with surface cubated with HRP-conjugated goat anti-mouse IgG1 (SouthernBiotech), http://www.jimmunol.org/ markers, fixed with 1% methanol-free formaldehyde (Thermo Fisher Sci- followed by development with 3-amino-9-ethylcarbazole (Sigma-Aldrich, entific) for 15 min on ice, permeabilized with the Permeabilization Buffer St. Louis, MO) and stopped by washing with PBS. The stained dots on (Thermo Fisher Scientific) for 15 min on ice, and incubated with rabbit the filters were quantified using an ImmunoSpot S6 Ultimate Analyzer Abs specific for mouse cleaved caspase-9 or cleaved caspase-3 (Cell (ImmunoSpot, Cleveland, OH). Signaling Technology, Danvers, MA) for 15 min on ice. Cells were then stained with Alexa Fluor 647–goat anti-rabbit IgG (Thermo Fisher Sci- Statistical analyses entific) for 15 min on ice. The cells were analyzed by flow cytometry on an LSRFortessa flow cytometer (BD Biosciences). Statistical analyses were performed by two-tailed Student t test or by Dunnett test (in Fig. 8) using GraphPad Prism 8 (GraphPad, San Diego, B cell purification and Western blot CA). The results of ELISA, ELISPOT, and flow cytometry were presented 6 , as the mean SD, with p 0.05 considered statistically significant. by guest on October 4, 2021 B cells were purified from splenocytes using CD19 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and stimulated with 20 ng/ml IL-4 and 1 mg/ml LPS in RPMI 1640 complete medium for 3 d (47). Cells Results were then cultured in RPMI 1640 medium with 0.5% FBS and har- Caspase-9 signaling in GC B cells vested at 0, 8, and 16 h for lysis in sample buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM zVAD-FMK, GC B cells are prone to caspase activation and apoptosis in the 3 and 1 protease/phosphayase inhibitor mixture [Cell Signaling absence of survival signals (46). To examine caspase-9 signaling in Technology]). Cell lysates were used for Western blot and probed with rabbit Abs against caspase-9, cleaved caspase-9, caspase-3, cleaved GC B cells, we cultured total spleen B cells from unimmunized caspase-3, caspase-1, cleaved caspase-1, gasdermin D, cleaved gasdermin D, mice under low serum conditions to induce spontaneous cell death Ripk1, phospho-Ripk3, Ripk3 (Cell Signaling Technology), MLKL (WuXi as described (46). We found that naturally occurring IgG1+ GC AppTec, Shanghai, China), or Pgam5 (Abcam, Cambridge, U.K.) followed B cells, but not naive or IgG1+ memory B cells, displayed the by incubation with HRP-conjugated goat anti-rabbit IgG (Abcam) and de- activation of caspase-9 and downstream caspase-3 (Fig. 1A). We velopment with SuperSignal Western Pico ECL substrate (Pierce Chemical, Dallas, TX). The blots were also probed with anti–b-Actin (Santa Cruz also examined caspase signaling in B cells from mice immunized Biotechnology) as loading controls. Wild-type (WT) and caspase-92/2 with NP-KLH. Consistently, the activation of caspase-9 and B cells were also cultured with 1 mg/ml biotinylated anti-Fas (BD Biosci- caspase-3 was detected in NP-specific IgG1+ GC B cells but not m ences) and 0.1 g/ml streptavidin (Jackson ImmunoResearch, West Grove, memory B cells (Fig. 1B). These data indicate that caspase-9 PA) for the indicated time. Cells were also used for staining with FITC- conjugated anti-Fas (BD Biosciences) for flow cytometry or lysed for signaling can be rapidly activated in GC B cells. Western blot to detect caspases. Alternatively, activated B cells were treated Reduced GC B cells and Ab production in the absence with or without 20 mM Z-IETD and 20 ng/ml TNF-a (R&D Systems, Minneapolis, MN) for 4 h. Z-IETD was added 30 min prior to TNF-a. Cells of caspase-9 were lysed for Western blot to detect necroptosis-related proteins. To investigate the function of caspase-9 in immune regulation, we Cell death analyses generated mice with a floxed allele of caspase-9 (Fig. 2A). These mice were bred with CD19-cre mice for B/Casp92/2.B/Casp92/2 To determine spontaneous cell death, splenocytes were collected and mice exhibited no significant differences in the numbers of tran- cultured in RPMI 1640 medium with 0.5% FBS for 4 h as described (46). Cells were then incubated with FITC-DEVD-FMK (SM Biochemicals, sitional 1 (T1), transitional 2 (T2), and mature naive B cells in the Anaheim, CA) at 37˚C for 30 min and then stained with biotin anti-CD4, spleen (Fig. 2B), indicating that caspase-9 is dispensable for biotin anti-CD8a, biotin anti-CD11b, biotin anti-CD11c, biotin anti–Gr-1, normal B cell development. To determine whether GC B cell biotin anti-IgD, biotin anti-CD138, Alexa Fluor 700 anti-B220, Alexa numbers were changed in the absence of caspase-9, we immu- Fluor 647 anti-IgG1, PerCP/Cy5.5 anti–GL-7, and PE/Cy7 anti-CD38, fol- 2 2 nized WT and B/Casp9 / mice with NP-KLH. Surprisingly, we lowed by staining with allophycocyanin/Fire-750 streptavidin (BioLegend). + + 2/2 Cells were then labeled with Alexa Fluor 350 Annexin V conjugate (Thermo found that NP IgG1 GC B cells were decreased in B/Casp9 Fisher Scientific) at room temperature for 15 min. Cells were analyzed by mice despite impaired caspase-9–dependent signaling (Fig. 2C). The Journal of Immunology 3

FIGURE 1. GC B cells are prone to caspase-9 signaling. (A and B) Intracellular staining of active caspase-9 and caspase-3 in (A) B220+CD23hiIgD+IgMlo naive B cells, IgD–IgM–CD11b–CD11c–Gr-1–CD4– CD8a–CD138– (DUMP–)B220+IgG1+GL-7+CD38– GC B cells, and DUMP–B220+IgG1+GL-7–CD38+ memory B cells in unimmunized mice and (B) NP- specific GC and memory B cells in mice immu- nized with NP-KLH. Cells were cultured in vitro for 0 h (gray background) or 4 h (black line). Percentages of naive, GC, and memory B cells with active caspase-9 or caspase-3 are also shown (lower panels). Data are representative of three indepen- dent experiments. **p , 0.01 (n = 3). Downloaded from

These data suggest that caspase-9 is not required for B cell de- absence of caspase-9. We found that B/Casp92/2 mice produce velopment but is important for maintaining normal GC B cell fewer Ag-specific ASCs by ELISPOT (Fig. 3B). Consistently, http://www.jimmunol.org/ numbers after immunization. Ag-specific CD138+ plasma cells were reduced in B/Casp92/2 We next measured Ab production in the absence of caspase-9. mice (Fig. 3C). Taken together, our data indicate that caspase-9 Consistent with a decrease in NP-specific GC B cells, B/Casp92/2 deficiency leads to a decrease in GC B cells, resulting in reduced mice exhibited a decrease in the production of NP-specific Abs 2 wk Ab response after immunization. after immunization with NP-KLH, including the production of high-affinity and total anti-NP IgG1 (Fig. 3A). Because class- Decreased apoptosis in GC B cells in the absence of caspase-9 switched Ab-secreting cells (ASCs) are generated by the GC re- To determine whether the caspase signaling cascade is indeed absent action (49), we next examined the numbers of these cells in the in caspase-9–deficient B cells, we purified and cultured B cells from by guest on October 4, 2021

FIGURE 2. GC B cells are decreased in the absence of caspase-9. (A) CD19-cre–me- diated excision of the Casp9 gene to generate a conditional knockout mouse model. Western blot of purified splenic B cell extracts from WT and B/Casp92/2 mice. (B) Flow cytometric analysis of DUMP–B220–CD23+IgMhiIgDlo T1, DUMP–B220+CD23–IgMhiIgDhi T2, and DUMP–B220+CD23+IgMloIgDhi ma- ture naive (M) B cells in the spleens of unimmunized mice. Bar charts show the number of T1, T2, and M B cells per 106 spleen cells. (C) Flow cytometric analysis of DUMP–B220+IgG1+NP+GL-7+CD38– GC B cells in the spleens of B/Casp92/2 or WT mice 2 wk after immunization. Bar charts show the number of NP-specific GC B cells per 106 spleen cells. Data in (B and C)are representative of three independent experi- ments with three to four mice per group as indicated. ns, statistically NS. **p , 0.01. 4 INHIBITION OF NECROPTOSIS BY CASPASE-9 IN B CELLS

FIGURE 3. Impaired primary Ab response in the absence of caspase-9. (A) ELISA for high-affinity

(NP5-BSA) anti-NP IgG1 and total (NP25-BSA) anti- NP IgG1 relative titers at 2 wk postimmunization. (B) ELISPOT for NP-specific IgG1 ASCs at 2 wk post- immunization. Bar charts show the numbers of ASCs 6 per 10 spleen cells. (C) Flow cytometric analysis of Downloaded from DUMP–B220+CD138+ IgG1+ NP+ plasma and plas- mablast cells in the spleen at 2 wk postimmunization. Data are representative of three independent experi- ments with three to five mice per group as indicated. *p , 0.05, **p , 0.01. http://www.jimmunol.org/ by guest on October 4, 2021

WT and B/Casp92/2 mice with reduced serum to induce cell decreased apoptosis under the condition of serum starvation death in vitro according to our previous protocol (46). We conditions in vitro. detected active caspase-9 and caspase-3 in WT B cells but not in in caspase-9–deficient B cells (Fig. 4A). Moreover, cas- Caspase-9–deficient B cells display decreased cleavage pase-9–deficient GC B cells showed less apoptotic activities of Ripk1 compared with WT controls as indicated by cleavage of DEVD Caspase-8 can inhibit necroptosis by cleavage of Ripk1s and (Fig. 4B). These data indicate that caspase-9 deficiency leads to caspase-8 deficiency leads to increased necroptotic cell death

FIGURE 4. Caspase-9 deficiency leads to decreased apoptosis in GC B cells. (A) Western blot analysis of apoptosis-related proteins in purified WT and Casp92/2 (knockout [KO]) B cells after 3 d of stimu- lation and culture in low serum for 0, 8, and 16 h. Data are representative of three inde- pendent experiments. (B)Flowcytometric analysis of DUMP–B220+IgG1+GL-7+CD38– GC B cells stained by DEVD-FITC after 9 h of low serum culture. Quantification of the percentage of cells positive for DEVD cleavage in WT and Casp92/2 GC B cells after3,6,or9hofculture.Dataarerep- resentative of three independent experi- ments with three mice per group. The Journal of Immunology 5

(50, 51). We therefore tested whether caspase-9 could similarly WT and caspase-9–deficient B cells (Fig. 5C, 5D). After Fas suppress necroptosis by examining necroptotic signaling mole- cross-linking, the activation of caspase-8 and caspase-3 was cules (35). We detected the cleaved and inactive form of Ripk1 in similar between WT and caspase-9–deficient B cells (Fig. 5E), WT B cells but not in caspase-9–deficient B cells after 8–16 h of suggesting that Fas-mediated apoptosis is normal in caspase- in vitro culture (Fig. 5A). Moreover, full-length Ripk1 was 9–deficient B cells. Together, our data indicate that caspase-9– higher in caspase-9–deficient B cells after in vitro culture deficient B cells display defects in intrinsic apoptosis but are (Fig. 5A), indicating that necroptosis is more active in the ab- normal Fas-mediated apoptosis. Caspase-9 deficiency does not sence of caspase-9. cause the activation of caspase-1–dependent pyroptosis in Caspase-1, an inflammatory caspase, has been shown to trigger B cells but decreases the cleavage of Ripk1, leading to increased pyroptosis, a form of inflammatory cell death mediated by cleavage Ripk1 levels. of gasdermin D (52–54). We determined whether pyroptosis might be responsible for reduced GC B cell numbers in the absence of Increased necroptosis signaling in the absence of caspase-9 caspase-9. However, we did not detect the activation of caspase-1 TNF-induced necroptosis regulated by Ripk3 is the primary or cleavage of gasdermin D in caspase-9–deficient B cells or WT pathway for necroptosis (26, 29). It has been shown that TNF-a controls following the induction of spontaneous cell death during can induce necroptosis in the presence of caspase inhibitors in vitro culture (Fig. 5B). These results indicate that pyroptosis (57, 58). We found that TNF-a could induce cell death in WT does not serves as an alternate cell death pathway in caspase-9– B cells in the presence of a caspase-8 inhibitor, Z-IETD deficient B cells. (Fig. 6A). Interestingly, caspase-9–deficient B cells under- It has been established that B cells are susceptible to Fas- went increased cell death (Fig. 6A). Moreover, a necroptosis dependent killing by Fas ligand–expressing T cells (48, 55, inhibitor, necrostatin-1 (59), significantly inhibited the cell Downloaded from 56). Because caspase-8 and caspase-9 are the initiator caspases death in caspase-9–deficient B cells (Fig. 6A), indicating cell in the extrinsic and intrinsic apoptosis pathways, respectively, death in caspase-9–deficient B cells involve necroptosis. Consis- caspase-9 deficiency is unlikely to affect caspase-8–dependent tently, we found that phosphorylation of Ripk3 was increased in extrinsic apoptosis mediated by Fas. We therefore determined caspase-9–deficient B cells compared with WT B cells (Fig. 6B), whether Fas-mediated activation of caspase-8 signaling is af- indicating an increase in necroptosis signaling. These results sug- fected in caspase-9–deficient B cells. We observed that Fas ex- gest that B cells display increased necroptosis in the absence of http://www.jimmunol.org/ pression and Fas-mediated cell death were comparable between caspase-9. by guest on October 4, 2021

FIGURE 5. Necroptosis signaling in Casp92/2 B cells. (A) Western blot for necroptosis-related proteins in purified WT and Casp92/2 (knockout [KO]) B cells. Relative levels of cleaved Ripk1 were analyzed by quantification of the density of the protein bands with National Institutes of Health ImageJ software. The ratios of intensities of cleaved Ripk1 versus b-Actin were as follows: 0.20 (0 h WT), 0.05 (0 h KO), 0.54 (8 h WT), 0.09 (8 h KO), 0.79 (16 h WT), and 0.09 (16 h KO); and full- length Ripk1 versus b-Actin: 0.79 (0 h WT), 0.82 (0 h KO), 0.27 (8 h WT), 0.44 (8 h KO), 0.22 (16 h WT), and 0.43 (16 h KO). (B) Western blot for pyroptosis-related proteins in samples as in (A). **Degraded or nonspecific band. (C) WT and Casp92/2 B cells were stained with FITC–anti-Fas (dark line) and analyzed by flow cytometry. Gray line, unstained control. (D) Induction of cell death in WT and Casp92/2 B cells after cross-linking with anti-Fas. ns, statistically NS (n = 3). (E) Western blot for caspase-8 and caspase-3 in WT and Casp92/2 B cells after different time of Fas cross-linking. Western blot data are representative of two independent experiments. 6 INHIBITION OF NECROPTOSIS BY CASPASE-9 IN B CELLS

FIGURE 6. Necroptosis in Casp92/2 B cells. (A) Quantification of the percentage of cell death in WT and Casp92/2 (knockout [KO]) B cells after incubation with Z-IETD and TNF-a with or without necrostatin-1 (Nec-1) for 20 h. Data are representative of two in- dependent experiments with three mice per group. **p , 0.01. (B) Western blot analysis of necroptosis- related proteins in WT and Casp92/2 (KO) B cells after 3 d of stimulation and treated with Z-IETD and TNF-a for 4 h. Data are representative of two inde- pendent experiments.

Deletion of Ripk3 rescues Ag-specific caspase-9–deficient GC numbers of NP-specific ASCs were also rescued in Ripk32/2B/ B cells Casp92/2 mice compared with B/Casp92/2 mice (Fig. 8B). + 2/2 The above data suggest that decreased cleavage of Ripk1 in cas- Consistent with these results, NP-specific IgG1 Casp9 GC pase-9–deficient B cells results in an increase in full-length Ripk1 B cells were also rescued by the deletion of Ripk3 (Fig. 8C). These Downloaded from compared with WT controls (Fig. 5A). Moreover, necroptosis is results indicate that caspase-9 plays a protective role for GC elevated in caspase-9–deficient B cells as shown by increased B cells by limiting necroptotic cell death. Our results reveal an phosphorylation of Ripk3 (Fig. 6B). It has been shown that nec- important role for caspase-9 in maintaining a balance between roptosis can be triggered in caspase-8–deficient T cells to com- apoptosis and necroptosis to protect the homeostasis of GC B cells pensate for impaired apoptosis (40). We investigated whether in Ab responses (Fig. 8D). necroptosis might contribute to the decreased numbers of caspase- http://www.jimmunol.org/ 9–deficient GC B cells in vivo. Because Ripk3 is a key mediator of Discussion necroptosis (29, 30), we crossed B/Casp92/2 mice with Ripk32/2 In our study, we found that GC B cell numbers are decreased in mice to generate mice with double knockout (DKO) of caspase-9 caspase-9–deficient mice despite a defect in apoptosis. We ob- and Ripk3 in B cells. We first determined whether Ripk3 affects served that the cleaved inactive form of the necroptosis mediator GC B cell apoptosis. We found that Ripk32/2 and WT GC B cells Ripk1 was decreased in caspase-9–deficient B cells. Moreover, showed similar levels of cell death after serum starvation in vitro caspase-9–deficient B cells display increased potential for nec- (Fig. 7). Moreover, GC B cells from B/Casp92/2 and DKO mice roptosis signaling. By comparison, we did not detect a significant also showed similar levels of reduction in cell death (Fig. 7). cleavage of the pyroptosis signaling molecules, including caspase-1 This indicates that GC B cell apoptosis after serum starvation and gasdermin D in WT and caspase-9–deficient B cells. Consistent by guest on October 4, 2021 in vitro is affected by caspase-9 but not Ripk3. We then immu- with increased necroptosis, deletion of an essential necroptosis nized B/Casp92/2, Ripk32/2, DKO mice, and WT controls with gene, Ripk3, rescued GC B cell numbers and Ab production in NP-KLH and examined anti-NP GC B cell numbers and primary B/Casp92/2 mice. Our findings suggest that caspase-9 pro- Ab responses. We found that deletion of Ripk3 rescued anti-NP motes apoptosis and inhibits necroptosis to maintain the ho- IgG1 Abs in Ripk32/2B/Casp92/2 mice (Fig. 8A). Moreover, the meostasis of GC B cells.

FIGURE 7. Deletion of Ripk3 has no effect on GC B cell apoptosis. Flow cytometric analysis of DUMP–B220+GL- 7+CD38– GC B cells stained by DEVD-FITC and Annexin V after 6 h of low serum culture. Quantifi- cation of the percentage of DEVD+Annexin V+ cells in WT, B/Casp92/2, Ripk32/2, and DKO GC B cells after 0, 3, 6, or 9 h of culture. Data are representative of two independent experiments with three mice per group. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ 2/2 FIGURE 8. Deletion of Ripk3 rescues Ag-specific Casp9 GC B cells. (A) ELISA for high-affinity (NP5-BSA) anti-NP IgG1 and total (NP25-BSA) anti-NP IgG1 relative titer in WT, B/Casp92/2, Ripk32/2, and DKO mice 2 wk after immunization with NP-KLH. (B) ELISPOT for NP-specific IgG1 ASCs at 2 wk postimmunization with NP-KLH. (C) Flow cytometric analysis of DUMP–B220+IgG1+NP+GL-7+ CD38– GC B cells in the spleen of WT, B/Casp92/2, Ripk32/2, and DKO mice at 2 wk postimmunization with NP-KLH. (D) Schematic mode of GC B cell death pathway. In response to cy- tochrome c release from mitochondria, caspase-9 cleaves executioner caspases to drive apoptosis while also inhibiting Ripk1/Ripk3 signaling to block necroptosis. In the absence of caspase-9, GC B cells shift from apoptotic to necroptotic cell death. Data in this figure were analyzed by Dunnett test from three independent experiments with three or more mice per group. *p , 0.05, **p , 0.01, ***p , 0.001. by guest on October 4, 2021 GC B cells can undergo apoptosis through either the cell intrinsic shown to play an important role in the suppression of necroptosis or extrinsic pathways. After engagement by Fas ligand expressed (23, 51). Our results suggest that caspase-9 also plays an important by activated T cells, Fas on GC B cells can trigger the extrinsic role in limiting necroptosis in GC B cells. apoptosis pathway through activation of caspase-8 (48, 55, 56, 60). Programmed cell death plays an essential role in the regulation of We found that the expression of Fas, as well as Fas-mediated GC responses. This may help to prevent the entry of deleterious GC caspase-8 activation and apoptosis, were normal in caspase-9– B cell clones generated from somatic hypermutation into the long- deficient B cells (Fig. 7C–E). This suggests that caspase-8–de- lived memory B cell and plasma cell pools. We have observed that pendent extrinsic apoptosis of B cells is not affected by the loss of GC B cells rapidly induce caspase-9 signaling during in vitro caspase-9. Caspase-9 that mediates the intrinsic pathway has been culture. However, the contributions of apoptotic and non- implicated in BCR-induced apoptosis (18). Therefore, caspase-9 apoptotic cell death in the regulation of GC B cell homeostasis is may be required for BCR-induced intrinsic apoptosis but not Fas- not clear. This study suggests that caspase-9 not only helps to delete mediated extrinsic apoptosis. GC B cells by apoptosis but also prevents excessive necroptosis to In addition to their long-established roles as mediators of the maintain the proper numbers of GC B cells for Ab responses. Our proapoptotic proteolytic cascade, caspases have more recently been data reveal an important role for caspase-9 in maintaining a balance recognized for other functions. Caspase-9 has been shown to inhibit between apoptosis and necroptosis to protect GC B cell survival type I IFN signaling, a mechanism of antiviral immunity that results and Ab production. in inflammatory cell death (43). Caspase-8 has been demonstrated to negatively regulate necroptosis in T cells, with its absence Acknowledgments resulting in decreased T cell numbers, a phenotype that is rescued We thank Min Li, Wei Liu, and Francis Chan for technical support, Vishva by the ablation of Ripk3 (40). Interestingly, however, GC B cell– Dixit for Ripk32/2 mice, and Xiulong Xu for discussions. specific deletion of Fas or caspase-8 has been shown to cause GC B cell accumulation (60, 61). This suggests that the extrinsic and intrinsic apoptotic caspases have distinct, nonredundant functions Disclosures in GC B cell homeostasis. The authors have no financial conflicts of interest. Necroptosis is a programmed form of necrosis that triggers inflammation (35). It may be favorable for a virus-infected cell to References die by necroptosis, both to eliminate the viral reservoirs and to 1. De Silva, N. S., and U. Klein. 2015. Dynamics of B cells in germinal centres. provoke a host antiviral immune response. However, excessive Nat. Rev. Immunol. 15: 137–148. 2. Stebegg, M., S. D. Kumar, A. Silva-Cayetano, V. R. Fonseca, M. A. Linterman, inflammation may be detrimental to the host. Apoptosis is an and L. Graca. 2018. Regulation of the germinal center response. Front. Immunol. immunologically silent form of cell death. Caspase-8 has been 9: 2469. 8 INHIBITION OF NECROPTOSIS BY CASPASE-9 IN B CELLS

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