Abstract
B cell activation by Tfh cells, i.e., through CD154 engagement of CD40 and IL-21, and survival within GCs are crucial for the T-dependent Ab response. LUBAC, composed of HOIP, SHARPIN, and HOIL-1, catalyzes linear ubiquitination (Linear M1-Ub) to mediate NF-κB activation and cell survival induced by TNF receptor superfamily members, which include CD40. As shown in this study, B cells expressing the Sharpin null mutation cpdm (Sharpincpdm) could undergo proliferation, CSR, and SHM in response to immunization by a T-dependent Ag, but were defective in survival within GCs, enrichment of a mutation enhancing the BCR affinity, and production of specific Abs. Sharpincpdm B cells stimulated in vitro with CD154 displayed normal proliferation and differentiation, marginally impaired NF-κB activation and survival, but markedly exacerbated death triggered by IL-21. While activating the mitochondria-dependent apoptosis pathway in both Sharpin+/+ and Sharpincpdm B cells, IL-21 induced Sharpincpdm B cells to undergo sustained activation of caspase 9 and caspase 8 of the mitochondria-dependent and independent pathway, respectively, and ultimately caspase 3 in effecting apoptosis. These were associated with loss of the caspase 8 inhibitor cFLIP and reduction in cFLIP Linear M1-Ub, which interferes with cFLIP poly-ubiquitination at Lys48 and degradation. Finally, the viability of Sharpincpdm B cells was rescued by caspase inhibitors but virtually abrogated – together with Linear M1-Ub and cFLIP levels – by a small molecule HOIP inhibitor. Thus, LUBAC controls the cFLIP expression and inhibits the effects of caspase 8 and IL-21-activated caspase 9, thereby suppressing apoptosis of CD40 and IL-21-activated B cells and promoting GC B cell survival.
Highlights
B lymphocytes are central to humoral immunity, as they differentiate into “effector” cells that secrete class-switched (IgG, IgA, and IgE) and high-affinity mature antibodies (Abs) or “helper” cells that produce regulatory cytokines [1,2,3]
We further addressed the B cell-intrinsic role of SHANK-associated RH domain interacting protein (SHARPIN) in mediating the germinal center (GC) B cell survival and Ab responses to a T-dependent Ag in vivo by generating mice with B cellspecific expression of Sharpincpdm (B-Sharpincpdm) and mice in which Sharpincpdm B cells directly competed against wildtype B cells within the same GC environment
Upon injection with alum-mixed NPCGG, which elicits a T-dependent Ab response, B-Sharpincpdm mice showed severe impairment in the GC development in the spleen and the output of FAShiGL-7hi GC B cells and CD138+ plasma cells despite normal GC and total B cell proliferation (Figures 1C, D and Supplementary Figure 1B), culminating in decreased formation of Ab secreting cells (ASCs) that produced NP-binding IgM and IgG1 Abs as well as much reduced titers of NP-specific IgM and high-affinity (NP7-binding) IgG1, IgG2b, IgG3 and IgA (Figures 1E, F) – total NP-specific (NP34-binding) IgG1 titers were reduced to similar extents (Figure 1G)
Summary
B lymphocytes are central to humoral immunity, as they differentiate into “effector” cells that secrete class-switched (IgG, IgA, and IgE) and high-affinity mature antibodies (Abs) or “helper” cells that produce regulatory cytokines [1,2,3]. IL-21 synergizes with CD154 to induce AID, an activated B cell-restricted cytidine deaminase that introduces DNA lesions in the immunoglobulin (Ig) genes to initiate class switch DNA recombination (CSR) and somatic hypermutation (SHM), which underpin Ab class-switching (e.g., from IgM to different IgG isotypes) and affinity maturation, respectively [2, 13]. Th2 cytokine IL-4 synergizes with CD154 to activate NF-AT to enhance B cell proliferation as well as induce AID expression and IgH Ig1-Cg1 and Ie-Ce germline transcription to initiate CSR to IgG1 and IgE [2, 14]
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