Abstract

Abstract Lifelong humoral immunity depends on the longevity of long-lived plasma cells (LLPC). We have shown that CD28 signaling in LLPC is critical to their survival but the molecular mechanisms are unclear. Here, we report that CD28 mediates a pro-survival response through autophagy in multiple myeloma (MM). As the transformed LLPC, MM is similar to LLPC in CD28 signaling and survival regulation. CD28 in MM is activated by antibody crosslinking or co-culture with dendritic cells that express CD80/CD86. With chemotherapy (melphalan) treatment or serum starvation, CD28 activation increases MM viability from 30% to 70%. Pharmacologic inhibition of autophagy (Baf-A1 or 3-MA) or ATG5 knockdown, a critical autophagy protein, abrogates the CD28 pro-survival effects. CD28 activation up-regulates the autophagy marker LC3II as well as increases autophagosome numbers assessed by Cyto-ID. Conversely, knockdown of CD28 decreases these effects. CD28 signals via GRB2-VAV-PLC in LLPC, and we found that blocking PKC activity in MM by inhibitor Gö6983 diminishes CD28 pro-survival effects and CD28-mediated LC3II up-regulation. Upon activation of CD28, ATG5 protein levels are elevated not through transcriptional up-regulation but enhanced by ATG5 protein modification. Taken together, these data suggest CD28 signaling via GRB2-VAV-PLC-PKC induces autophagy by ATG5 induction. CD28 regulates metabolic fitness in T cells, and we also found inhibition of autophagy by 3-MA minimizes CD28-mediated increase of oxidative phosphorylation. This indicates that CD28-mediated autophagy maintains metabolic homeostasis to improve survival. Our findings suggest a novel mechanism by which CD28 signaling regulates metabolism, survival and function in LLPC.

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