The role of autophagy in plasma cells is unknown. Here we found notable autophagic activity in both differentiating and long-lived plasma cells and investigated its function through the use of mice with conditional deficiency in the essential autophagic molecule Atg5 in B cells. Atg5(-/-) differentiating plasma cells had a larger endoplasmic reticulum (ER) and more ER stress signaling than did their wild-type counterparts, which led to higher expression of the transcriptional repressor Blimp-1 and immunoglobulins and more antibody secretion. The enhanced immunoglobulin synthesis was associated with less intracellular ATP and more death of mutant plasma cells, which identified an unsuspected autophagy-dependent cytoprotective trade-off between immunoglobulin synthesis and viability. In vivo, mice with conditional deficiency in Atg5 in B cells had defective antibody responses, complete selection in the bone marrow for plasma cells that escaped Atg5 deletion and fewer antigen-specific long-lived bone marrow plasma cells than did wild-type mice, despite having normal germinal center responses. Thus, autophagy is specifically required for plasma cell homeostasis and long-lived humoral immunity.

IntroductionMultiple myeloma (MM) is a frequent and still incurable plasma cell malignancy, causing 2% of all cancer deaths. In recent years, treatment of MM has improved remarkably. For example, the proteasome inhibitor (PI) bortezomib (PS-341) proved effective even in the context of heavily pretreated, relapsed, and refractory MM, 1-3 although more than 50% of patients fail to respond to second-line treatment. 4 The molecular bases of different individual responsiveness to bortezomib remain unclear. Age (Ͻ 65 years) and extent of bone marrow plasma cell infiltration (Ͻ 50%) are the conventional factors for successful treatment identified so far. [5][6][7] Identifying the molecular bases underlying PI sensitivity would provide the framework for their improved clinical application.Bortezomib targets the proteasome, a 2.4-MDa multicatalytic protease complex ubiquitously expressed in eukaryotic cells. 1,8 Crucial for degrading proteins involved in cell cycle, angiogenesis, adhesion, cytokine production, and apoptosis, 3,9,10 proteasome inhibition can affect tumor cell growth via direct and indirect mechanisms (eg, by blocking interactions with endothelial and bone cells). 8,11 Proteasomes also dismantle damaged and misfolded/unfolded proteins, which are potentially harmful for the cell. 8 As a result, proteasome impairment causes buildup of polyubiquitinated proteins and eventual cell death. 3 Proteasomes also degrade a significant proportion of newly synthesized proteins in mammalian cells (rapidly degraded polypeptides [RDPs]). 12 Thus, increased protein synthesis or other metabolic unbalances could increase proteasome workload.We recently showed that plasma cell differentiation in vitro, ex vivo, and in vivo entails a dramatic decrease in proteasome expression and activity, correlating with increased sensitivity to PIs. 13,14 Indeed, PIs reduce antibody (Ab) responses in vivo. 14,15 Moreover, inducible expression of orphan Ig-chains sensitizes nonlymphoid tumor cells to PI-induced toxicity. 13 In MM cells (MMCs), the levels of both Ig synthesis and retention correlate with apoptotic sensitivity to PIs, and manipulating Ig synthesis alters sensitivity. 16,17 Altogether, these data suggest that the exquisite sensitivity of certain MMCs to PIs could stem from decreased proteasomal capacity, increased proteasomal workload, or both (ie, an adverse load-versus-capacity ratio).In this study, we exploited MM lines with differential apoptotic sensitivity to PIs to address if proteasome expression and degradative workload vary among different clones, and defined their role in The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 9, 2018. by guest www.bloodjournal.org From determining apoptotic sensitivity to PIs. Moreover, using primary patient-derived MMCs, we revealed ...

Macroautophagy/autophagy is a cellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane organelles called autophagosomes. Lipidation of ubiquitin-like LC3/ GABARAP proteins on the autophagosome membrane is important for autophagy. The cysteine protease ATG4 executes 2 LC3/GABARAP processing events: priming of newly synthesized pro-LC3/ GABARAP to enable subsequent lipidation, and delipidation/deconjugation of lipidated LC3/ GABARAP (the exact purpose of which is unclear in mammals). Four ATG4 isoforms (ATG4A to ATG4D) exist in mammals; however, the functional redundancy of these proteins in cells is poorly understood. Here we show that human HAP1 and HeLa cells lacking ATG4B exhibit a severe but incomplete defect in LC3/GABARAP processing and autophagy. By further genetic depletion of ATG4 isoforms using CRISPR-Cas9 and siRNA we uncover that ATG4A, ATG4C and ATGD all contribute to residual priming activity, which is sufficient to enable lipidation of endogenous GABARAPL1 on autophagic structures. We also demonstrate that expressing high levels of pre-primed LC3B in ATG4deficient cells can rescue a defect in autophagic degradation of the cargo receptor SQSTM1/p62, suggesting that delipidation by human ATG4 is not essential for autophagosome formation and fusion with lysosomes. Overall, our study provides a comprehensive characterization of ATG4 isoform function during autophagy in human cells.