Little is known about how cells regulate and integrate distinct biosynthetic pathways governing differentiation and cell division. For B-lineage cells it is widely accepted that activated cells must complete several rounds of mitosis before yielding antibody-secreting plasma cells. However, we report that marginal zone (MZ) B cells, innate-like naïve B cells known to generate plasma cells rapidly in response to blood-borne bacteria, generate functional plasma cells despite cell cycle arrest. Further, short-term Notch2 blockade in vivo reversed division-independent differentiation potential and decreased transcript abundance for numerous mTORC1-and Myc-regulated genes. Myc loss compromised plasma cell differentiation for MZ B cells, and reciprocally induced ectopic mTORC1 signaling in follicular B cells enabled division-independent differentiation and plasma cell-affiliated gene expression. We conclude that ongoing in situ Notch2/mTORC1 signaling in MZ B cells establishes a unique cellular state that enables rapid division-independent plasma cell differentiation.

Proteasome inhibition is a useful therapeutic strategy against plasma cell disorders in autoimmunity, neoplasias, and transplant rejection. However, how bortezomib induces cell death and the transcription factors that are involved in its execution are not well characterized. In this study, we aim to address the mechanism of action of proteasome inhibitors in plasma cells and in early precursor activated B-cells. We immunized mice with the hapten-protein conjugate nitrophenyl chicken-gamma-globulin (NP-CGG) and validated depletion of plasma cells and germinal center B-cells in vivo. At steady-state, we utilized B220 expression as a surface marker for newly-formed plasma cells and determined that bortezomib primarily depletes newly-formed plasma cells. To gain more mechanistic insight on how bortezomib affects newly-formed plasma cells, we employed an in vitro culture system of murine-derived primary follicular B-cells that were stimulated with CPG, IL-4, and IL5, to promote differentiation into the plasma cell fate. We find that deletion of the ER-stress induced transcription factor CHOP rescued in vitro generated plasma cells from low-dose bortezomib treatment. In addition, we identify the transcription factor p53 as a key inducer of apoptosis in in vitro generated early activated B-cells. Deletion of p53 rescued cells from apoptosis and reduced the number of cells expressing cleaved caspase-3 in response to bortezomib treatment. In conclusion, we have identified two transcription factors that are involved in bortezomib-induced cell death in B-lineage cells. Supported by NIAID Diversity Supplement: R01AI139123

Pre-existing allo-antibodies (allo-Abs), that preclude transplant due to the risk of hyperacute rejection, lead to prolonged wait times and high mortality rates. Current desensitization approaches are ineffective as they do not adequately deplete allo-specific B cells and plasma cells (PCs). We hypothesize that stringent depletion of these cells is required to eliminate pre-existing allo-Abs. We leverage the exquisite ability of CAR T cells to eliminate target cells to desensitize transplant candidates. We constructed CARs targeting murine CD19 or BCMA, which cover the entire B cell-PC continuum. We first evaluated the function of CAR T cells against B cells and PCs in vitro. C57BL/6 mice were sensitized with BALB/c skin grafts. After skin rejection, sensitized mice received total body irradiation followed by treatment with either control T cells, CART-19 T cells, or a combination of CART-19 and CART-BCMA T cells (combo-CART). Allo-Abs, total Ig, and B cells were measured over 13 weeks. Functional desensitization was then assessed by induction of diabetes followed by BALB/c-derived islet cell transplant and glucose were measured to assess graft survival. CD19- and BCMA-targeted CARs effectively depleted primary B cells and PCs in vitro and in vivo. Control and CART-19 T cells were ineffective at desensitizing mice, but combo-CART treatment resulted in significant decrease of allo-Abs. Islet cell grafts succumbed to hyperacute rejection in 80% of control and CART-19 treated mice. However, combo-CART treatment resulted in prolonged graft survival in all mice (mean 35 days, range 16–60). Thus, CAR T cells targeting B cell and PC antigens represent a promising approach to desensitization and could enable lifesaving transplantation.