Abstract
B cells play a major role in the antibody-mediated rejection (AMR) of solid organ transplants, a major public health concern. The germinal center (GC) is involved in the generation of donor-specific antibody-producing plasma cells and memory B cells, which are often poorly controlled by current treatments. Myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the B-cell lymphoma-2 family, is essential for maintenance of the GC reaction and B-cell differentiation. During chronic AMR (cAMR), tertiary lymphoid structures resembling GCs appear in the rejected organ, suggesting local lymphoid neogenesis. We report the infiltration of the kidneys with B cells expressing Mcl-1 in patients with cAMR. We modulated GC viability by impairing B-cell receptor signaling, by spleen tyrosine kinase (SYK) inhibition. SYK inhibition lowers viability and Mcl-1 protein levels in Burkitt’s lymphoma cell lines. This downregulation of Mcl-1 is coordinated at the transcriptional level, possibly by signal transducer and activator of transcription 3 (STAT3), as shown by (1) the impaired translocation of STAT3 to the nucleus following SYK inhibition, and (2) the lower levels of Mcl-1 transcription upon STAT3 inhibition. Mcl-1 overproduction prevented cells from entering apoptosis following SYK inhibition. In vitro studies with primary tonsillar B cells confirmed that SYK inhibition impaired cell survival and decreased Mcl-1 protein levels. It also impaired B-cell activation and immunoglobulin G secretion by tonsillar B cells. These findings suggest that the SYK–Mcl-1 pathway could be targeted, to improve graft survival by manipulating the humoral immune response.
Highlights
B cells play a major role in acute and chronic antibody-mediated rejection (AMR) and allograft survival
Anti-Myeloid cell leukemia-1 (Mcl-1) (S-19; Santa Cruz Biotechnology Cat# sc-819 RRID:AB_2144105), anti-B-cell lymphoma-2 (Bcl-2) (C-2; Santa Cruz Biotechnology Cat# sc-819 RRID:AB_2144105), anti-Bcl-xS/L (S-18; Santa Cruz Biotechnology Cat# sc-819 RRID:AB_2144105), anti-GAPDH (Sigma-Aldrich Cat# G9545 RRID:AB_796208), and anti-PARP (Cell Signaling Technology Cat# 9542 9542S, 9542L, 9542P RRID:AB_2160739) antibodies were used for immunoblotting and anti-hCD19 (HIB19; BD Biosciences Cat# 555409 RRID:AB_395809), anti-hMcl-1 (Sigma-Aldrich Cat# HPA008455 RRID:AB_1079334), and anti-signal transducer and activator of transcription 3 (STAT3) (K-15; Santa Cruz Biotechnology Cat# sc-483 RRID:AB_632441) Abs were used for immunohistology
According to the mature B-cell (BM) 1-BM5 classification [expression of surface IgD and CD38 [31]], we determined Mcl-1 expression profiles for different B-cell populations based on the expression of sIgD and CD38: sIgD+CD38−/low cells are naïve B cells that are undifferentiated or in the early stages of differentiation (BM1-BM2), sIgD+CD38high cells are pre-Germinal centers (GC) cells (BM2’), sIgD−CD38high cells are GC cells (BM3 + BM4), and sIgD−CD38−/ low cells are terminally differentiated B cells
Summary
B cells play a major role in acute and chronic antibody-mediated rejection (AMR) and allograft survival. Targeting SYK-Mcl-1 in Germinal Center B-Cells [5, 6], proteasome inhibitors [4, 7], and complement inhibitors [8, 9] These treatments frequently fail to control AMR, donor-specific antibody (DSA) production, and memory B-cell formation. Following T cell-dependent B-cell activation and GC formation, B cells undergo clonal expansion, isotype class switching, somatic hypermutation [16,17,18], and affinity maturation and selection [19,20,21]. They leave the GC as highly specific long-lived memory B-cells and antibody (Ab)-producing plasma cells
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