Abstract
The transcriptional repressor Bcl6 is a master regulator of the germinal center (GC) reaction through directing naïve B cells and CD4+ T cells to differentiate into GC B cells and follicular T helper (TFH) cells respectively. Bcl6 mediates its action largely by recruitment of co-repressors through its N-terminal BTB domain and its middle second repression domain (RD2). The BTB domain repression function is critical for GC B cell survival and proliferation, but not important for TFH cell differentiation. However, the in vivobiological function of RD2 remains unknown.
To explore the specific role of RD2 transcriptional repression in the GC reaction, we generated a knockin mouse model in which the endogenous Bcl6 locus encodes a mutant form of the protein that specifically disrupts RD2 mediated transcriptional repression. RD2 mutant mice were developmentally indistinguishable from wild-type mice and displayed normal B cell development prior to the GC phase. However, these mice failed to accumulate GCs after immunization with sheep blood cells and exhibited remarkably impaired production of high-affinity antibodies 21 days after T-cell dependent antigen immunization, indicative of severe deficiency of the GC reaction. Mixed bone marrow transplantation experiments showed that RD2 loss of function led to complete loss of GC B cells and partial impairment of TFH cell differentiation in cell-intrinsic manner. Intravital imaging analysis indicated that RD2-deficent antigen-engaged B cells migrate normally to the inter-follicular zone of lymph nodes and interacted normally with cognate T helper cells. To further understand the nature of the functional defect of RD2 mutant B-cells, hen egg lysosome (HEL)-specific RD2-deficient GFP B cells and wild type RFP B cells (with the ratio 1:1) were transferred together with non-fluorescent ovalbumin (OVA)-specific T cells into SMARTA hosts, which were then immunized at the footpad with HEL-OVA two days later. On day 5 after immunization, draining popliteal lymph nodes were harvested and subjected for immunofluorescence histology analysis. At this time point, wild-type RFP B cells have started to cluster into tiny GC, whereas RD2-deficient GFP B cells did not form GCs. Moreover, wild-type B cells in the follicular interior were predominantly Bcl6hi, a characteristic of pre-GC B cells, suggesting that they could serve as a source of GC B cells. By contrast, RD2-deficient GFP B cells were primarily extra-follicular, and infrequently observed in the follicle interior. Most importantly, these cells were typically Bcl6lo, demonstrating that RD2 repression function is essential for pre-GC B cell differentiation.
BCL6 knockout mice display a lethal inflammatory phenotype due to aberrant T-cell and macrophage activation. In striking contrast, RD2-deficient mice experienced normal healthy lives with no inflammation, and had nearly normal inflammation cytokine production in B cells and macrophages as well as differentiation of Th1,Th2 and Th17 subtypes. Hence the RD2 repression domain is specifically involved in humoral immunity but has minimal participation in the anti-inflammatory functions of BCL6. Instead we observed that the BCL6 zing finger domain plays the key role in anti-inflammatory functions in macrophages, and through ChIP-competition assays show that this is mediated by directly competing with STATs for binding to chemokine genes.
These results highlight an essential role of RD2-mediated transcriptional repression in pre-GC B cell development specifically at the early B-cell activation phase. This is different than mice with BCL6 BTB mutations where early activation is normal and the defect occurs later on in the proliferative phase of GCs. The data suggest a surprising development and cellular context-specific biochemical functions of Bcl6 governing each distinct phase of the humoral immune response and inflammation.
Disclosures:
No relevant conflicts of interest to declare.
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