SummaryGerminal center (GC) B cells cycle between the dark zone (DZ) and light zone (LZ) during antibody affinity maturation. Whether this movement is necessary for GC function has not been tested. Here we show that CXCR4-deficient GC B cells, which are restricted to the LZ, are gradually outcompeted by WT cells indicating an essential role for DZ access. Remarkably, the transition between DZ centroblast and LZ centrocyte phenotypes occurred independently of positioning. However, CXCR4-deficient cells carried fewer mutations and were overrepresented in the CD73+ memory compartment. These findings are consistent with a model where GC B cells change from DZ to LZ phenotype according to a timed cellular program but suggest that spatial separation of DZ cells facilitates more effective rounds of mutation and selection. Finally, we identify a network of DZ CXCL12-expressing reticular cells that likely support DZ functions.

T follicular helper (TFH) cells are the prototypic helper T cell subset specialized to enable B cells to form germinal centers and produce high-affinity antibodies. We found that miRNA expression by T cells was essential for TFH cell differentiation. More specifically, we show that after protein immunization the microRNA cluster miR-17~92 was critical for robust TFH cell differentiation and function in a cell-intrinsic manner that occurred regardless of changes in proliferation. In a viral infection model, miR-17~92 restrained the expression of TFH subset-inappropriate genes, including the direct target RAR-related orphan receptor alpha (Rora). Genetically removing one Rora allele partially rescued the inappropriate gene signature in miR-17~92-deficient TFH cells. Our results identify the miR-17~92 cluster as a critical regulator of T cell-dependent antibody responses, TFH cell differentiation and the fidelity of the TFH cell gene expression program.

The seminal discovery by Eisen that antibodies undergo improvements in antigen-binding affinity over the course of an immune response led to a long running search for the underlying mechanism. Germinal centers in lymphoid organs are now recognized to be critically involved in this phenomenon, known as antibody affinity maturation. As well as improving in affinity for specific epitopes, some antibody responses maintain or even increase their breadth of antigen-recognition over time. This has led to another intense line of research aimed at understanding how broadly neutralizing anti-pathogen responses are generated. Recent work indicates that germinal centers also play an important role in the diversification process. We discuss current understanding of how germinal centers are programmed to support both affinity maturation and antibody diversification.