It should serve as a valuable tool for future studies, in particular, with respect to the phenotypic transition from GC B cells to plasma cell precursors. Materials and Methods Purification of tonsillar centroblast B cells Human tonsils were obtained as discarded material from routine tonsillectomies with approval of the Institutional Review Boards of AECOM and Montefiore Medical Center in accordance with Helsinki protocols. and duration of IL-21 triggered Jak-STAT3 signaling. In the human locus, CD40L treatment enhanced the ability of STAT3 to upregulate Blimp-1 by removing BCL6, a potent inhibitor of Blimp-1 expression, from a shared BCL6/STAT3 site in intron 3. Thus, IL-21 and CD40L collaborate through at least two distinct mechanisms to synergistically promote Blimp-1 activation and PC differentiation. Introduction A key aspect of the humoral immune response is terminal differentiation of activated B cells into antibody secreting plasma cells (PCs). Although Blimp-1 upregulation is necessary and sufficient for the appearance of functional PCs (1), PC differentiation starts prior to Blimp-1 activation and can give rise to the so-called pre-plasmablast TNFSF8 (Pre-PB) in a Blimp-1-independent manner (2, 3). The pre-PB is a CD138 (Syndecan) Cnegative cell characterized by low level Ig secretion, compromised Pax5 function, and expression of two PC-associated transcription factors, XBP-1 and IRF4 (2, 3). Believed to be developmentally plastic, pre-PB represents a transient and yet important step in PC differentiation. The initial identification and functional characterization of pre-PB took advantage of an elegant Blimp-1 knock-in mouse model (3). In human, existence of the pre-PB has not been rigorously Azaphen (Pipofezine) defined. Nevertheless, in both mouse and human, the term plasmablast Azaphen (Pipofezine) is reserved for the dividing PC precursors that express CD138 (Syndecan) and bone marrow homing receptors, including CD44, VLA-4, and LFA-1 (2, 4). One of the goals of the current study is to better define human pre-PB in molecular terms. In vivo, PCs can be generated through the extra follicular route as well as the GC response (1). While both pathways share a strict requirement for IRF4 and Blimp-1, the GC-associated PC differentiation has additional requirements and is subject to more elaborate control. Presumably, this is due to the fact that only the affinity matured GC B cells can give rise to long-lived PCs as well as memory B cells (1, 2) so that, if dysregulated, these GC offspring will cause more damage to the organism compared to the short-lived extrafollicular antibody response (5). Three major differences exist between the two pathways of PC development. First of all, STAT3 is dispensable for T cell-independent, extrafollicular Ab response but crucial for post-GC differentiation of IgG PCs (6). Nevertheless, the reason for this pathway-specific function of STAT3 Azaphen (Pipofezine) is unknown; the specific stage of PC development that requires STAT3 function is also not defined. Secondly, initiation of PC differentiation within a GC B cell requires the downregulation of BCL6, a transcriptional repressor that inhibits the expression of three critical transcription factors for PC development, e.g., STAT3, IRF4, and Blimp-1 (7C11). This BCL6-imposed barrier for PC differentiation is much lower for the extrafollicular pathways since na?ve B cells have very little BCL6 protein (12). Lastly, unique to the GC-associated PC development is the role played by follicular T helper (Tfh) cells, which regulate all aspects of the GC response (13). Recent multi-photon microscopy studies have suggested that GC B cells compete for limited Tfh help signals within the GC light zone (14, 15). A combination of this cognate B-T interaction and a direct contribution from the follicular dendritic cells (FDCs) (16) presumably provides the cellular basis for positive selection that licenses affinity matured GC B Azaphen (Pipofezine) cells into the long-lived PC pools. Tfh cells provide help to B cells through a variety of molecules.