We previously reported that RO ؉ expression correlated with increased mutation, activation, and selection among human germinal center (GC) B cells. Here, we subdivided human tonsillar B cells, including IgD ؊ CD38 ؉ GC B cells, into different fractions based on RB expression. Although each subset contained RB ؉ cells, when used as an intrasubset marker, differential RB expression effectively discriminated between phenotypically distinct cells. For example, RB ؉ GC B cells were enriched for activated cells with lower AID expression. RB inversely correlated with mutation frequency, demonstrating a key difference between RB-and RO-expressing GC B cells. Reduced RB expression during the transition from pre-GC (IgM ؉ IgD ؉ CD38 ؉ CD27 ؊ ) to GCB cells was followed by a dramatic increase during the GC-to-plasmablast (IgD ؊ CD38 ؉؉ CD27 ؉ ) and memory (IgD ؊ CD38 ؊ CD27 ؉ ) transition. Interestingly, RB ؉ GC B cells showed increased signs of terminal differentiation toward CD27 ؉ post-GC early plasmablast (increased CD38 and RO) or early memory IntroductionImmune function is uncompromisingly governed by at least 3 interdependent principles including recognition, effector function, and transition. Early naive B and T cells that successfully bind foreign antigen become activated and undergo developmental transition toward more mature, faster responding memory subsets. In contrast, lymphocytes that bind self-antigen in the periphery are usually counterselected against and undergo a different type of transition, one toward apoptosis or anergy. Ineffectual negative selection against self-specific B cells has been associated with the development of autoimmune and neoplastic diseases. Unfortunately, potential factors responsible for the redirection or abrogation of transitions toward cell death and anergy have not been conclusively determined. Antibody sequence analyses revealed that B cells participating in autoreactive responses are often mutated and show signs of selection for self-antigen. 1,2 This suggests that somatic hypermutation (SHM) during proliferation and activationbased transitions in the dynamic germinal center reaction may play a role (whether direct or indirect) in disease pathogenesis.We therefore sought to develop an expedient approach that would allow for the reproducible identification of B-cell populations that exist at different transitional stages based on their states of activation, SHM-dependent diversification, and developmental progression toward adjacent downstream subsets. Delineating how surface marker profiles change as B cells transition between key stages (especially when immunoregulatory markers such as CD45 protein tyrosine phosphatase are included in such profiles) should increase our understanding of B-cell development and ultimately how to circumvent dysregulated processes that could lead to disease.Over the past 3 decades, our research laboratory has investigated multiple aspects of human B-cell development and the molecular processes that collectively shape the peripheral BCR repertoi...

To date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD(-)CD38(+)) were subdivided into 3 surface CD45RO fractions: RO(-), RO(+/-), and RO(+). We show here that the average number of mutations per IgV(H) transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO(+) GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO(+) GC B cells were negative for Annexin V, comprised mostly (93%) of CD77(-) centrocytes, and were enriched for CD69(+) cells. Collectively, RO(+) GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker.

We previously showed that IgH sequence alone minimally influenced germinal centre (GC) B‐cell survival fate. As end‐stage effector B cells are typically more mutated than founder GC B cells, we worked to develop an assay that would enrich for populations of GC B cells with progressively increasing numbers of somatic mutations, which could potentially be used as an indicator of positive selection. We targeted CD45 as it has been shown to influence activation‐induced cytidine deaminase (AID) expression. In this study, anti‐CD77 and anti‐CD45RO (RO) were used to subdivide CD19+IgD−CD38+CD77+ centroblasts (CB) and CD19+IgD−CD38+CD77− centrocytes (CC) into three contiguous RO fractions (RO−, RO+/− and RO+) and assessed whether mutation frequency and characteristics associated with selection varied with respect to increasing RO expression. Here, we show that the average number of mutations per IgVH4 transcript increased concordantly with RO for CC, but not for CB. CC also exhibited an RO‐associated increase in replacement mutations. Comparative analysis of clonally related sequences revealed that increased mutations were not due to the exclusive persistence of surface RO on highly mutated cells. RO‐expressing CC and CB pools showed increased signs of activation (CD69+) and were enriched for surface Ig+ cells. BCR‐crosslinking induced a significant increase in surface RO on total tonsillar and GC B cells, which collectively suggests that the RO‐associated increase in mutations is attributable, at least in part, to the cycling of cells that may have recently undergone BCR‐mediated selection, or are potentially in developmental transition between CC and CB stages.