Key developmental transitions in human germinal center B cells are revealed by differential CD45RB expression

Abstract

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...

Chronic airway inflammation provides a unique environment for B cell activation and antibody production

“…In addition, while we found no differences in the frequencies of memory B cells (CD19 + IgD neg CD27 + CD38 neg ), the frequency of naïve B cells (CD19 + IgD + CD27 neg ) was significantly higher in tonsils (p<0.0001; Figure 1B). We next characterized the frequencies of highly activated B cell subsets, including GC B cells (CD19 + IgD neg CD38 + ) [14] and plasmablasts (CD19 + IgD neg CD27 + CD38 hi ). Interestingly, while the frequency of GC B cells was significantly higher in tonsils (p<0.0001), the frequency of plasmablasts was significantly higher in NP (p<0.0001; Figure 1C).…”

“…14 RNA quality was assessed using the Agilent Bioanalyzer 2100 system, and only samples with a RIN >7 were used for cDNA synthesis and qRT-PCR. The following primer pairs were used 15, 16, 17 : aicdaF: 5’-agaggcgtgacagtgctaca-3’, aicdaR: 5’-tgtagcggaggaagagcaat-3’, g1F: 5’-ccagggcagggtcagca-3’, g1R: 5’-ggtgctcttggaggaggg-3’, g4F: 5’-ccagggcagggtcagca-3’, g4R: 5’-ggcagcccagggcg-3’, a1F: 5’-ctcagcactgcgggccctcca-3’, a1R: 5’-gttcccatctggctgggtgctgca-3’, a2F: 5’-ctcagcactgcgggccctcca-3’, a2R: 5’-gttcccatcttggggggtgctgtc-3’, eF: 5’-ggccacacatccacaggc-3’, eR: 5’-ggggtgaagtccctggagc-3’, GAPDHF: 5’-gaaggtgaaggtcggagtc-3’, GAPDHR: 5’-gaafatggtgatgggatttc-3’.…”

“…RNA and cDNA were isolated from tissue samples as previously described [14]. RNA quality was assessed using the Agilent Bioanalyzer 2100 system, and only samples with a RIN > 7 were used for cDNA synthesis and qRT-PCR.…”

KSHV infects a subset of human tonsillar B cells, driving proliferation and plasmablast differentiation

“…In contrast to our findings with Ki67 and IgM, both the level and likelihood of CD27 expression were approximately the same regardless of the number of LANA dots/cell, indicating that CD27 expression did not correlate with intracellular viral load. Of note however, between 7% and 36% of LANA + cells were CD27 + , a finding that mirrors the phenotype of KSHV-infected B cells in MCD (48).…”

The myeloma stem cell concept, revisited: from phenomenology to operational terms

“…The very early B-cell precursors develop into pro-and pre-B cells before they migrate as immature B cells into the blood to reach peripheral lymphoid organs as naive B cells. [24][25][26][27][28][29][30][31] Germinal and post-germinal-center centrocytes, centroblasts, memory cells, plasmablasts, and end-stage plasma cells (PCs) are included in the later stages of the mature B-cell differentiation hierarchy. Most malignant B-cell lymphomas, chronic lymphoblastic leukemias, and MMs are considered to originate from these cells following analyses of the somatic hypermutation and class switch-recombination status of the gene encoding the immunoglobulin heavy chain (IgH) which defines the hierarchical status of any clonotypic cell.…”