TLR9 Ligand Induces the Generation of CD20+ Plasmablasts and Plasma Cells from CD27+ Memory B-Cells

Abstract

Plasma cells (PCs) have a heterogeneous phenotype in humans. While bone marrow PCs are CD20−CD138+, tonsil PCs are CD20+CD138+/− and peripheral plasmablasts (PBs) are CD20−CD138−. In vitro, PCs are mainly generated by the activation of CD27+ memory B-cells through transient stimulation of CD40, and their phenotype appears similar to that of bone marrow PCs. While CD20 expression is lost at the plasmablastic stage, CD138 expression appears only at the PC stage. Thus, the CD20+CD138± phenotype of tonsil PCs does not represent an intermediate stage in the differentiation of memory B-cells into PCs. Because it has been previously shown that TLR9 activation was more able than CD40 stimulation to induce the differentiation of IgM+ CD27+ B-cells, we wondered whether TLR9 or CD40 stimulation would induce the same phenotype of PCs. Thus, we compared the differentiation of CD27+ B-cells isolated from either the tonsils or peripheral blood and stimulated with either CD40L-expressing fibroblasts or a TLR9 ligand, CpG oligodeoxynucleotide (CpG ODN). We observed that CpG ODN mainly induced CD27+ B-cell differentiation into CD20+CD38+CD138− PBs and CD20+CD38+CD138± PCs, which appear similar to tonsil PCs. Removal of CpG ODN during differentiation induced a decrease in the CD20+ plasmablastic population, and, conversely, stimulation of CD40L-induced pre-plasmablasts with CpG ODN increased the population of CD20+CD38+ PBs. Analysis of Ig secretion showed that CpG ODN induced increased IgM secretion compared to CD40L. PCs from patients with multiple myeloma, the malignant counterpart of bone marrow PCs, rarely express CD20. We show that CpG ODN did not induce or increase CD20 in nine IgG or IgA myeloma cell lines. These data strongly suggest that CpG ODN mainly targets CD27+ IgM+ B-cells.

Characterization of proposed human B-1 cells reveals pre-plasmablast phenotype

“…However, it is important to note that CD20 1 plasmablasts have been described in tonsils as well as after in vitro differentiation experiments. [14][15][16] Moreover, in agreement with the study of Sushanek et al, 32 we observed low expression of CD20 in the proposed B-1 cells relative to both naive and memory Bcell subsets (as measured by the median fluorescence intensities by flow cytometry; data not shown). The higher expression levels of both CD43 and CD27 and the lower expression levels of CD20 relative to memory B cells (and comparable to plasmablasts, this work, and Verbinnen et al 11 ) indicate a pre-plasmablastic phenotype.…”

“…[14][15][16][17] We therefore further characterized the proposed human B-1 cells and compared them directly with circulating CD20 2 plasmablasts. Spontaneous antibody production of different isotypes as well as the induced production of antigen-specific antibodies after vaccination with a TD antigen did not reveal differences between the proposed B-1 cells and genuine plasmablasts.…”

Enhanced B‐cell differentiation driven by advanced cirrhosis resulting in hyperglobulinemia

“…In fact, other groups reported lipopolysaccharide and CpG‐ODN increase and are significant in cirrhotic patients and murine models . Increased TLR9 expression of memory B cells could enhance sensitivity to CpG‐ODN, a well‐described B‐cell stimulant previously shown to induce CD27 + B‐cell differentiation into plasmablasts after B‐cell receptor cross‐linking or cytokine activation …”

Distinctions among Circulating Antibody-Secreting Cell Populations, Including B-1 Cells, in Human Adult Peripheral Blood

“…Putative pre-plasmablasts in tonsillar tissue were described as CD20 negative and CD38 low expression; however, these B cells were looked for but not found in peripheral blood (39). CD20 + B cells that express a high level of CD38 have been generated following memory B cell culture with a CpG-containing cytokine cocktail, but it is not clear whether such putative pre-plasmablasts occur normally or circulate (40, 46). A minor subpopulation of peripheral blood plasma cells that is CD20+ has been noted in a study post-immunization, but little is known about these cells (38).…”

“…Most prominently, it has been suggested that CD20 + CD27 + CD43 + B-1 cells represent plasmablasts that, like B-1 cells, spontaneously secrete antibody, or represent B cells related to plasmablasts, such as pre-plasmablasts (36). It should be noted that, to date, pre-plasmablasts have been poorly defined, and/or undetectable in peripheral blood (38-40). Overall, the existence of a distinct, circulating B-1 cell population in humans, which is separate from immunoglobulin-secreting pre-plasmablasts/plasmablasts remains controversial.…”