The Use of Novel Antibodies to Characterize B cells in Health and Disease

CD38, a glycoprotein ectoenzyme found on the surface of majority of hematopoietic cells, can exist in monomeric or multimeric conformations and enzymatic activity correlates with multimerization. During human B cell differentiation, CD38 expression levels are low on naïve and memory B cells, intermediate on transitional cells, and high on antibody-secreting plasmablasts and plasma cells. Conventional anti-CD38 (C-CD38) antibodies, recognize all CD38 formations and are not selective for identification of plasmablasts, thus requiring the use of additional antibodies including CD27. In contrast, a novel sea lamprey derived monoclonal VLRB antibody, tetVLRB MM3 (V-CD38), recognizes an epitope likely located within the catalytic site of active multimeric CD38 and therefore might be more specific to plasmablasts, which express active CD38. Characterize the B cells recognized by V-CD38 in the peripheral blood of individuals with or without inborn errors of immunity (IEI). B cell subsets were characterized as described in figure 1 using conventional antibodies to CD3, CD19, IgM, CD27, C-CD38, and by V-CD38 generated by a collaborator. Results were presented as means with standard deviations (SD). Correlations were calculated using the Pearson’s Correlation Coefficient formula. To validate our identification of plasmablasts with V-CD38, samples from patients with X-linked agammaglobulinemia lacking B cells, and tonsils rich in plasmablasts were used as negative and positive controls, respectively. Analysis of samples from 16 individuals revealed nearly identical (r = 0.99, p <.00001) proportions of CD27hi plasmablasts recognized by V-CD38 (0.51%+/-0.74%) and C-CD38 (0.49%+/-0.70%), suggesting that all cells expressed multimeric CD38. In contrast, only 45.28% ±16.19% of transitional IgMhighCD27-C-CD38int B cells were also recognized by V-CD38, (2.52%+/–3.62% and 1.15%+/–1.75%, respectively), a finding that was consistent across samples (r = 0.98, p <.00001). Preliminary studies in three IEI patients demonstrated markedly reduced plasmablasts (0.035%+/–0.026%), class-switched memory B cells (1.25% +/–1.52%) and transitional cells (1.25%+/–1.06%) compared to healthy controls (0.59%+/–0.74%, 15.29%+/–8.95%, and 2.81%+/–3.96% respectively).Download : Download high-res image (343KB)Download : Download full-size imageFigure 1. (abstract: 92). Gating strategy to identify CD3-CD19+CD27hiC-CD38hiV-CD38+ Plasmablasts and CD3-CD19+IgMhiC-CD38intV-CD38+ Transitional Cells. A representative example of the plasmablast and transitional cells gating strategy is shown. Plasmablasts and transitional B cells were gated from the singlets, viable, CD3 negative CD19 positive cells. Plasmablasts were gated as CD27hiC-CD38hi or CD27hiV-CD38+ (middle left). Transitional cells were gated as IgMhiC-CD38int or IgMhiV-CD38+. Left-hand side arrows point to populations of C-CD38 and V-CD38 plasmablasts gated on CD27/V-CD38 or CD27/C-CD38 graphs, respectively, demonstrating overlap of gated cells. Right-hand side arrows point to populations of C-CD38 and V-CD38 transitional cells gated on IgM/V-CD38 or IgM/C-CD38 graphs, respectively, demonstrating partial overlap of gated cells. The bottom middle panels depict staining with VLRB B7, a monoclonal antibody against the spike protein of SARS-CoV-2 as a negative control. Figure 1. (abstract: 92). Gating strategy to identify CD3-CD19+CD27hiC-CD38hiV-CD38+ Plasmablasts and CD3-CD19+IgMhiC-CD38intV-CD38+ Transitional Cells. A representative example of the plasmablast and transitional cells gating strategy is shown. Plasmablasts and transitional B cells were gated from the singlets, viable, CD3 negative CD19 positive cells. Plasmablasts were gated as CD27hiC-CD38hi or CD27hiV-CD38+ (middle left). Transitional cells were gated as IgMhiC-CD38int or IgMhiV-CD38+. Left-hand side arrows point to populations of C-CD38 and V-CD38 plasmablasts gated on CD27/V-CD38 or CD27/C-CD38 graphs, respectively, demonstrating overlap of gated cells. Right-hand side arrows point to populations of C-CD38 and V-CD38 transitional cells gated on IgM/V-CD38 or IgM/C-CD38 graphs, respectively, demonstrating partial overlap of gated cells. The bottom middle panels depict staining with VLRB B7, a monoclonal antibody against the spike protein of SARS-CoV-2 as a negative control. While C-CD38 and V-CD38 antibodies can identify plasmablasts in peripheral blood, V-CD38 allows more selective detection of CD38 on plasmablasts. Some circulating transitional cells also express multimeric CD38. Additional studies will determine whether V-CD38 can better identify patients suffering from abnormal plasmablast formation and impaired production of antibodies.