Characterization of rotavirus specific B cells and their relation with serological memory

Abstract

We quantified circulating total, rotavirus (RV) and Tetanus toxin (TT) memory B cells (mBc) in healthy adults using a limiting dilution assay (LDA) and a flow cytometry assay (FCA) that permit evaluation of both CD27+ and CD27- mBc. RV mBc were enriched in the CD27-, IgG+ and in the CD27+, IgM+ subsets. The numbers of RV mBc were higher by FCA than by LDA and results of the two assays did not correlate. TT IgGmBc and RV IgA mBc determined by FCA and by LDA correlated with TT plasma IgG and RV plasma IgA, respectively. The mean ratio of specific mBc/mug/ml of the corresponding plasma immunoglobulin was lower for TT IgG than for RV IgA mBc. Our studies contribute to understand the relationship between circulating mBc and serological memory, and enhance our capacity to develop better correlates of protection against RV disease.

Figure 1

(a) Higher or comparable numbers of RV IgM/IgG/IgA mBc were detected in CpG+IL-2 stimulated PBMC than with other stimulation methods. PBMCs (empty symbols) or purified B cells (filled symbols) were polyclonally stimulated with CpG+IL-2 (circles), CpG+SAC+PWM (triangles), CpG+IL-2+ autologous CD4+ T cells (diamonds) or CpG+IL-2/6/10 +NIH/3T3 murine fibroblasts as feeder cells (stars). Specific mBc precursors were detected by ELISA after 10 days of culture. Bars represent the median. * Statistical differences by Mann Whitney test p<0.05. (b) Representative LDA experiments to determine total, TT and RV-specific mBc in PBMC polyclonally stimulated with CpG+IL-2, as described in Materials and Methods. mBc frequencies were calculated from the 37% intercept. Correlation coefficients (R²) were > 0.75 and p < 0.05, except for the TT IgA mBc that were below the sensitivity of the assay.

Figure 2

Frequencies of mBc were determined by LDA and FCA (analyzing CD27+ and CD 27− mBc subsets together), as described in Figs 1 and 3 respectively. (a) Total IgM, IgG and IgA (n= 11-14). (b) TT and (c) RV-specific IgM, IgG and IgA (n= 8-10). Median values are represented by a line. *: Significant difference (Wilcoxon test, p< 0.03).

Figure 3

Representative FCA dot plots of TT and RV-specific mBc from one volunteer. Purified B cells were gated on CD20+/CD27+ and on CD20+/CD27− subsets (left dot plot). On these gates we performed: (a) Analysis of the expression of IgM+, IgG+, IgA+ on CD20+/CD27+ mBc. (b) Analysis of the expression of IgM (naïve), IgG+ and IgA+ (mBc) on CD20+/CD27− naïve B cells and mBc. The first left dot plots in each row are controls stained with the anti-IgG antibody, and only streptavidin as a control for TT-biotin, and no antigen in the case of RV VLPs. Numbers in each quadrant are the percentage of CD20+, CD27+ or CD27− B cells.

Figure 4

Frequencies (%) of mBc subsets considering (a) total mBc; (b) total and RV CD27−, IgG+ and IgA+ mBc and (c) CD27+ mBc. *: significant differences (Wilcoxon Test, p<0.05). Median values are represented by a line.

Figure 5

Correlations between plasma antibody concentrations and frequency of mBc. (a) Significant correlations were found between TT-specific IgG or RV-specific IgA mBc determined by LDA and their corresponding TT-specific IgG, and RV-specific IgA levels in plasma (n=8-10, Spearman test, p<0.01). (b) Significant correlations were found between plasma antibody levels and the frequency of CD27+ mBc detected by FCA for TT-specific IgG and RV-specific IgA (n= 8, Spearman test, p<0.04). After Bonferroni correction for multiple comparisons the correlations between LDA, but not FCA mBc, are still significant (p<0.01).