Simultaneous assessment of rotavirus-specific memory B cells and serological memory after B cell depletion therapy with rituximab

Abstract

The mechanisms that contribute to the maintenance of serological memory are still unclear. Rotavirus (RV) memory B cells (mBc) are enriched in IgM(+) and CD27- subpopulations, which are associated with autoimmune diseases pathogenesis. In patients with autoimmune diseases treated with Rituximab (RTX), some autoantibodies (auto-Abs) decrease after treatment, but other auto-Abs and pathogen-specific IgG Abs remain unchanged. Thus, maintenance of autoimmune and pathogen-specific serological memory may depend on the type of antigen and/or Ab isotype evaluated. Antigen-specific mBc and antigen-specific Abs of different isotypes have not been simultaneously assessed in patients after RTX treatment. To study the relationship between mBc subpopulations and serological memory we characterized total, RV- and tetanus toxoid (TT)-specific mBc by flow cytometry in patients with autoimmune diseases before and after treatment with RTX. We also measured total, RV- and TT-Abs, and some auto-Abs by kinetic nephelometry, ELISA, and EliA tests, respectively. Minor differences were observed between the relative frequencies of RV-mBc in healthy controls and patients with autoimmune disease. After RTX treatment, naïve Bc and total, RV- and TT-specific mBc [IgM(+), switched (IgA(+)/IgG(+)), IgM(+) only, IgD(+) only, and CD27- (IgA(+)/IgG(+)/IgM(+))] were significantly diminished. An important decrease in total plasma IgM and minor decreases in total IgG and IgA levels were also observed. IgM rheumatoid factor, IgG anti-CCP, and IgG anti-dsDNA were significantly diminished. In contrast, RV-IgA, RV-IgG and RV-IgG1, and TT-IgG titers remained stable. In conclusion, in patients with autoimmunity, serological memory against RV and TT seem to be maintained by long-lived plasma cells, unaffected by RTX, and an important proportion of total IgM and serological memory against some auto-antigens seem to be maintained by short-lived plasma cells, dependent on mBc precursors depleted by RTX.

Figure 1. RV-mBc are enriched in the IgM^hiIgD^low, IgM^lowIgD^hi, and IgM⁺ only mBc subsets.

Summary of the frequencies of seven subsets of total and RV-mBc assessed by multiparametric flow cytometry. All p values reported are 2-tailed (p<0.05, Wilcoxon test). Lines and error bars denote the median and interquartile range, respectively. A. Healthy volunteers (n = 10). B. Patients (n = 10) with autoimmune diseases before RTX treatment.

Figure 2. Comparison of selected total B cell subpopulations among the study groups.

Healthy volunteers (HV) (n = 10), patients before RTX treatment (prior-RTX) (n = 10) and patients after RTX treatment (after-RTX) (n = 10). For total CD19⁺ B cells, the dashed line represents the clinical depletion limit after RTX treatment (less than 5,000 CD19⁺ cells/mL). The dotted lines represent the estimated flow cytometry detection limit of 3.5 CD19⁺ B cells/mL. Solid lines and error bars denote the median and interquartile range, respectively. Differences between HV and patients prior-RTX were evaluated with Mann–Whitney tests and between patients prior-RTX and patients after-RTX with Wilcoxon tests. All p values reported are 2-tailed.

Figure 3. Comparison of RV-specific B cell subpopulations among the study groups.

Solid lines and error bars denote the median and interquartile range, respectively. Differences between HV (n = 10) and patients prior-RTX (n = 10) were evaluated with Mann–Whitney tests and between patients prior-RTX and patients after-RTX (n = 10) with Wilcoxon tests. All p values reported are 2-tailed.

Figure 4. Comparison of total immunoglobulins in plasma among the study groups.

Levels of total IgA (A), IgG (B) and IgM (C) in plasma determined by kinetic nephelometry. The dotted lines represent the lower and upper values of the normal reference range (IgA: 82–453 mg/dL; IgG: 751–1560 mg/dL; IgM: 46–304 mg/dL). Solid lines and error bars denote the median and interquartile range, respectively. Differences between HV (n = 10) and patients prior-RTX (n = 14) were evaluated with Mann–Whitney tests and between patients prior-RTX and patients after-RTX (n = 14) with Wilcoxon tests. All p values reported are 2-tailed.

Figure 5. Comparison of RF, anti-CCP and anti-dsDNA autoantibodies levels in plasma among the study groups.

Levels of rheumatoid factor (RF) determined by kinetic nephelometry (A), anti-cyclic citrullinated peptide (anti-CCP) (B) and anti-doubled stranded DNA (anti-dsDNA) (C) determined by a fluorescence enzyme immunoassay (EliA test) in patients diagnosed with RA (A and B) (n = 11) or with SLE (C) (n = 7) (some patients had both diagnoses). Solid lines and error bars denote the median and interquartile range, respectively. The dotted lines correspond to the clinical limits below which a sample is considered negative according to the technique used for its detection (RF: <20 IU/mL, anti-CCP: <7 U/mL and anti-dsDNA: <10 IU/mL). Differences between HV and patients prior-RTX were evaluated with Mann–Whitney tests and between patients prior-RTX and patients after-RTX with Wilcoxon tests. All p values reported are 2-tailed.

Figure 6. Comparison of RV- and TT-specific immunoglobulins titers in plasma among the study groups.

Titers of TT-IgG (A), RV-IgA (B), RV-IgG (C) and RV-IgG₁ (D) in plasma determined by ELISA. Solid lines and error bars denote the median and interquartile range, respectively. Differences between HV (n = 10) and patients prior-RTX (n = 14) were evaluated with Mann–Whitney tests and between patients prior-RTX and patients after-RTX (n = 14) with Wilcoxon tests.