Human rotavirus-specific IgM Memory B cells have differential cloning efficiencies and switch capacities and play a role in antiviral immunity in vivo

Abstract

Protective immunity to rotavirus (RV) is primarily mediated by antibodies produced by RV-specific memory B cells (RV-mBc). Of note, most of these cells express IgM, but the function of this subset is poorly understood. Here, using limiting dilution assays of highly sort-purified human IgM(+) mBc, we found that 62% and 21% of total (non-antigen-specific) IgM(+) and RV-IgM(+) mBc, respectively, switched in vitro to IgG production after polyclonal stimulation. Moreover, in these assays, the median cloning efficiencies of total IgM(+) (17%) and RV-IgM(+) (7%) mBc were lower than those of the corresponding switched (IgG(+) IgA(+)) total (34%) and RV-mBc (17%), leading to an underestimate of their actual frequency. In order to evaluate the in vivo role of IgM(+) RV-mBc in antiviral immunity, NOD/Shi-scid interleukin-2 receptor-deficient (IL-2Rγ(null)) immunodeficient mice were adoptively transferred highly purified human IgM(+) mBc and infected with virulent murine rotavirus. These mice developed high titers of serum human RV-IgM and IgG and had significantly lower levels than control mice of both antigenemia and viremia. Finally, we determined that human RV-IgM(+) mBc are phenotypically diverse and significantly enriched in the IgM(hi) IgD(low) subset. Thus, RV-IgM(+) mBc are heterogeneous, occur more frequently than estimated by traditional limiting dilution analysis, have the capacity to switch Ig class in vitro as well as in vivo, and can mediate systemic antiviral immunity.

Fig 1

Sort-purified total and RV IgM⁺ mBc switch isotype to IgG in vitro. (A) Starting from rosette-enriched B cells, naive, IgM⁺, and switched mBc were sort purified based on their expression of CD19, CD27, IgG, and IgA. Results of 1 representative experiment of 14 performed experiments is shown. Serial dilutions (24 replicate wells per dilution) of purified naive, IgM⁺ mBc, and switched mBc were stimulated as described in Materials and Methods and cultured for 7 days. Total and RV-specific IgM, IgG, and IgA were measured in the supernatants by ELISA. (B, left panel) Number of total IgM-, IgG-, and IgA-secreting B cells in the indicated cultures. (Right panel) Number of RV-specific IgM-, IgG-, and IgA-secreting B cells in the indicated cultures. The lines represent the medians. An asterisk indicates a statistically significant difference (P ≤ 0.03, Wilcoxon test). (C) Frequencies of total, RV, and tetanus toxoid (TT) IgM⁺ mBc that switched isotype to IgG in vitro. The medians and ranges are shown. An asterisk indicates a statistical differences (P ≤ 0.02, Mann-Whitney test). ns, nonsignificant difference.

Fig 2

IgM⁺ mBc have lower cloning efficiencies than switched mBc. (A) Cloning efficiencies of total and RV IgM⁺ mBc and switched mBc. Total mBc cloning efficiencies were calculated based on the percentage of the numbers of mBc obtained in the LDA. The cloning efficiency of RV-mBc was calculated as the fraction of the cells used for the sorting experiments stained with GFP-VLP (shown in panel B). Lines represent the medians. One and two asterisks represent statistically significant differences in the frequency of mBc between IgM⁺ mBc and switched mBc (P = 0.04 and 0.02, respectively; Wilcoxon test). (C) The expression of plasmablast markers in naive, IgM⁺ mBc, and switched mBc after 7 days of culture was evaluated by FC. One representative experiment of seven performed is shown. (D) Frequencies of total IgM, IgG, and IgA ASCs generated after culture of each indicated subset, as detected by two-color ELISPOT. Medians and ranges of nine experiments are shown. The asterisk indicates a statistically significant difference (P = 0.01, Mann-Whitney test).

Fig 3

Total and RV IgM⁺ mBc switch to IgG isotype frequencies in vivo. (A) Immunodeficient NOD/Shi-scid IL-2Rγ^null adult mice were i.p. inoculated with PBS (□, dashed line), PBMCs (△, continuous line), Bc-dPBMCs (▽, dashed line), or Bc-dPBMCs plus highly purified IgM⁺ mBc (♢, continuous line) and immediately infected orally with the murine EC_WT strain RV. Fifteen days after infection, relative quantities of human total, RV-specific, and tetanus toxoid (TT)-specific (as a control Ag) IgM, IgG, and IgA were determined in the serum by ELISA. Optical densities (at 450 nm) of serial dilutions of serum are presented on a log₂ scale. Each point represents the median (n = 4), and the lines represent the range for each dilution. An asterisk represents a statistically significant difference (P < 0.05, Mann-Whitney test). (B) Human IgM⁺ mBc are involved in clearing RV antigenemia. Relative quantities are shown for RV VP6 detected by ELISA (optical density at 450 nm [OD₄₅₀]) in the serum of mice that were transferred human cells (or PBS as a control). The bars represent the medians, and lines represent the ranges (n = 4). An asterisk indicates a significant difference (P = 0.02, Mann-Whitney test).

Fig 4

Transferred human B cells do not affect RV shedding in the stool. (A) Little or no human total (upper panel) or RV IgM, RV IgG, or RV IgA (lower panel) was detected in feces of immunodeficient mice after transfer. Relative quantities of total and RV IgM, RV IgG, and RV IgA were detected by ELISA in fecal samples collected 1 to 21 days postinfection in the four indicated groups of mice. (B) Relative quantities of RV VP6 detected by ELISA in fecal samples collected 1 to 21 days postinfection in the four indicated groups of mice. Immunodeficient NOD/Shi-scid IL-2Rγ^null mice were intraperitoneally inoculated with PBS (□, dashed line), PBMCs (△, continuous line), Bc-dPBMCs (▽, dashed line), or B-dPBMCs plus highly purified IgM⁺ mBc (♢, continuous line) and immediately infected orally with the murine RV strain EC_WT. The median (n = 4) for each data point is presented.

Fig 5

IgM⁺ mBc are heterogeneous, and RV-specific mBc are enriched in the IgM^hi IgD^low subset. Rosette-enriched human B cells, like those used for the sorting experiments, were stained with GFP-VLPs, anti-CD20, anti-CD27, anti-IgD, and anti-IgM. (A) B cells (CD20⁺) and mBc (CD20⁺ CD27⁺), total and RV-specific cells (GFP-VLP⁺) were gated, and expression levels of IgD and IgM were analyzed for each subpopulation. One of five experiments performed is shown. (B) Summary of the frequencies (as percentages) of the five subsets of CD27⁺ total and RV-mBc: IgM⁻ IgD⁺, IgM⁺ IgD⁻, IgM⁻ IgD⁻, IgM^hi IgD^low, and IgM^low IgD^hi. An asterisk indicates a significant difference (P < 0.05, Wilcoxon test). Median values are represented by the lines.