Subpopulation of CD19 negative B-cells partially contribute to natural and elicited humoral responses against porcine antigens in the non-human primate system

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Abstract 129
Introduction Characterization of the B-cell repertoire responsible for the development of xenogeneic humoral responses has tremendous therapeutic implications to overcome humoral reactions against porcine tissues. In this report we used a novel anti-CD20 monoclonal antibody to study xenogeneic B-cells and show that a subpopulation of CD19 negative B-cells is partially responsible for the production of natural and elicited xenoantibodies to pig tissue.
Materials and methods Eight baboons were used for these studies. In vivo, B-cells were depleted with the use of Rituximab, an anti-CD20 monoclonal antibody. Six baboons were given Rituximab, and the levels of anti-Galα1-3Gal IgM were followed prospectively for one month. From this group, 2 baboons were randomly selected for sensitization studies. Baboons were administered Rituximab followed at 48 h by an intramuscular injection of 1 cm3 of pig red blood cells (pRBCs). Titers of anti-Galα1-3Gal IgM/IgG and B-cells were prospectively followed for a month. These were compared to a control group (n = 2) which were immunized solely with pRBCs. Fixed pig endothelial cell ELISAs were used to measure relative concentrations on anti-Galα1-3Gal IgM antibodies. B-cells were sorted by way of forward- and side- scattering characteristics as well as by two-colored flow cytometry. Flow cytometry markers included: CD19, CD20, sIgG, sIgM, and Via-probe.
Results Levels of anti-Galα1-3Gal IgM decreased in 50% of baboons treated with Rituximab. Anti-Galα1-3Gal IgM titers in this group decreased from an average of 58.01 μg/ml ± 49.42 to an average of 22.33 μg/ml ± 18.21 within a period of 9.7 days (±0.57). Within a 14-day period the titers of anti-Gal α1-3Gal IgM rose but not above baseline levels. Immunization with pRBCs stimulated an elicited anti-Galα1-3Gal IgM and IgG response in both Rituximab treated and control groups. With a raise in the antibody titer there was a concomitant rise in the percent of CD19(−)/IgM(+) and CD19(−)/IgG(+) B-cells. These events occurred despite effectively eliminating circulating CD19(+)/CD20(+) B-cells.
Conclusion A subpopulation of CD19(−)/IgM(+), IgG(+) B-cells partially contributes to humoral responses against xenogeneic tissues. Findings in this study suggest that therapies targeting more than one B-cell antigen may be need to overcome xenogeneic humoral barriers.
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