T-Cell Dependency of T-Independent (TI) Antigens: Antibody Response to Blood Group A-Antigen Requires CD4 T-Cells and CD40/CD40L Interaction

Abstract

Introduction: Polysaccharides are considered TI antigens capable of eliciting antibody secretion without T-cell help. TNP-Ficoll is used as a model TI (type 2) antigen, but detailed mechanisms of B-cell responses remain unclear. We studied a T-cell role in the antibody response to polysaccharides, using a clinically relevant ABO antigen in its natural form. Methods: Mice (4-8 week-old) received three standardized immunizations (days 0- 7- 14) with human blood group-A erythrocyte membranes, TNP-Ficoll or sperm whale myoglobin (SPWM; protein); some groups received first injection in complete Freund's adjuvant then two boosts without adjuvant. For T-cell depletion studies, anti-CD4 (clone GK1.5) or isotype control were injected on day -1-0- +1. To further characterize direct and indirect interaction of CD40/CD40L, CD40L KO mice were injected with anti-CD40 (clone IC10) or isotype control in order to activate or block CD40 on B cells; CD40 KO mice were injected with mouse rCD40-Fc fusion protein to activate T-cells which in turn may activate B-cells through soluble factors. TLR contribution was studied in MyD88 KO. Anti-A antibodies were measured in plasma at day -1-14-21 by hemagglutination and ABO-ELISA, and TNP and SPWM antibodies by ELISA. Results: Natural anti-A antibodies were undetectable on day -1 in all groups, whereas anti-TNP antibody was present in young mice. A-erythrocytes elicited a substantial antibody response, which was abrogated by CD4 T-cell depletion (fig 1), including IgM (fig 2), whereas of SPWM antibodies, only IgG was absent with normal or low IgM and IgA. Antibodies to TNP-Ficoll were comparable in CD4-depleted vs. control mice. Similarly there were no differences in response to secondary A-antigen immunization when CD4 cells were depleted in pre-immunized mice. Anti-A antibodies were significantly reduced in CD40L KO, and further diminished with anti-CD40 antibody; no reduction was seen in MyD88 KO mice. Conclusions: This study clearly demonstrates that antibody response to A-antigen requires CD4 T-cells. Although polysaccharides may not stimulate MHC class II-dependent T-cell help, the presence of activated CD4 T-cells and their interaction with B-cells through CD40-CD40L (fig 2) is critical for B-cells to become antibody secreting cells. These findings have major implications for ABO-incompatible and xenotransplants.[Figure 1][Figure 2]