Preferential Production of Distinct IgG Subclasses in Response to Anterior Chamber Immunization With Allosplenocytes

Abstract

Abstract: : Purpose: A selective deficiency in antibodies that fix complement, specifically IgG2, is a well documented characteristic of ACAID and a potential contributor in the inhibition of delayed–type hypersensitivity. The initial studies establishing this concept, however, assessed ACAID induced with soluble antigen resulting in the preferential stimulation of IgG1. The current study has re–investigated this utilizing genetically–mismatched allogeneic splenocytes to determine whether ACAID induced with cell–associated antigens results in the similar preferential stimulation of IgG1. Methods: Female C57BL/6 (H–2^b) mice were immunized with 10⁶ BALB/c (H–2^d) splenocytes either subcutaneously (SC) or via the anterior chamber (AC), (n=5 per group). Sera were collected weekly from individual mice for 4 weeks post–immunization to determine the kinetics of antigen–specific IgG production and titers of each IgG subclass. Titers were measured using FACS analysis of P815 cells (H–2^d) incubated with recipient sera and compared against naïve sera, then developed with FITC–conjugated IgG isotype–specific antibodies. Standard curves were created using a best–fit nonlinear regression consisting of data points determined by sequential 1:4 dilutions of sera. Titers were defined as the serum dilution at which 33% of P815 cells remained positively stained. Results: Overall production of IgG two weeks post immunization was higher in AC–immunized mice with a titer of 1:563, as compared to SC–immunized mice at 1:461. The highest IgG subclass titers were IgG1 and IgG2b in both AC and SC–immunized mice. However, IgG1 and IgG2b percentages demonstrated significant differences between the groups. Of the total IgG produced in AC–immunized mice, 34% was IgG1 and 38% was IgG2b, while SC–immunized mice produced 21% IgG1 and 65% IgG2b. Conclusions: These data demonstrate that ACAID induced with cell–associated antigen, similar to soluble antigen–induced ACAID, results in the preferential stimulation of IgG1 production, while inhibiting IgG2b production. Higher IgG titers in these mice indicate that ACAID favors a T_H2–mediated response when induced with allogeneic splenocytes. Since T helper cell cytokine milieu plays a major role in the preferential production of certain IgG subclasses, our data suggests the probable up–regulation of IL–4 and/or TGF–ß and down–regulation of IFN–γ.

Keywords: ACAID • immune tolerance/privilege • anterior chamber