Modulation of the T cell response to β-lactoglobulin by conjugation with carboxymethyl dextran

Abstract

We have previously prepared β-lactoglobulin (β-LG)-carboxymethyl dextran (CMD) conjugates with water-soluble carbodiimide and achieved reduced immunogenicity of β-LG. In the present study, to elucidate the mechanism for the reduced immunogenicity of β-LG, we investigated changes in the T cell response to β-LG after conjugation with CMDs differing in molecular weight (about 40 and 162 kDa). Lymph node cells from BALB/c, C3H/He, and C57BL/6 mice that had been immunized with β-LG or the conjugates were stimulated with β-LG, and the in vivo T cell response was then evaluated by BrdU (5-bromo-2′-deoxyuridine) ELISA as the ex vivo proliferative response. T cells from the conjugate-immunized mice showed a lower proliferative response than those from the β-LG-immunized mice. T cell epitope scanning, using synthesized peptides, showed that the T cell epitope profiles of the conjugates were similar to those of β-LG, whereas the proliferative response to each epitope was reduced. These results indicate that the lower in vivo T cell response with the conjugates was not due to induction of conjugate-specific T cells, but due to a decrease in the number of β-LG-specific T cells. After the lymph node cells from β-LG-immunized mice had been stimulated with β-LG or the conjugates, the efficiency of the antigen presentation of the conjugate to β-LG-specific T cells was evaluated by BrdU ELISA as the in vitro proliferative response. The antigen presentation of β-LG to the T cells was reduced by conjugation with CMD. In addition, conjugation with CMD enhanced the resistance of β-LG to cathepsin B and cathepsin D, which suggest that conjugation with CMD inhibited the degradation of β-LG by proteases in APC and led to suppression of the generation of antigenic peptides including T cell epitopes from β-LG. It is therefore considered that the suppressive effect on the generation of T cell epitopes reduced the antigen presentation of the conjugates and that this reduction led to a decrease in the number of β-LG-specific T cells in vivo. As a result, the decreased help to B cells by T cells would have reduced the antibody response to β-LG. We conclude that suppression of the generation of T cell epitopes by conjugation with CMD is important to the mechanism for the reduced immunogenicity of β-LG.