SUMMARY AND CONCLUSIONS

Transcription

1 SUMMARY AND CONCLUSIONS

2 Summary and Conclusions This study has attempted to document the following effects of targeting protein antigens to the macrophage scavenger receptors by maleylation. 1. Modification of antigenic epitopes due to maleylation Maleylation drastically modifies the B cell epitopes of antigens; the antisera generated by immunization with native antigen are almost entirely non-crossreactive with those generated by maleylated antig~n immunization. The 'new' epitopes appear to be maleylation-restricted to some extent, as antisera from one maleylated protein can recognize other, unrelated maleylated proteins. The T cell epitopes of maleylated proteins remain essentially unaltered by the chemical modification as priming of T cells with maleylated antigens results in an enhanced B cell response to native antigen. The lack of B cell crossreactivity between native and maleylated antigens has enabled us to study the contribution of the overlapping T cell epitopes in generating an antibody response to native antigens. Since, native antigen immunizations in PBS do not induce antibody responses, priming with maleylated antigens followed by a single dose of native antigen in PBS ['cross-priming'] leads to the generation of a significant anti-native antigen B cell response. This antibody response to native antigen, after 'cross-priming' is not due to a minor crossreactivity between maleylated and native antigens at the B cell level as they show antibody responses to a hapten [arsonate] after priming with maleyl-antigen. This suggests clearly that T cell crossreactivity allows maleylated antigens to prime for an antibody response to native antigen. 148

3 Summary and Concl~ions 2. Modulation of quantitative and qualitative immunogenicity Targeting of maleylated antigens to macrophage scavenger receptors enhances immunogenicity at the B cell level. Native antigens without adjuvant, with the exception of TT, are unable to generate any antibody responses in vivo, while maleylated antigens in the absence of adjuvants induce significant antibody responses. There is a likelihood that the antibody response is, at least partially, T-independent as maleylated antigens are polyvalent in nature. This could also explain the enhancement in immunogenicity. However, analysis of the isotypes of the antibodies generated by native and maleylated antigen immunizaton suggest that there is a T cell component in the immune response. In addition, there is a difference in the cytokine profile in vivo of responder T cells from native and maleylated antigen-immune animals. The IgG1/IgG2a ratios are lower in maleylated antigen immunized mice than in native antigen immunized animals indicating a rise in the IFN-y levels in the maleylated antigen immunized animals. It appears therefore that macrophage targeting skews the T cell responses toward a Th-1-like or typel pattern. 3. Maleylation and alum complement each other resulting in enhanced immunogenicity The use of alum as an adjuvant in the 'cross-priming' experiments, only for the priming immunizations, drastically enhances the antibody response to native antigen implying that for effective B cell immunogenicity, it is the T cell epitopes which require adjuvant and 149

4 Summary and Conclusions not the B cell epitopes. Adequate T cell help is critical in the generation of an antibody response. The duration of the B cell response again depends on T cell priming and the use of maleylated antigen on alum for primary immunizations leads to a long lasting antibody response to native antigen. Maleylated proteins adsorbed on alum are more immunogenic than native proteins so adsorbed as they are far superior at recalling in vitro T cell responses than native antigens on alum. This suggests that maleylation and subsequent targeting to the scavenger receptors enhances the adjuvantic potential of alum. 4. Break in immune tolerance due to macrophage scavenger receptor targeting Maleylation of murine self antigens enables targeting of these proteins to macrophages, which are professional APCs. As in the case of foreign proteins, targeting of maleylated self antigens enhances B cell immunogenicity leading to the induction of an antibody response, in the presence or absence of adjuvant. Native self proteins do not generate a B cell response even when administered with adjuvant. There are fairly high levels of IgM in the anti-maleyl antibody response, but there are significant amounts of IgG antibodies as well suggesting that T cell-mediated help is availal?le for mounting a B cell response. T cell proliferation assays demonstrate conclusively that maleylated self antigen-immune mice mount a decent response when recalled in vitro with native self antigen but recall a better response when stimulated with maleylated antigen. 150

5 Summary a11d Co,clusio11s Further studies with purified self antigens show unequivocally that there is a T cell response generated against native self antigens resulting in a break in T cell tolerance to self antigens. The break in tolerance suggests that under normal conditions, tolerance is most probably maintained by anergy and not deletion. Experiments using a scavenger receptor ligand as a competitor in vivo show conclusively that macrophage targeting is essential for inducing B cell immunogenicity and for the break in tolerance. The peptide repertoires between native and maleylated antigens are not dramatically different as a scavenger receptor ligand can act as a competitor in 'Qitrp and block recall responses of maleyl-antigens to maleyl-antigen-immune T cells in T cell proliferation assays. The break in tolerance appears to be due to the generation of a higher ligand density of self peptides rather than the formation of new epitopes.or the unmasking of 'cryptic' epitopes. One target self peptide of MSA which may be partially responsible for the break in tolerance has been identified, by its ability to stimulate maleyl-msa-specific T cells. Tolerance to circulating self proteins is normally maintained by anergy, and the generation of a greater density of self peptides due to enhanced processing/presentation may lead to an abrogation of anergy. Therefore, scavenger receptor-mediated targeting of antigens to macrophages alters the immunological response dramatically and is of consequence in evaluating the role of macrophages in regulating immunological phenomena. 151