Effects Of High Dose Heat Shock Protein Gp96 And Its Terminal Fragments On CTL Response Mediated By Regulatory T Cells

Previous studies showed that heat shock protein gp96, when complexed with virus- or tumor-derived antigens, induced specific T cells responses and humoral immune responses in immunized mice, but higher amounts (100μg)of gp96 were detrimental to its adjuvant effect. The mechanisms are not fully understood. Since an intact gp96 was obtained from liver of mice by three step method.The amino(N)-terminal fragments of gp96, which was believed to contain the putative peptide-binding domain,were cloned and expressed in E.coli. BALB/c mice were vaccinated with an H-2Kd restricted epitope HBcAg87-95 and gp96 or its N-terminal fragments as adjuvant. Mice were sacrificed 7 days after the third immunization. Splenocytes were dispersed with a syringe plunger. We found that immunization of gp96 simultaneously stimulated both CTLs and Treg activities were detected by ELISA, ELISPOT, Tetramer, In this study, we monitored the activities of T cells and activation of regulatory T cells (Treg) populations upon immunization of different doses of gp96 as adjuvant. In vitro suppression assays. CD4+CD25- or CD8+ T cells that were stimulated with T-cell expander, cultured alone or with Treg. Its activation of CTLs at low dose was far more pronounced than Treg, but with increasing dose of gp96, amplification of Treg both in number and suppressive function eventually abrogated induced T cell responses by itself. Activating Treg by gp96 may be partly independent in CTL activation as its terminal fragment N355 was not as effective in activation of Treg as in activation of CTL. Low-dose of cyclophosphamide treatment could restore T cell responses by suppression of Treg. We further addressed the clinical relevance of the finding by examining gp96 expression were detected by immunoperoxidase in chronic severe hepatitis B (CSHB) patients with vigorous CTL activation in liver. Our results provide new insights into the mechanisms of gp96 mediated balance between regulatory and responder T cells.Since gp96 based vaccines have been widely used in rodent models, as well as clinical trials, better understanding of its immunomodulatory role will help to design more effective strategies for treatment of cancer and infectious diseases.