Inhibition of allogeneic cytotoxic T cell (CD8+) proliferation via polymer-induced Treg (CD4+) cells.

T cell-mediated immune rejection remains a barrier to successful transplantation. Polymer-based bioengineering of cells may provide an effective means of preventing allorecognition and the proliferation of cytotoxic (CD8+) T lymphocytes (CTL). Using MHC-disparate murine splenocytes modified with succinimidyl valerate activated methoxypoly(ethylene glycol) [SVA-mPEG] polymers, the effects of leukocyte immunocamouflage on CD8+ and CD4+ alloproliferation and T regulatory (Treg) cell induction were assessed in a mixed lymphocyte reaction (MLR) model. Polymer-grafting effectively camouflaged multiple leukocyte markers (MHC class I and II, TCR and CD3) essential for effective allorecognition. Consequent to the polymer-induced immunocamouflage of the cell membrane, both CD8+ and CD4+ T cell alloproliferation were significantly inhibited in a polymer dose-dependent manner. The loss of alloproliferation correlated with the induction of Treg cells (CD4+CD25+Foxp3+). The Tregs, surprisingly, arose primarily via differentiation of naive, non-proliferating, CD4+ cells. Of biologic importance, the polymer-induced Treg were functional and exhibited potent immunosuppressive activity on allogeneic CTL proliferation. These results suggest that immunocamouflage-mediated attenuation of alloantigen-TCR recognition can prevent the tissue destructive allogeneic CD8+ T cell response, both directly and indirectly, through the generation/differentiation of functional Tregs. Immunocamouflage induced tolerance could be clinically valuable in attenuating T cell-mediated transplant rejection and in the treatment of autoimmune diseases. STATEMENT OF SIGNIFICANCE: While our previous studies have demonstrated that polymer-grafting to MHC disparate leukocytes inhibits CD4+ cell proliferation, the effects of PEGylation on the alloproliferation of CD8+ cytotoxic T cells (CTL) was not examined. As shown here, PEGylation of allogeneic leukocytes prevents the generation of the CTL response responsible for acute rejection. The loss of CTL proliferation is consequent to the polymer-based attenuation of allorecognition and the induction of T regulatory cells (Tregs). Interestingly, the Tregs are primarily generated via the differentiation of non-proliferating naive T cells. Importantly, the Tregs are functional and effectively induce a tolerogenic environment when transferred to an alloresponsive environment. The use of polymer-modified leukocytes provides a unique approach to effectively maximize the biologic production of functional Tregs both in vitro and in vivo. By using this approach it may be possible to attenuate unwanted alloresponses (e.g., graft rejection) or to treat autoimmune diseases.