Induction of immunotolerance via mPEG grafting to allogeneic leukocytes

The induction of anergy or tolerance to prevent allorecognition is of clinical interest. To this end, the effects of methoxypoly(ethylene glycol) [mPEG] grafting to allogeneic lymphocytes on proliferation and phenotype (Th17 and Treg) was examined in vitro and in vivo. Control studies demonstrated that PEGylation did not affect cells viability or proliferation (mitogen) potential. Conditioned media (1° MLR) collected at 72 h from resting PBMC demonstrated no immunomodulatory effects whereas the control MLR demonstrated significant (p < 0.001) pro-proliferative potential and significantly increased in IL-2, TNF-α, and INF-γ. However, 1° media from either resting mPEG-PBMC or the PEGylated MLR resulted in a significant inhibitory effect (p < 0.001) in the 2° MLR and no increase in cytokines. PEGylation of donor murine splenocytes resulted in significant in vivo immunosuppressive effects in H2-disparate mice. While unmodified allogeneic splenocytes resulted in a significant in vivo decrease in Treg and increased Th17 lymphocytes, PEGylated allogeneic splenocytes yielded significantly increased Tregs and baseline levels of Th17 lymphocytes. This effect was persistent to at least 30 days post challenge and was not reversed by unmodified allogeneic cells. These studies demonstrate that PEGylation of allogeneic lymphocytes induced an immunoquiescent state both in vitro and in vivo.