Immunocamouflage: prevention of transfusion-induced graft-versus-host disease via polymer grafting of donor cells.

Graft-versus-host disease (GVHD) can occur following the transfer of allogeneic lymphocytes into immunosuppressed and, in rare cases, immunocompetent recipients. The initiation of GVHD requires the allorecognition of the recipient's disparate MHC molecules by the donor T lymphocytes (T cell). Currently, GVHD is controlled by cyclosporine administration--a potent, but toxic, T-cell suppressing agent. To determine if the nontoxic grafting of methoxypoly(ethylene glycol) (mPEG) to immunologically foreign lymphocytes could prevent allorecognition and GVHD, in vitro and in vivo murine studies were performed. In vitro studies utilizing mixed lymphocyte reactions (MLRs) demonstrate that mPEG modification effectively prevented allorecognition and subsequent T-cell proliferation. The loss of cellular proliferation was not due to mPEG cytotoxicity but rather to the inhibition of cell-cell interactions. Flow cytometric studies showed that T-cell and antigen-presenting cell adhesion molecules (CD2, CD11a), signaling (CD3epsilon, T-cell receptor), and costimulatory molecules (CD28, CD80) were efficiently immunocamouflaged by mPEG derivatization. Interestingly, upon antigenic stimulation mPEG-modified cells demonstrate enhanced apoptosis as evidenced by DNA laddering. In vivo studies using immunocompetent and immunosuppressed mice established that mPEG modification of donor lymphocytes effectively attenuated the in vivo proliferation of donor cells and the initiation of GVHD.

Beyond the red cell: pegylation of other blood cells and tissues.

Immunological recognition of allogeneic tissue is of critical concern in transfusion and transplantation medicine. While the major emphasis of our work on the immunocamouflage of cells has been focused on the erythrocyte, we have extended this research beyond the red blood cell (RBC) to other tissues. Our studies from blood transfusion (i.e., a specialized form of cellular transplantation) suggest that covalent modification of cells and tissues with methoxypoly(ethylene glycol) mPEG can significantly diminish immunologic recognition of other allogeneic tissues and, furthermore, may enhance the induction of tolerance. The mechanisms underlying the mPEG-mediated immunocamouflage of alloantigens is the global camouflaging of antigenic sites, membrane surface charge and the attenuation of receptor-ligand and cell-cell interactions. As a consequence of the immunocamouflage imparted by the grafted mPEG, weak costimulation of alloreactive T cells is observed which subsequently induces apoptosis of these reactive cells. As a result of this clonal deletion, a pro-tolerance state is induced. The potency of immunocamouflage is readily observed in in vivo murine models of transfusion-associated graft versus host disease. Furthermore, initial studies on the in vivo transplantation of pegylated rat and murine pancreatic islets have demonstrated that mPEG-derivatization does not impair the finely tuned signaling necessary for glucose homeostasis. Finally, in contrast to the pharmacological inhibition of the immune response by agents such as cyclosporine, mPEG-mediated immunocamouflage directly attenuates the inherent antigenicity and immunogenicity of the donor tissue itself while leaving the recipient a fully competent immune system.

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.

Comparative analysis of polymer and linker chemistries on the efficacy of immunocamouflage of murine leukocytes.

Membrane grafting of methoxypoly(ethylene glycol) [mPEG] to allogeneic leukocytes attenuates allorecognition and significantly reduces the risk of graft-versus-host disease in mice. To optimize the immunological efficacy of polymer grafting, murine splenocytes were modified using three differing linker chemistries: CmPEG (5 kDa), BTCmPEG (5 and 20 kDa) and TmPEG (5 kDa). In vitro immunocamouflage efficacy was examined by flow cytometic analysis of leukocyte markers and mixed lymphocyte reactions (MLR). In contrast to CmPEG and BTCmPEG, TmPEG exerted significant cellular toxicity. Flow cytometric analysis demonstrated that both CmPEG and BTCmPEG were highly effective at camouflaging cell surface markers while TmPEG was ineffective. Furthermore, CmPEG and BTCmPEG dramatically blocked MLR allorecognition and cellular proliferation. Polymer length was the most critical factor in the immunocamouflage of cells with the BTCmPEG 20 kDa being the most effective. In contrast to other immunomodulatory approaches, immunocamouflage of leukocytes yields a multivalent effect globally interfering with attachment, allorecognition, presentation and costimulation pathways.

Virus-specific and bystander CD8 T cells recruited during virus-induced encephalomyelitis.

Neurotropic coronavirus-induced encephalitis was used to evaluate recruitment, functional activation, and retention of peripheral bystander memory CD8+ T cells. Mice were first infected with recombinant vaccinia virus expressing a non-cross-reactive human immunodeficiency virus (HIV) epitope, designated p18. Following establishment of an endogenous p18-specific memory CD8+ T-cell population, mice were challenged with coronavirus to directly compare recruitment, longevity, and activation characteristics of both primary coronavirus-specific and bystander memory populations trafficking into the central nervous system (CNS). HIV-specific memory CD8+ T cells were recruited early into the CNS as components of the innate immune response, preceding CD8+ T cells specific for the dominant coronavirus epitope, designated pN. Although pN-specific T-cell numbers gradually exceeded bystander p18-specific CD8+ T-cell numbers, both populations peaked concurrently within the CNS. Nevertheless, coronavirus-specific CD8+ T cells were preferentially retained. By contrast, bystander CD8+ T-cell numbers declined to background numbers following control of CNS virus replication. Furthermore, in contrast to highly activated pN-specific CD8+ T cells, bystander p18-specific CD8+ T cells recruited to the site of inflammation maintained a nonactivated memory phenotype and did not express ex vivo cytolytic activity. Therefore, analysis of host CD8+ T-cell responses to unrelated infections demonstrates that bystander memory CD8+ T cells can comprise a significant proportion of CNS inflammatory cells during virus-induced encephalitis. However, transient CNS retention and the absence of activation suggest that memory bystander CD8+ T cells may not overtly contribute to pathology in the absence of antigen recognition.