Retroviral expression of MIR2 decreases both surface MHC class I and the alloimmune CTL response

Abstract

The immune response to allogeneic cells in tissue-engineered constructs is a major barrier to their successful application in the treatment of many human diseases. Specifically, the T cell-mediated immune response, initiated through the recognition of cell surface MHCI molecules, is the primary cause of acute cellular allograft rejection. In this study, we altered expression of MHCI through viral immunomodulatory mechanisms to examine whether allogeneic cells could be made to 'mimic' viral evasion of a host CTL response. We demonstrate the successful application of a retroviral vector in vitro to overexpress the Kaposi's sarcoma-associated herpesvirus immunomodulatory protein, MIR2, in human monocyte-like myeloid progenitor cells. This approach led to differential downregulation of cell surface MHCI, ICAM-1 and B7-2 molecules. We also demonstrate that downregulation of immunoactive molecules has the functional effect of significantly reducing T cell-mediated cytotoxicity without altering NK-mediated cytotoxicity in vitro. These results provide proof-of-concept that viral immune evasion strategies allow cell-based tissue-engineered constructs to delay or even prevent acute cellular immune rejection in vivo. Importantly, this methodology could facilitate the development of universal donor cells for tissue engineering applications.

Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells

“…The difference between the two values (control and test sample) gives the specific (effector cell-mediated) target cell death [Eq. 1, calculating percent specific death in cell-mediated cytotoxicity assays (Thakur et al 2011)]. We refer to this calculation as the 'Calcein-loss' method for estimating specific cell death.…”

“…The CTL assay was conducted as previously described by our laboratory (Thakur et al 2011). An E:T ratio of 4:1 was used in this assay (Fig.…”