Raising Peptide/allo-MHC Complex-specific CTLs By Autologous/syngeneic Macrophage Bearing The H-2K~d/IgG2aFc Molecules In Vitro And In Vivo

CD8+ cytotoxic T lymphocytes (CTLs) are major effectors in immune defence. However, their response to chronic viral infection and tumor are often less vigorous, resulting in CTLs of lower avidity, especially when antigens on the surface of chronic viral infection and tumor are represented by normal self-proteins, for the T cells specific to the antigens with high affnity are likely to be deleted from repertoire.Alloreactivity refers to the ability of T cells to recognize peptide–allogeneic-MHC complexes (pMHC) and manifests itself clinically as allograft rejection response. It has been clearly shown that graft versus host disease (GVHD) is accompanied by a graft versus leukemia reaction (GVLR) which reduces the incidence of leukemia relapse. Furthermore, many evidences confirm that alloreactive T cell recognition is pMHC-specific. Therefore, allogeneic CTLs with defined specificity is expected to circumvent the tolerance. However, preparation of alloreactive T cells with a given specificity is still under investigation.In the study, we describe a novel protocol to generate pMHC specific CTLs. An allogeneic MHC class I molecule associated with its restricted peptide is attached to Mφ, the latter"presents"the allogeneic epitope and induces autologous/syngeneic splenocytes to generate pMHC-specific CTLs. This method might be applied for immunotherapy of chronic viral infection and tumor.1. Generation of H-2Kd/IgG2aFc molecule and its binding to macrophage An divalent peptide/H-2Kd/IgG2aFc molecule was constructed by fusing extracellular domains of H-2Kd with murine Fc fragment of IgG2a, consisting of divalent TCR-ligands and an FcRⅠbinding moiety, which allows us to attach an allogeneic MHC molecule (H-2Kd) to Mφ(H-2Kk).An H-2Kd/IgG2aFc hybrid gene was constructed by recombining cDNA coding for extracellular domains of H-2Kd with that for Fc fragment of IgG2a. cDNA encoding the H-2Kd and the Fc fragment were cloned from total mRNA of BALB/c mouse splenocytes and HB-95 cell line, respectively. The pcDNA3.1(+)-[H-2Kd/IgG2aFc] plasmid was generated by inserting the hybrid sequences into pcDNA3.1(+) vector, and then transfected into J558L cell line by electroporation. The highest expressing clone is named as Kd-Fc-J558L.The supernatant of Kd-Fc-J558L was collected and passed over a Staphylococcal Protein A (SPA) column to purify the products.The peptide/H-2Kd/IgG2aFc molecules (the dimer) includes a divalent TCR-ligands and a Fc receptor (FcR) reactive moiety, which was confirmed by analysis of ELISA, flow cytometry and Western-blotting. Furthermore, the binding efficiency of the dimer to macrophage (Mφ) was detected by measuring H-2Kd molecules on H-2Kk Mφ. The Mφdisplayed significant amount of H-2Kd molecules after loaded with the dimer. Therefore, the Mφcould"presents"the single allo-pMHC epitope.2.Raising peptide-specific, alloreactive CTLs from C3H splenocytes co-cultured with autologous macrophage loaded with the dimer Splenocytes of C3H (H-2Kk) was co-cultured with the dimer-loaded autologous macrophages for raising alloreactive T cells. Splenocytes of C3H segregated were stimulated cells. The C3H macrophages loaded with dimer (pulsed the specific peptide) were stimulating cells.After 6 days co-culture, the splenocytes show an elevated proliferation after stimulation by the dimer-loaded Mφcompared with the control. Furthermore, after 14 days co-culture, not only the splenocytes stimulated by autologous Mφloaded with the-dimer are more frequently stained with the specific-tetramer than that with the irrelavent-tetramer, but also the splenocytes exhibit an elevated cytotoxicity against specific target (pMHC-specific target ), compared with that against the irrelavent targets. The results of staining are consistent with those of cytotoxicity assay, suggesting the alloreactive T cells should be peptide-specific and functional.3 . Raising of peptide-specific, alloreactive CTLs from C3H mouse immunized by syngeneic macrophages loaded with the dimer in vivo.The syngeneic macrophage loaded with the dimer was used to elicit peptide-specific, alloreactive CTL response in vivo. After immunized with the dimer-loaded Mφinjected through vena caudalis, the splenocytes of C3H mouse were tested for their cytotoxicity against specific target and binding ability to specific tetramer. Similar to the results of the co-culture in vitro, the findings also support that the response in vivo induced by the dimer-loaded Mφshould be peptide-specific and functional.These data suggest that the dimer molecule may be useful to provide a strategy for boosting pMHC restricted CTLs which might add to our arsenal for adoptive immunotherapy to eliminate chronic viral infection and tumor cells.