| Although the T-cell response to allogeneic cells is typically regarded as a detrimental phenomenon responsible for rejection of transplanted allografts and graft-versus-host disease following haematopoietic stem cell transplantation, beneficial components also exist within the alloreactive population. The graft-versus-leukemia (GVLR) or graft-versus-tumor reaction (GVTR) accompanying with the graft-versus-host disease (GVHD) suggests the dramatic effect of the allogeneic T cells on eradicating malignant disease. And our previous studies suggest that it is the peptide/MHC complex as a whole recognized by allogeneic T cells and the recognition is pMHC specific. So it is possible to discriminate the good from the bad depending on their pMHC disparities. In this study, we aim to investigate alloreactivity at a single pMHC level and provide the in vivo experimental evidence that the transfused single pMHC specific allogeneic CTLs could target eliminating specified antigens without GVHD, which would pave the way for further exploring the mechanism and regulatory mode of allo-reactivity, in addition, add to our arsenal for breaking pathological immune tolerance.However, because there are exceedingly wide range of cell surface pMHC complexes, it is essentially the combination of allogeneic T cells against multiple epitopes prepared with traditional access. Therefore, we set up a "single pMHC induction system " to prepare single pMHC specific CTLs (InsB/H-2Kb specific) and transfused the induced allogeneic CTLs to target imparing isletsβcells with InsB/H-2Kb expression. 1. Preparation of divalent H-2Kb/IgG2aFc and its binding to H-2Kk macrophages.The H-2Kb/IgG2aFc hybrid gene was constructed by recombining cDNA coding for the extracellular domains (al,a2,a3) of H-2Kb with that for Fc fragment (Hinge,CH2,CH3) of murine IgG2a heavy chain. Then the hybrid H-2Kb/IgG2aFc sequence and mouseβ2m sequence were respectively inserted into the double promoters of pFastBacTMual vector for divalent H-2Kb/IgG2aFc expression (short for H-2Kb dimer).The H-2Kb dimer consists of a divalent TCR-ligands and an IgG2aFc receptor (FcR) conjunctive moiety. And the H-2Kb dimer can be introduced as an allogeneic MHC molecule to Macrophages (H-2Kk) via the Fc and FcR interaction, which paves the way for generation of allogeneic CTLs with single pMHC specificity.2. Preparing pMHC-specific, alloreactive CTLs through an in vivo primed plus ex vivo enhanced way.Splenocytes of C3H (H-2Kk) were stimulated cells, while the H-2Kk macrophages (M(?)) bearing InsB/H-2Kb dimer were stimulating cells. The stimulating cells were i.p. injected into mouse C3H (H-2Kk) for InsB/H-2Kb specific, allogeneic CTLs induction. Splenocytes stimulated by autologous Mcp bearing the InsB/H-2Kb dimer were more frequently stained with the corresponding tetramer than those with irrelavent-tetramer and exhibited an elevated cytotoxicity against specific targets with InsB/H-2Kb epitope.3. An insulin-producing P-cell targeted damage caused by i.v. adoptive transfusion of the induced allogeneic T cellsThe InsB/H-2Kb-specific allogeneic T cells were i.v. transfused into recipient mice C57BL/6 (H-2b),B6C3F1(H-2b×k) or SCID beige (H-2d). Then we monitored the variation in blood glucose concentration (BGC), serum insulin level and took out immuno-histochemical detection of pancreatic islets with anti-mouse CD3 or anti-mouse insulin antibody which turned out to be that on day 3 ater transfusion with InsB/H-2Kb-specific allogeneic T cells there was an increased BGC, decreased serum insulin, visible T cells infiltration and isletsβcells great loss in pancreatic islets of C57BL/6 and B6C3F1. While there was no corresponding changes in recipient SCID beige on the whole. Taken together, the target damage toβcells offered the in vivo evidence that the transfused allogeneic T cells could traffic to target sites and carry out the exact cytolytic effects in a pMHC-specific manner.
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