Wt1-specific Monitoring Of The Gvl Response After Non-myeloablative Allogeneic Hematopoietic Stem Cell Transplantation And Clinical Research

Objective: NST, a promptly established form in curing leukemia in last decade, has been citified to be an effective therapy platform on which elimilation of MRD depends more on the strong graft-versus-leukemia (GVL) effect mediated by donor lymphocytes, owing to the reduced cytotoxity of conditioning regimes. So far, monitoring GVL effect should be an important content for prognostication and instruction after NST. Although be recorgnized in 1956, for a long period, the determination of GVL effect has been still based on the clinical therapeutic effects that lack of sensitive and reliable detective evidences and quantitative indexes, and no more profound studies were performed consequently. Until recently, Rezvani K et al. analyzed the relationship between the expression of WT1 gene and WT1 specific CD8+CTLs numbers of 10 ALL patients after allo-HSCT which were detected by HLA-tetramer and ICCS, and then suggested that post-transplant WT1+CD8+ CTL numbers could be used as an indirect index for reflecting GVL effects. Other previous studidies had showed that Tc1 and Th1 subpopulations were the main effector cells in mediating GVL effect, and so, although Rezvani K assayed the effects of CD8+T ceγlls on inducing post-SCT specific T cell response, similary influence from CD4+T cells were neglected. Pentamer, a quantitive detection method, is more sensitive than tetramer staining, and ICCS emphasized on functional detection and T-cell subsets assay, so we performed this study aiming to quantitate WT1+CTL, WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells in PB of leukemic patients, and to compare the formation of these cells between HLA-identical SCT and haaplo-identical SCT, CML and acute leukemia patients, then to assay the relationships between WT1 specific immune response and GVL effect after NST.Methods: HLA-typing results of 35 leukemic patients and respective donors were followed up before transplant, and WT1 expression and donor-recipient chimerism levels pre- and post-NST were assayed by PCR. Meanwhile, WT1 specific CD8+CTL, Tc1/Tc2 and Th1/Th2, and subpopulations of lymphocytes in PB were detected by pentamer, ICCS and FACS before and after transplant, respectively. Based on the proportions of CD4+ and CD8+T cells, we calculated CD4/8 ratio and further analyzed the following items: (1) detection of WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells in donors and patients; (2) the comparison of pentamer and ICCS in the detection of WT1-specific T cells; (3) disparity in the detection of WT1 specific T cells pre and post NST; (4) generation phase of WT1 specific T cell and its occurrence of peak level after transplant; (5) relationship between WT1 expression and proportions of WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells; (6) disparities in generation of WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells between HLA-identical and haplo-identical transplantation, and relations to reconstitution of lymphocyte subsets; (7) disparities in generation of WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells between AL and CML patients in HLA-identical transplant cohorts, and relations to reconstitution of lymphocyte subsets.Results: Main results were showed as follows: (1) WT1-specific CD8+CTL was detected only in patients and donors with WT1+ HLA-A*0201+, but not in those with WT1+ HLA-A*0201-, WT1-HLA-A*0201+ and WT1-HLA-A*0201-. Similarly, WT1+CD8+IFNγ+T-cell was detected only in donors with WT1 expression, but not in those without WT1 expression.(2)Although no obvious difference were found between the detection rate, primary detection time and detection time of peak levels of WT1- specific CD8+CTL and WT1+CD8+IFNγ+T-cell(P>0.05), positive rate of WT1+CD8+IFNγ+T-cell within 30 days after transplant was slightly higher than that of WT1-specific CD8+CTL, and the peak levels of the latter was significantly higher than that of WT1+CD8+CTL(P<0.01)(.3) the respective detection rates of WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells of patients post-NST were significantly higher than those of donors and patients pre-transplant(P<0.01).(4) WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells were detected abundantly around 30 and 90 days after transplant .(5) on the whole, WT1 gene expressions were negatively correlated with the proportions of WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells(.6) rations of WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells of which primary detection time and detection time of peak levels within 30 days in HLA-identical transplant cohorts were all higher than those in haplo-identical group, but all showed no statistics difference(P>0.05).(7) no statistic differences were observed in respective peak levels of WT1+CTL, WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells between HLA-identical and haplo-identical transplant(P>0.05). In addition, after HLA-identical NST, no statistics differences were found in the reconstitution of T cells subpopulations and CD4/CD8 ratios between HLA-identical and haplo-identical transplant early after transplant. (8) No significant differences were observed in positive rates of WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells between AL and CML patients treated with HLA-identical NST(P>0.05).(9) the peak values of WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells in CML patients were all higher than those of AL, but the difference was not statistically significant (P>0.05). Moreover, no significant differences were observed in the responsiveness of AL and CML patients to GDMI. (10) in earlier stage after NST, no statistics differences were found in the reconstitution of T cells subpopulations and CD4/CD8 ratios between AL and CML patients.Conclusion: NST could promotes the forming of WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells , and these cells were induced mainly within 30 days after transplant. Meanwhile, WT1+CTL,WT1+CD8+IFNγ+T and WT1+CD4+IFNγ+T-cells may indirectly reflect WT1 specific GVL effect mediated by donor T cells. No obvious differences were observed in generation of WT1 specific GVL effects between HLA-identical and haplo-identical transplant early aster NST, which maybe attribute to the difference in application of immunosuppressive agents. No obvious differences were observed in generation of WT1 specific GVL effects between AL and CML, so we speculated that the differences in SCT and GDMI on AL and CML patients were related to the different sensitivity of these two type of leukemic patients to a similary GVL effect, but not to the different generation of GVL. Together, we raised a new method to indirectly reflect allogeneic GVL effect by LAA-specific-T-cell which was detected by the combined application of pentamer and ICCS, and based on which proper adjustment were performed accordingly on treatment after NST.