WT1-Specific T Cell Responses Are Frequent after Allogeneic Stem Cell Transplantation for Acute and Chronic Myeloid Leukemia.

The transcription factor WT1 is often overexpressed in leukemic cells and thus represents a tumor specific antigen which has already been used as a target for immunotherapeutic approaches. To investigate its role in graft-versus-leukemia effects after allogeneic stem cell transplantation (SCT), the frequency of T cells specific for WT1 was determined in 52 patients with myeloid malignancies before and after allogeneic SCT.

Methods: Using unseparated PBMCs, T cell frequencies were assessed by the IFN-gamma ELISPOT assay at continuous intervals after dose reduced (n=15) or conventional transplantation (n=37). In case of HLA-A2.1-positive pts, a panel of 4 peptides (Db126_126-134, WH187_187-195, WH242_240-250 and WT1 37_37-45), known to represent HLA-A2.1-restricted epitopes of the WT1 protein was applied. In addition, we made use of a pool of overlapping peptides derived from the WT1 protein to assess responses in HLA-A2.1-negative patients and against other epitopes. HIV-specific (ILKEPVHGV) and CMVpp65-specific (NLVPMVATV) peptides served as negative and positive controls. In case of positive results in the ELISPOT, cells were further analyzed in tetramer binding assays. In order to detect the expression of the WT1 gene in tumor cells, a quantitative RT-PCR was carried out.

Results: 11 patients with CML and 41 patients with AML/MDS were analyzed. In contrast to 17 conventionally treated patients with CML who showed no WT-1-specific reactivity, antigen-specific T cells were present in high frequency (up to 356:100.000) in 7 of 11 pts with CML up to 780 days after allogeneic transplantation. Further, T cell-responses against WT-1 peptides were found in 18 of 41 patients with AML after transplant with frequencies between 21 and 322 spots/100 000 PBMC (median 70) for Db126 and 20-356 spots/100 000 PBMC (median 93) for WH187. In contrast, only 20% of patients pre allo SCT (4/20) had a positive response against Db126 (median 144, range 111–160) and WH187 (median 151, range 122–174). Only 2 out of 17 normal subjects showed Db126 and WH187 specific CTLs with very weak frequencies (maximal T cell response 40 and 41 spots /100 000 PBMC). Reactivity against WT1 37 or WH 242 was exclusively observed following transplantation (in 2 and 5 out of 16 patients). In HLA A2.1 positive as well as negative recipients, the pool of overlapping WT1 peptides allowed us to detect WT1-reactive T cells in 3 of 9 patients. The number of WT1-reactive T cells increases soon after transplantation, and is maintained at different levels over a longer period of time. Concomitantly, the proportion and number of CD3⁺/Foxp3⁺ regulatory T cells was shown to be significantly reduced in all patients tested so far.

Conclusion: T cell responses against WT1 are elicited short after allogeneic SCT. We speculate that the lack of regulatory T cells during the early phase after HSCT could be responsible for this phenomenon. To further enhance WT1 specific immune reactivity during this phase after HSCT, we have started a phase II clinical trial, in which patients with AML and MDS are vaccinated with the Db126 peptide.