Abstract
The Wilms tumor gene WT1 is overexpressed in hematopoietic malignancies such as Myelodysplastic syndromes and leukemias and the WT1 protein was demonstrated to be an attractive target antigen for an immunotherapeutic approach to these diseases. Most of the efforts have been focused to the search for immunogenic peptides suitable for inducing cytotoxic T lymphocytes (CTLs) and to less extent for CD4+ T lymphocytes with potential cytotoxic activity. On this matter, in our previous experience with a p210-derived peptide vaccine developed for chronic myeloid leukemia patients with minimal residual disease, the main immune and therapeutic effect observed after vaccinations appeared to be mediated by peptide-specific CD4+ T cells induced by the longest peptide (25 mer) included in our vaccine. CML-peptide specific T cells were found to be either CD4+/perforin+ or CD4+/CD25+/Foxp3+ and we recently showed their direct cytotoxicity against a CML cell line. Thus to pursue a vaccine strategy mainly devoted to a similar CD4+ T cell immune response, we screened WT1 protein through Syfpeithi database to identify original peptides with a suitable length (23–25 amino acids) to be processed by several HLA class II molecules and to induce a strong CD4+ T cell stimulation. Additionally, in order to maximize the immunogenic potential of the novel peptides, we focused our attention on areas of the protein with known CTLs/CD4 T cells immunogenic epitopes. We identified two peptides that fulfilled these requirements: SEPQQMGSDVRDLNALLPAVPSLGG (WT1-iso5 64–88) which includes 5 amino acid from the alternative splicing derived isoform 5 of WT1 and the first 20 aa of “canonical” WT1 sequence and RPFMCAYPGCNKRYFKLSHLQMHSR (WT1321–345). Both 25mer peptides showed strong HLA binding properties for HLA-DRB1*0101, HLADRB1* 0401, HLA-DRB1*0701, HLA-DRB1*1101, HLA-DRB1*1501 and HLADRB1* 0301( DR17). We first tested them in vitro for their capability to induce peptide-specific CD4+ T cells. Briefly, CD4+ T cells freshly isolated from PBMC were cultured for 21 days in 5% AB human serum media while undergoing to 3 rounds of stimulation with autologous CD14+ cells and both WT1-iso5 64–88 and WT1 321–345 peptides at 20μg/ml in the presence of IL-15. This in vitro stimulation was performed in 3 normal subjects and in 3 MDS patients with high levels of bone marrow WT1 transcript (2 patients presenting a low-International Prognostic Scoring System (IPSS) refractory anemia (RA) and 1 with intermediate IPSS RA). In all 3 healthy donors tested, both peptides were able to induce peptide specific CD4+ T cell proliferation as measured by standard 3HThymidine assay, with a stimulation index (SI) ranging from 2.0 to 2.5 regardless of their HLA-DR phenotype ( SI= cpm CD4+ T cells plus test peptides/CD4+ T cell alone or CD4+ T cells plus control peptides; peptide-specific T cell proliferation was considered positive for SI≥2). Similar results were obtained in all 3 MDS patients in which WT1-iso5 64–88 and WT1 321–345 induced peptide-specific CD4+ T cell proliferation with a SI value of 2.5, 2.9 and 3.0 respectively. In conclusion the present study identified 2 novel WT1-derived 25 mer peptides which were able to easily induce in vitro a peptide-specific CD4+ T cell response in MDS patients. WT1-specific CD4+ T cells proliferated with similar SI values in normal donors and in WT1 positive MDS patients, the latter being highly exposed to this antigen and thus potentially tolerant to it. A possible cytotoxic activity of these WT1-specific CD4+ T cells is under evaluation and in vivo vaccinations of low-intermediate IPSS MDS patients with these peptides are planned.
CD98 Heavy Chain Is a Potent Positive Regulator of CD4+ T Cell Proliferation and Interferon-γ Production In Vivo