1) Leukemia antigens: Two antigens which induce powerful and leukemia-specific cytotoxic T cell responses have been extensively studied by our group: PR1, a nonapeptide sequence shared by proteinase-3 and human neutrophil elastase, first described in our laboratory, and peptide sequences from the tumor antigen Wilms tumor-1 (WT-1). We found low numbers of circulating antigen-specific T-cells recognizing WT-1 and PR1 in normal donors, higher numbers in patients and highest numbers in patients post-transplant. These T-cells are largely of memory phenotype implying that they have escaped central tolerance mechanisms and importantly represent a T cell population that has the potential to rapidly expand upon antigen stimulation. Following SCT, the higher numbers of such antigen-specific T-cells suggests that post-transplant conditions are favorable for expansion of T cells with potential GVL reactivity. We have developed combined WT-1 and PR1 peptide vaccine protocols to enhance the chance of an antileukemic response. A safety study to evaluate toxicity of a single dose of each vaccine has accrued 8 patients with myelodysplastic syndrome. Only local reactions to the vaccine were noted, and in all patients there was an immediate increase (doubling or greater) in the frequency of PR1 or WT1 tetramer specific T-cells as well as antigen-induced IFN-gamma producing T cells, together with a brief fall in WT-1 mRNA expression (used as a measure of disease burden). We initiated a phase II study evaluating 6 two-weekly vaccinations in patients with hematological malignancies at risk from relapse or disease progression. Patients with more aggressive leukemias and those who relapse after SCT, are entered into a separate study exploring the use of WT-1 vaccine following fludarabine-induced lymphopenia and infusion of autologous lymphocytes (previously collected by apheresis). The trial is ongoing. It will determine whether transfused lymphocytes boosted by vaccine expand more readily in a lymphopenic environment and provide better immune control of hematological malignancies. A trial of PRI and WTI vaccine given both to the donor and leukemia transplant recipients will also be submitted for regulatory approval. 2) Transfer of immunity from donor to recipient and lymphocyte recovery post-transplant: We found expansions of cytomegalovirus (CMV) and leukemia-specific T- cells early post-transplant indicating that the homeostatic drive from the lymphopenic environment can induce massive expansions of transplanted donor T cells. We found that higher donor regulatory T cell (TREG) frequencies were associated with significantly lower risks of severe GVHD. Therefore determining the TREG levels in the donor and manipulating TREG early after transplantation may provide a new approach to controlling GVHD but may also modulate GVL effects. To study immune recovery we explored the impact of the day 30 lymphocyte count on transplant outcome. In 54 patients transplanted for myeloid or lymphoid malignancies we found a favorable effect of above median day 30 natural killer (NK) cell count on transplant outcome. We found that occurrence in the donor of three killer immunoglobulin-like receptor (KIR) genes: 2DLA5, 2DS1 and 3DS1 was also associated with significantly less relapse, less acute GVHD, and reduced TRM. Patients with these genotypes were also significantly more likely to have NK cell counts above the median. In a multivariate analysis, NK cell count was the only factor significant for outcome (survival, relapse, TRM). We found that donors with favorable genotypes have NK cells which mature more rapidly but were otherwise functionally identical to NK cells from unfavorable KIR genotypes. 3) Translational research and clinical trials: A long-term project of our group has been to develop a technique for the selective depletion (SD) from the graft of donor T cells recognizing GVHD-associated antigens to allow post-transplant reconstitution of immunity without GVHD, thereby avoiding the need for immunosuppression. The first SD study, completed in 2006, found a reduced risk of severe GVHD affecting the gastrointestinal tract. Lymphocyte recovery was prompt and TRM was low. Overall survival was 40%, associated with high disease relapse rates. This SD technique used had many limitations. We therefore developed an improved SD approach using photodepletion in collaboration with Kiadis Inc. (Amsterdam, Netherlands). This approach is now being tested in a clinical trial to develop an immunosuppression-free SCT. Seventeen patients have been transplanted in the first cohort of this trial. No patient developed clinically severe GVHD and a second cohort of patients receiving shorter post transplant immunosuppression has been enrolled. 4) Clinical trials with T cell depleted SCT from HLA identical sibling donors for patients with hematological malignancy: The current protocol, is designed to evaluate safety and efficacy of the Miltenyi CliniMACs system to achieve T cell depletion. The protocol will accrue up to 68 subjects aged 10-75 years. Subjects receive a conditioning regimen of cyclophosphamide, fludarabine and total body irradiation 1200cGy (or 400cGy if over 55 years), followed by an infusion of a selected CD34+ cells , low dose cyclosporine and a donor lymphocyte infusion on day 90. Currently 30 patients have been transplanted. A notable finding is the favorable outcome for patients over 55 years receiving a reduced intensity conditioning regimen. The long-term follow-up protocol of our own transplant survivors has enrolled 110 patients transplanted 3-14 years. The study has proved to be richly rewarding for new findings. Notably we found that osteopenia is extremely common in long-term survivors, pulmonary function defects persist and may worsen and more than 30% of patients have hypothyroidism. A five year follow-up study of transplant survivors has been completed. These individuals have a low subsequent mortality and a quality of life comparable to the general population.
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