1. Immunological profiles affecting transplant outcome. We studied T and NK cell subsets in the donor and in the patient during the early post-transplant period together with plasma cytokine profiles and showed that the lymphocyte profile of the donor can influence GVHD, relapse, and survival. Results indicate ways to improve outcome by modifying the donor T cell repertoire in vivo, or in the transplanted product: We have now completed a series of analyses of T cell and NK cell recovery after T cell depleted SCT. Outcome after SCT can be predicted by a number of characteristics in the donor and in the early pattern of immune recovery afte transplant. In the donor: higher T reg frequencies and in particular higher frequencies of natural Tregs (CD45 RA+, Helios+) correlated with higher survival, less acute GVHD and less chronic GVHD. In the patient by day 30 higher NK cell counts predicted less leukemic relapse and higher day 30 lymphocyte counts predicted higher overall survival. After SCT the most prominent change in cytokines was a rise in IL-15 which related inversely to the lymphocyte count suggesting that IL-15 is an importan growth factor during immune recovery. Understanding the relationship between lymphocyte counts and lymphocyte growth factors could lead to developing lymphokine treatments to favor rapid lymphocyte recovery. 2. Long term follow-up: While most stem cell transplant survivors have an excellent quality of life and a high probability of disease free survival, mostly without chronic GVHD beyond 5 years from transplant, late complications including relapse, second malignancies and death from consequences of chronic GVHD can occur. The post-SCT complications we identified (pulmonary function defects, thyroid failure, defective spermatogenesis, HPV-related cancers and precancerous lesions and osteopenia) all were more common in patients who had experienced chronic GVHD. In a group of survivors in their second decade after SCT we have made three recent observations: (i) males have an increased risk of cardiovascular events and have a cardiovascular age 10 years over their actual age, (ii) Patients with abnormal pulmonary function after SCT tend to recover full pulmonary function beyond 5 years from transplant (iii) the immune profile of long-term survivors is normal but the T cell repertoire has been remolded in the recipient. B lymphocyte counts are higher than the normal range. Because post SCT cancers involve squamous cell malignancy associated with HPV, we initiated in conjunction with Dr Pam Stratton (NICHD) a preventitive screening program for post transplant females with an HPV vaccine trial using Gardesil vaccine to generate immunity to the common oncogenic HPV strains 11 and 16. This study is ongoing. 3.Optimizing immune reconstitution after SCT: We previously reported on a technique to selectively deplete alloreacting T cells from the donor transplant to prevent GVHD while conserving useful immune function using a photodepletion technique. This transplant approach failed because the depletion approach wes not specific for alloreacting T cells and also depleted CD4 T cells from the graft leading to delayed immune recovery and late transplant related mortality from infectious causes amongst other complications. Concurrently we have now completed a T cell depletion (protocol 07-H-0248): sixty patients (median age 43 years) with hematologic malignancies underwent SCT from their HLA-identical siblings. Subjects received myeloablative conditioning with cyclophosphamide (60 mg/kg/dose x 2), fludarabine (25 mg/m2/dose x 5) and total body irradiation (12 Gy divided in 8 fractions, with lung shielding to 6 Gy). Subjects 55 years of age and older received 4 Gy divided in 8 fractions without lung shielding. G-CSF mobilized peripheral blood stem cells from the donor were CD34+ selected using the Miltenyi CliniMacs system, with infusion of a target CD34+ dose of 6 x 106/kg (range 3 to 10 x 106/kg) and a fixed CD3+ dose of 5 x 104 /kg. GVHD prophylaxis was reduced to low-dose CSA alone day -6 to +21. DLI (5 x 106 CD3+/kg) was given day 90 in the absence of grade >II GvHD. At a median follow up of 3.6 years, Kaplan-Meier estimates of relapse, nonrelapse mortality and overall survival were 35%, 33% and 44% respectively. Since these results were not significantly different from the selective depletion approach we elected to conduct a further T cell depletion study to address some of the shortcomings of the trial 07-H-0248. (i) The T cell depletion technique was changed so as to negatively select for CD34 cells using a CD3 CD19 antibody magnetic separation approach from Miltenyi Corporation under an investigator IND. The transplant product contains a mixed population of CD34 cells more mature progenitors and NK cells. It was anticipated that hematological reconstitution would be more rapid and robust with this method. (ii) No planned DLI to be given;the use of DLI was reserved only for patients with graft failure or relapsed leukemia (iii) Older patients receive 600cGy instead of 400cGy to improve engraftment since a number of older ptients had engraftment failure in the previous protocol. To date 9 patients have been transplanted. Hematological recovery is prompt and all patients have engrafted. Currently all survive. We will use this transplant approach as the platform for introducing vaccines and cell therapies in the early post-SCT period to prevent GVHD, infection and relapse. 4. Prevention of viral infection after SCT. In collaboration with Dr Catherine Bollard (Baylor College) and Dr David Stroncek (DTM) we have developed a robust system to expand multivirus specific T cells recognizing CMV, EBV, Adenovirus and BK virus which commonly complicate the outcome after SCT. Using a pepmix of common immunodominant antigens from these viruses we have induced multivirus specific T cells in high frequencies. The technique is now being scaled up to make a clinic 4. Prevention and treatment of GVHD and post transplant complications with mesenchymal stromal cells: Using NIH generated third party mesenchymal stromal cells (MSC) we have initiated a phase I safety study in patients undergoing SCT at NIH. The protocol evaluate safety and efficacy of MSC (i) to treat steroid resistant acute GVHD, (ii) to treat organ damage (pulmonary, hepatic, hemorrhagic cystitis) and (iii) to improve poor marrow function. Alongside clinical evaluation of therapeutic success, cytokine profiles during and after MSC infusion, changes in T cell and NK phenotype and homing of MSC in biopsy samples of affected tissues will be measured. Eight patients have been treated. The safety profile of MSC infusions is excellent. Some responses in GVHD but not in poor marrow function have been observed. The study is ongoing. 5. Prevention of GVHD through induction of Tregs by IL-2: Our observations that donors with a higher Treg frequency had a lower risk of developing aGVHD (19,20) validate current attempts to use Treg to prevent or treat GVHD. One approach to enhance Treg would be to treat the donor with low dose IL-2. We have completed a study of low dose IL-2 in healthy volunteers to determine the kinetics of Treg and NK expansion as a prelude to a study in HLA mismatched SCT where donors are administered IL-2 before stem cell harvest so as to create Treg and NK rich cell products to reduce the risk of GVHD and boost GVL. We found that there is a twofold or greater increase in Tregs and also in natural Tregs by 7 days and that IL-2 at the lowest dose of 100,000u/m2 daily for 5 days was sufficient. This schedule was without significant side effects. We now plan to explore lower doses of IL-2 and evaluate the impact of addition of the stem cell mobilizing agent G-CSF in a group of volunteers prior to initiating a trial in stem cell donors.
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