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 showed that lymphocyte counts around day 30 post transplant are strongly predictive for outcome. Specifically, higher NK cell counts favored early molecular remission and relapse free survival in CML and higher total lymphocyte count following T cell depleted SCT predicts less relapse and superior survival in myeloid malignancies in general. Understanding the relationship between lymphocyte counts and lymphocyte growth factors could lead to developing lymphokine treatments to favor rapid lymphocyte recovery. Interleukin-15 (IL-15), and IL-7 (amongst other cytokines) stimulate lymphocyte recovery after SCT. 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. Since many 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 explored two SCT approaches that could (a) optimize transplant outcome by minimizing transplant-related mortality and (b) serve as immunosuppression-free platforms for combining with immunotherapy strategies such as vaccination and T cell therapy to enhance GVL. (i) T cell depletion (protocol 07-H-0248): Thirty-six 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. Day 200 overall survival was 79%. One patient rejected the transplant but was successfully retransplanted. 34/36 subjects achieved complete donor (>95%) myeloid chimerism by day 14 and the median time to complete donor CD3+ chimerism was 45 days. The incidence of acute GVHD grade II, III and IV were 23%, 2.9% and 0%, respectively. The incidence of chronic GVHD was 34.3%. 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. Future studies will use this transplant approach as the platform for introducing vaccines and cell therapies in the early post-SCT period to enhance GVHD effects. (ii) Selective depletion (SD) of alloreactive cells (Protocol 07-H-0136): We scaled up and validated an SD technique to specifically eliminate alloreacting lymphocytes from the transplant inoculum. Twenty-four patients with hematological malignancies received myeloablative conditioning with fludarabine, cyclophosphamide and total body irradiation followed by a T cell depleted stem cell transplant from an HLA identical sibling (CD34 dose >5x106/kg;CD3 dose <104/kg) together with 5 x 106/kg SD T cells. The primary aim of the study was to determine whether immunosuppression with CSA was necessary for GVHD prevention. The first cohort of 17 patients received low dose CSA until day 90 post transplant as the sole GVHD prophylaxis, No severe grade II GVHD occurred and the second cohort therefore received CSA stopping at d45 post SCT. Although the procedure appeared to protect from severe GVHD (13+7% grade III-IV aGVHD) and relapse was low in this high risk patient group (27+10%) an unexpected complication was an increased risk of CMV reactivation and occurrence of late complications, mainly infections, resulting in delayed transplant-related mortality and an overall survival of 39+12%(We developed a technique to selectively deplete alloreactive T cells from the transplant have completed two selective depletion SCT trials, confirming the potential of the approach. Further studies now seek to improve the quality of SD T cell products, exploring variables in the depletion step, the type of antigen presenting cell used to elicit the alloresponse, different degrees of HLA disparity between donor and recipient, and different culture conditions. 4. Prevention and treatment of GVHD and post transplant complications with MSC: In collaboration with Prof Katarina Le Blanc (Karolinska Institute), we found that while MSC do not harbor most viruses they are occasionally a reservoir for the B19 parvovirus, but in the rare examples where the virus was identified in MSC products, there was no transmission of the virus to the recipient. Lymphocytes from patients receiving MSC to treat GVHD after SCT remained anergic to MSCs but not lymphocytes from the MSC donor (15). These results justify the use of random third party MSC to treat complications in SCT recipients. Three clinical trials using NIH generated third party MSC are now open for accrual. All patients undergoing SCT at NIH are eligible. The protocols evaluate 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. In collaboration with Karolinska Institute, we found that MSC express small amounts of foxP3 a gene essential for regulatory T cell function. So far the function of foxP3 in MSC is unclear. We next plan to insert the foxP3 gene into MSC to explore whether this improves the immunosuppressive function of MSC. 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. Low doses ( 0.5 x 106 units/day) are well tolerated with no significant adverse effects and studies suggest that IL-2 given to the donor could induce selective expansion of regulatory T cells and NK cells which could reduce GVHD and enhance GVL. We have therefore initiated 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.

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Anand, Ankit; Anandi, Prathima; Jain, Natasha A et al. (2016) CD34+ selection and the severity of oropharyngeal mucositis in total body irradiation-based allogeneic stem cell transplantation. Support Care Cancer 24:815-22
Ishii, Kazusa; Barrett, Austin J (2016) Novel immunotherapeutic approaches for the treatment of acute leukemia (myeloid and lymphoblastic). Ther Adv Hematol 7:17-39
Hourigan, Christopher S; Goswami, Meghali; Battiwalla, Minoo et al. (2016) When the Minimal Becomes Measurable. J Clin Oncol 34:2557-8
Anandi, P; Tian, X; Chinian, F et al. (2016) Improved reproducibility and quality of GvHD biomarker assay: application of multiplex microfluidic channel system. Bone Marrow Transplant :
Whitehill, Greg D; Amarnath, Shoba; Muranski, Pawel et al. (2016) Adenosine Selectively Depletes Alloreactive T Cells to Prevent GVHD While Conserving Immunity to Viruses and Leukemia. Mol Ther :
Goswami, M; McGowan, K S; Lu, K et al. (2015) A multigene array for measurable residual disease detection in AML patients undergoing SCT. Bone Marrow Transplant 50:642-51
Ito, Sawa; Barrett, A John (2015) ST2: the biomarker at the heart of GVHD severity. Blood 125:10-1
Barrett, A John; Ito, Sawa (2015) The role of stem cell transplantation for chronic myelogenous leukemia in the 21st century. Blood 125:3230-5
Jain, N A; Ito, S; Tian, X et al. (2015) Clinical and biological predictors of outcome following relapse of CML post-allo-SCT. Bone Marrow Transplant 50:189-96
Amarnath, Shoba; Foley, Jason E; Farthing, Don E et al. (2015) Bone marrow-derived mesenchymal stromal cells harness purinergenic signaling to tolerize human Th1 cells in vivo. Stem Cells 33:1200-12

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