panded CD4ϩ25ϩ cells prevent BM graft rejection in sublethally irradiated recipients via a TGF␤-independent mechanism. Hanash and Levy have shown that the addition of donor CD4ϩ25ϩ cells resulted in greater lineage committed and multi-potential donor progenitors in recipient spleens one week post-transplant and significantly increased long-term multi-lineage donor chimerism in sublethally irradiated recipients. A major limitation for the clinical application of human CD4ϩCD25ϩ cells has been

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... ir paucity in peripheral blood. Only the CD4ϩ25bright cells are potent suppressors and such cells typically are hypoproliferative. Although exogenous IL-2 can support human CD4ϩCD25ϩ cell proliferation the initial protocols published for human CD4ϩCD25ϩ T cells resulted in limited expansion. However, recent publications have solved this problem. Studies by Godfrey et al have used a non-cell sorter, bead-based approach to isolate CD4ϩ25hi cells from adult peripheral blood mononuclear cells that were able to be expanded Ͼ100-fold in 2-3 weeks. These expanded CD4ϩ25ϩ cells were far more potent suppressors of MLR cultures than fresh cells and retained the expression of L-sel hi and other LN homing receptors. Hoffmann et al have shown that CD4ϩ25bright cells can be isolated using cell sorting approaches and that such isolated cells can be expanded in great numbers while still retaining expression of L-sel hi . Recent studies by Godfrey et al have shown that cord blood cell CD4ϩ25ϩ cells are more readily isolated and more often lead to high levels of suppression as compared to adult peripheral blood mononuclear cells. These preliminary findings demonstrating a high degree of MLR suppression now have been extended in subsequent studies using Ͼ100 cord blood lines generated to date (B. Blazar and S. Porter, unpublished data). Thus, the stage is set for clinical trials of CD4ϩ25ϩ cells for GVHD inhibition and therapy and engraftment promotion. Umbilical cord blood (CB) from unrelated donors is increasingly used to restore hematopoiesis after myeloablative or more recently, nonmyeloablative therapy. CB transplants are associated with higher rates of delayed and failed engraftment than are bone marrow transplants, particularly for adult patients. At the University of Colorado, we studied the ex vivo expansion of CB in an attempt to improve time to engraftment and reduce the graft failure rate in the recipients. In the study, 43 patients with hematologic malignancies (n ϭ 40) or breast cancer (n ϭ 3) received high-dose therapy followed by unrelated allogeneic CB transplantation. A fraction of each patient's CB allograft was CD34-selected and cultured ex vivo for 10 or 14 days prior to transplantation in defined media with stem cell factor (SCF), granulocyte colonystimulating factor (G-CSF), and megakaryocyte growth and differentiation factor (MGDF). The remainder of the CB graft was infused without further manipulation. Two sequential cohorts of patients were accrued to the study. The first cohort had 40% and the second cohort had 60% of their CB graft expanded. This study demonstrated that the CD34 selection and ex vivo expansion of CB prior to transplantation is feasible. The engraftment failure rate was relatively low, and the time to neutrophil engraftment in large adults was in the range reported for pediatric recipients of much larger CB cell doses. In the successor cord expansion trial being conducted at the MD Anderson Cancer Center, several changes in the approach have been made. Patients with hematologic malignancies are now being randomized to receive either two unmanipulated CB units or one unmanipulated unit and one unit from which all the cells are expanded ex vivo. For the patients randomized to expansion, on day Ϫ14 one of their cord units is CD133selected using the CliniMACS device. The CD133-negative fraction containing the T cells is frozen and the CD133ϩ fraction is cultured ex vivo for 14 days in media containing SCF, G-CSF and thrombopoietin (TPO). Following administration of the preparative regimen, on Day 0, the second unmanipulated CB unit is infused, followed by the thawed CD133-negative fraction and finally, the fraction which has been in culture for two weeks. This trial was recently initiated and accrual is in progress. Another expansion trial technology involves the use of a copper chelating agent, which has been shown by Peled et al to enhance the expansion of a more primitive CD34ϩ CB population when combined with early acting growth factors. A clinical trial employing this technology is in progress at MD Anderson. Patients with hematologic malignancies undergoing CB transplantation, with a unit that is cryopreserved in two fractions are eligible for the study. Twenty one days prior to infusion, the CD133ϩ cells are isolated from the smaller cord fraction using the CliniMACS device and cultured for three weeks in media containing interleukin-6 TPO, FLT-3, SCF, and the copper chelator tetraethylenepentamine (TEPA). The patients then receive high-dose therapy with the infusion of the larger, unmanipulated CB fraction on day 0, and infusion of the smaller expanded fraction of day ϩ1. Thus far 10 patients have been enrolled on the study. Evaluable patients engrafted neutrophils a median of 27 (range 16-46) in and platelets in a median of 52 (range 27-105) days. An alternative approach for ex vivo expansion is the co-culture of CB cells with bone marrow mesenchymal stem cells (MSCs) which provide a microenvironment for the complex molecular cues that direct self-renewal/proliferation and regulate the differentiation and maturation of hematopoietic progeny. McNiece et al have reported impressive expansion of CB on MSC stroma. Robinson et al have shown superior expansion of CB in MSC co-cultures when compared to liquid cultures of the same CD133ϩ CB fractions. Infusion of MSCs has been shown to promote engraftment of human CD34 ϩ cells in NOD/SCID mice and fetal sheep. Importantly MSC have been shown to have immunomodulatory activity which may reduce graft versus host disease and enhance engraftment in patients. Clinical evaluation of CB expansion on MSCs is planned. The pre-clinical data supporting this approach as well as updates on the two clinical CB expansion trials described above will be presented at the Los Angeles meeting.