R. Handgretinger, X. Chen, M. Pfeiffer, M. Schumm, I. Mueller, T

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Cellular Immune Reconstitution after Haploidentical Transplantation in Children R. Handgretinger, X. Chen, M. Pfeiffer, M. Schumm, I. Mueller, T. Feuchtinger, G. Hale, P. Lang Biology of Blood and Marrow Transplantation Volume 14, Issue 1, Pages 59-65 (January 2008) DOI: 10.1016/j.bbmt.2007.10.015 Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 The fundamental difference between CD34+ positive selected (left panel) and CD3/C19-depleted (right panel) mobilized peripheral stem cells from haploidentical donors is shown. Although CD34+ positive selection results in a homogenous population of highly purified CD34+ stem cells, CD3/C19 depletion results only in a low enrichment of CD34+ cells, but retains large numbers of CD56+ NK cells and other myeloid cells. Biology of Blood and Marrow Transplantation 2008 14, 59-65DOI: (10.1016/j.bbmt.2007.10.015) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Comparative analyis of CD4+ T-helper cells and CD56+ NK cells after transplantion with haploidentical CD3-depleted PBSCs after TBI-containing myeloablative regimen (circle) or RIC regimens (triangle) (upper panel). In the middle panel, the recovery of the TRECs and the V-beta spectratyping score is depicted. In the lower panel, the copy numbers as determined by real-time PCR in patients with reactivation of cytomegalovirus (CMV) or adenovirus (ADV) is shown. Biology of Blood and Marrow Transplantation 2008 14, 59-65DOI: (10.1016/j.bbmt.2007.10.015) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 The in vitro purification steps of ADV-specific donor T cells are shown. Mononuclear cells from the haploidentical stem cell donor were isolated and incubated overnight with viral antigen. After stimulation, 0.7% of the CD3+ T cells were found to secrete interferon-gamma and thus specific for ADV (A, left panel). These ADV-specific T cells are composed of CD4+ and CD8+ T cells (A, middle panel). After isolation using the interferon-gamma capture assay, the interferon-gamma secreting CD3+ T cells are highly enriched and ready for infusion into the patient (C, right panel). In (B), the expansion of ADV-specific T cells in 5 patients with posttransplant ADV reactivation (left panel) and the concomitant decrease of the ADV copy numbers or the complete clearance (right panel) is shown. Biology of Blood and Marrow Transplantation 2008 14, 59-65DOI: (10.1016/j.bbmt.2007.10.015) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Comparative retrospective analysis of the cumulative incidence of lethal viral infections after myeloablative conditioning regimens and CD34+ positive selection and after RIC regimen and CD3/CD19 depletion. In the myelablative group (upper panel), almost 20% of the patients died from a lethal infection. Half of these patients had a lethal ADV infection, whereas no death from ADV or any other viral, bacterial, or fungal infection was seen in the RIC group (lower panel). Biology of Blood and Marrow Transplantation 2008 14, 59-65DOI: (10.1016/j.bbmt.2007.10.015) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Flowcytometric analysis of peripheral blood chimerism posttransplant. In this example, the donor (D) is HLA-A2/28 positive, whereas the recipient (R) is HLA-A2/28 negative. At day +200 posttransplant, all NK cells (A) and T cells (B) are donor-derived. The HLA-A2/28 negative population stained positive for CD45dim, whereas all donor-derived hematopoietic cells were CD45+ bright (C). Further analysis (D) showed that most of the HLA-A2/28 negative cells were CD34+ and had the same leukemic phenotype as the patient's blasts prior to transplant. Biology of Blood and Marrow Transplantation 2008 14, 59-65DOI: (10.1016/j.bbmt.2007.10.015) Copyright © 2008 American Society for Blood and Marrow Transplantation Terms and Conditions