Summary:Conventional hematopoietic stem cell cryopreservation methods use a DMSO concentration of 10%. However, cells manipulated ex vivo may require more refined freezing protocols adapted to the specific cell suspension. In this retrospective study, we evaluated the results obtained with CD34 ؉ cells purified from peripheral blood of 39 patients on the CEPRATE SC System and frozen in 7.5% DMSO with a view to transplantation. ؉ cells and CFU-GM were significantly related to hematological recovery. Our data suggest that purified CD34 ؉ cells can be successfully cryopreserved in 7.5% DMSO and may represent a first step in establishing freezing parameters for selected CD34 ؉ cells.

No abstract

No abstract

Summary:Previous studies demonstrated that ex vivo IL-2-activated PBSC could generate cytotoxic effectors without impairing haematopoietic reconstitution. Clinical experience and previous studies indicated that children with solid tumours could benefit from high-dose chemotherapy with immune modulation. We studied the generation of cytotoxic effectors from growth-factor ؎ chemotherapy-mobilised PBSC from 10 patients (five adults and five children) with different solid tumours. Cells were placed in culture in serum-free culture medium supplemented with IL-2 1000 U/ml ؎ IL-12 for 1, 2, 4 or 7 days. Anti-tumour cytotoxicity was tested against K562, Daudi and two neuroblastoma cell lines (Gau, NB91). Cultured adult PBSC in the presence of IL-2 (1000 U/ml) showed marked cytotoxicity against all the cell lines tested from day 1. At day 2, with an E:T ratio of 25:1, cytotoxicity was 53% ؎ 10.4, 63.2% ؎ 23.8, 38% ؎ 9.1, and 39% ؎ 15.7 against K562, Daudi, Gau and NB91, respectively. Cytotoxic activity of child PBSC was significantly lower (P Ͻ 0.05) and was displayed after longer culture times (day 4). No difference was found in the phenotype analysis of lymphoid subsets before and after IL-2 activation between adult and child PBSC. Haematological properties of the graft were not significantly impaired by IL-2 activation. Bone Marrow Transplantation (2001) 27, 869-875. Keywords: PBSC; interleukin 2; neuroblastoma; children High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) has been demonstrated to improve the prognosis of various solid tumours in children. 1,2 However, the risk of relapse remains high. Relapses may be attributed in part to residual disease in the patient and/or in the graft 3 and to the lack of a 'graft-versus-tumour effect' (GVT). 4 As the escalation of HDC is hampered by excessive visceral toxicity, other strategies must be devised to eradicate residual disease.Post-transplant immunotherapy using ex vivo-activated PBSC in combination with HDC may constitute an effective strategy for improving clinical outcome without the drawback of overlapping toxicities. Interleukin-2 (IL-2) therapy instituted immediately after IL-2-activated autologous stem cell transplantation (ASCT) proved effective in inducing graft-versus-leukaemia (GVL) effects in an acute myeloid leukaemia (AML) murine model. 5 This therapeutic approach was attempted in autologous bone marrow (ABM) and then in PBSC collected from cancer patients. The latter study demonstrated that ex vivo IL-2-activated PBSC could generate cytotoxic effectors without impairing haematopoietic reconstitution. 6 The early administration of low-dose IL-2 after transplantation of IL-2-activated PBSC was then demonstrated to be feasible in women with poor risk breast cancer. [7][8][9] In the light of these preliminary results we felt that children with solid tumours could benefit from HDC combined with immune modulation. The occurrence of spontaneous regressions, 10 intratumour lymphocyte infiltrates with T cell clonal expansion, 1...

To characterize circulating T cell subpopulations in B chronic lymphocytic leukemia patients, TCR V alpha and V beta gene-segment use was analyzed by PCR using a panel of V gene-segment subfamily-specific oligonucleotide primers (V alpha 1-29/V beta 1-24). Virtually all V alpha and V beta subfamily specificities were expressed in these patients (nine stage A and four stage C), and the mean values obtained for each specificity were similar to those of a group of 13 healthy donors. Nonetheless, individual analysis revealed that unique V alpha or V beta gene-segment transcripts were overrepresented in patients compared with the control group. Overrepresentation of some TCR V beta chains was also detected by cytofluorometric analysis using a panel of 18 anti-V beta-specific mAbs. To further characterize these T cell subpopulations, we sequenced five different V beta-C beta PCR products in two selected stage A patients and found highly predominant recurrent transcripts in each of the five V beta specificities (50% to 100% of the analyzed sequences with identical V(D)J regions). These results were confirmed on bulk cDNA (i.e., without cloning) and extended to other V beta specificities (up to nine clonal expansions of 24 V beta specificities in one patient) and two other patients using a PCR-based method that determines V(D)J junction size patterns. Finally, it was observed that a V beta 19+ T cell subpopulation was clonally expanded in one patient to up to 30% of circulating T cells. This V beta 19+ CD8+ T cell clone was shown to specifically recognize the autologous tumor cells in vitro, as determined in cytokine release assays. Together, these results support the view that multiple expansions of unique T cell clones may derive in vivo from B chronic lymphocytic leukemia tumor-associated Ag stimulation.