Andreas Geiselhart

Megadose transplantation of purified peripheral blood CD34(+) progenitor cells from HLA-mismatched parental donors in children.

We performed HLA-mismatched stem cell transplantation with megadoses of purified positively selected mobilized peripheral blood CD34+ progenitor cells (PBPC) from related adult donors in 39 children lacking an otherwise suitable donor. The patients received a mean number of 20.7 ± 9.8 × 106/kg purified CD34+ and a mean number of 15.5 ± 20.4 × 103/kg CD3+ T lymphocytes. The first seven patients received short term (<4 weeks) GVHD prophylaxis with cyclosporin A, whereas in all the following 32 patients no GVHD prophylaxis was used. In 38 evaluable patients, five patients experienced primary acute GVHD grade I and one patient grade II. In 32 patients, no signs of primary GVHD were seen and GVHD only occurred after T cell add backs. T cell reconstitution was more rapid if the number of transplanted CD34+ cells exceeded 20 × 106/kg. Of the 39 patients, 15 are alive and well, 13 died due to relapse and 10 transplant-related deaths occurred. We conclude that the HLA barrier can be overcome by transplantation of megadoses of highly purified mismatched CD34+ stem cells. GVHD can be prevented without pharmacological immunosuppression by the efficient T cell depletion associated with the CD34+ positive selection procedure. This approach offers a promising therapeutic option for every child without an otherwise suitable donor. Bone Marrow Transplantation (2001) 27, 777–783.

Isolation of Highly Purified Autologous and Allogeneic Peripheral CD34+ Cells Using the CliniMACS Device

The CliniMACS CD34+ selection device was used for positive selection of apheresis products for autologous transplantation from 10 patients with malignant diseases and for allogeneic transplantation from 26 healthy donors. A total of 71 separations were performed. In 1 allogeneic donor, CD34+ progenitors were also isolated from bone marrow. Between 0.27 and 8.9 x 10(10) nucleated cells (median 4.9 x 10(10)) containing 0.09%-10.8% (median 0.67%) CD34+ progenitor cells were separated. After separation, a median number of 227 x 10(6) mononuclear cells (MNC) (51-524) were recovered, with a median viability of 99% (22%-100%) and a median purity of 97.0% (68.3%-99.7%) CD34+ cells. Depletion of T cells was extensive, with a median of 0.04% residual CD3+ cells (range <0.01%-0.92%). Residual CD19+ cells were between <0.01% and 17%, including CD34+CD19+ cells. Recovery of CD34+ cells was calculated according to the ISHAGE guidelines and ranged from 24% to 105% (median 71%). We conclude that with the CliniMACS device CD34+ cells with high purity and recovery can be isolated with concomitant effective T cell depletion in the allogeneic setting and with a high purging efficacy in the autologous setting.

Comparative analysis of the immunophenotypes of decidual and peripheral blood large granular lymphocytes and T cells during early human pregnancy.

PROBLEM: The functional role of the leukocytes in the decidua is not clear. They may regulate the maternal immune response to the fetal allograft. However, the factors controlling maternal and fetal communication have not yet been identified.

Detection of circulating melanoma cells by immunomagnetic cell sorting.

We developed a cellular approach to the identification of circulating melanoma cells in peripheral blood using immunomagnetic cell sorting. One hundred seventy‐eight blood samples from 129 melanoma patients and 30 samples from healthy persons and nonmelanoma patients were examined. After density gradient centrifugation the interphase was incubated with the mAb 9.2.27. Positive cells were labeled with magnetic microbeads and enriched by immunomagnetic cell sorting. Cells were stained using an alkaline phosphatase–antialkaline phosphatase assay and examined by light microscopy. In spiking experiments, melanoma cells seeded at a concentration of one melanoma cell per ml whole blood could be detected reliably with the assay. Circulating melanoma cells were not found in 30 controls examined, nor were 9.2.27‐positive cells found in 41 patients with primary malignant melanoma. In patients with regional lymph node metastases and in patients with disseminated disease, circulating 9.2.27‐positive cells could be detected in 3 out of 22 patients (13.6%) and 10 out of 66 patients (15.2%) examined. We present a sensitive and specific immunocytological approach to detect circulating melanoma cells in peripheral blood. The method is not suitable for early detection of metastases but is a valuable tool for further investigating biological characteristics of circulating melanoma cells. J. Clin. Lab. Anal. 13:229–233, 1999.

Isolation and transplantation of highly purified autologous peripheral CD34(+) progenitor cells: purging efficacy, hematopoietic reconstitution and long-term outcome in children with high-risk neuroblastoma.

We have investigated the purging efficacy of positive selection of autologous mobilized CD34+ peripheral stem cells in 22 children with high-risk neuroblastoma. CD34+ cell selection was performed using the method of magnetic-activated cell sorting (MACS). The median purity of the CD34+ cells post selection was 97.6% (range 81.7–99.7). For detection of contaminating neuroblastoma cells before and after CD34+ selection, the chimeric anti-disialoganglioside GD2 antibody delta ch 14.18 was used. Prior to positive selection, various numbers of contaminating neuroblastoma cells were found in 17 patients. After positive CD34+ cell selection, low numbers of neuroblastoma cells were only detectable in four patients. In 18 patients, high-dose chemotherapy was performed and the isolated CD34+ cells were reinfused. In all patients, a rapid neutrophil recovery was seen with a median time to reach 0.5 × 109/l neutrophils of 12 days (range 8–24 days). Nine of the 18 patients are free of progression with a median follow-up of 55 months (range 45–70 months). Two patients are alive with relapse, six patients died due to progression or relapse and one patient died due to secondary AML 10 months after transplant while in remission from neuroblastoma. In summary, we show that, through a highly effective positive selection method, a high purging efficacy can be obtained without compromising the hematopoietic reconstitution capacity of the graft.

Human γδ T Lymphocytes Exert Natural and Il-2–induced Cytotoxicity to Neuroblastoma Cells

Human γδ T lymphocytes play an important role in nonadaptive reactions to infection and early tumor defense. This is the first report that freshly isolated, native γδ T cells of some healthy donors can kill human neuroblastoma cells to varying degrees. Their killing ability was increased and maintained during expansion and cultivation with interleukin-2 (IL-2; 400 IU/mL) for as long as 30 days (100% specific lysis at an effector-to-target cell (E:T) ratio of 20:1). γδ T lymphocytes without this spontaneous killing ability gained a specific cytolytic activity of 81% ± 10.4% SD after stimulation with IL-2 for 24 hours. γδ cells were isolated from peripheral blood by positive enrichment (using a magnetic cell sorting system; purity, 95.2% ± 3.2% SD, n = 21). High natural cytotoxic activity against human neuroblastoma cell lines (>50% specific lysis at an E:T ratio of 20:1) was exhibited by one of 11 donors, whereas two of 11 showed medium cytotoxicity (30% to 50% specific lysis). Eight of 11 donors showed very slight or no lytic activity against human neuroblastoma cells (<30% specific lysis). γδ T cells were also cytotoxic against Daudi (32.7% specific lysis at an E:T ratio of 20:1), Raji (10.3%), Colo 205 (23.1%), A 204 (54%), K 562 (100%), and SK-N-MC (100%) cells. Isolated γδ T cells were grown in Iscove modified Dulbecco medium with IL-2 (400 IU/mL). Increased cell proliferation (38.5% to 182%) was induced with phytohemagglutinin, IL-15, Clodronat, OKT3, or various combinations of these. Results of cold target inhibition assays suggest a natural killer-like activity of the γδ T-cell killing mechanism. Peptidase or papain render neuroblastoma cells unsusceptible to γδ T-cell killing, suggesting the involvement of antigen peptide(s) in the process of neuroblastoma cell killing. Treatment with acid phosphatase reduced specific lysis by 66.5% ± 34.1% SD, which suggests a binding to phosphorylated neuroblastoma cell-surface structures in the killing mechanism of γδ T cells. Heat shock did not affect the extent of neuroblastoma killing by γδ cells. Recognition of neuroblastoma cells by γδ cytotoxic T lymphocytes is negatively regulated by major histocompatibility complex I receptors. Evidence for natural and inducible cell cytotoxicity of γδ T cells against human neuroblastoma cells, easy propagation, purification, and missing alloreactivity in mixed lymphocytes cultures indicates a role for this subpopulation of T lymphocytes in adoptive immunotherapy.

Expression of CD44 isoforms by highly enriched CD34-positive cells in cord blood, bone marrow and leukaphereses

CD34-positive cells were isolated from cord blood (n = 8), bone marrow (n = 4) and leukapheresed material (n = 7), using an immunomagnetic isolation technique, MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). In flow cytometric analysis, cell populations after enrichment revealed a fraction of 96.1% (cord blood), 96.2% (bone marrow) and 98.6% (leukapheresis material) CD34-positive cells. Cells were further stained with antibodies specific for CD44 isoforms: CD44s (SFF-2), CD44v5 (VFF-8) and CD44v6 (VFF-18). CD44-positive cells were detected by directly (FITC, fluorescein isothiocyanate) or indirectly (streptavidin-PE, phycoerythrin)-conjugated fluorochromes in flow cytometric analysis. Analysis was restricted to CD34-positive cells. A high expression of CD44s was noted in all kinds of material under investigation with mean values in the range of 98.6–100%. There was little expression of CD44v6 (mean values in the range of 1.5–3.6%) and very slight expression of CD44v5 (mean values in the range of 0.6–1.4%). The finding that CD34-positive hematopoietic stem cells express CD44v5 and CD44v6 to a very small extent offers the possibility of using antibodies specific to CD44v5 and CD44v6 in immunopurging in the course of autologous stem cell transplantation.

Isolation and phenotypic characterization of CD117-positive cells.

Mononuclear cells derived from cord blood were stained using the CD1 17-specific, fluorochrome-labeled monoclonal mouse antibody 95C3. Additional staining was performed using an isotype-specific rat-anti-mouse antibody, labeled with supermagnetic microparticles. Target cells were enriched by the technique of magnetic cell separation, MACS. The resulting cell population contained 96.5% (+/-1.7% S.D.) CD1 17-expressing cells (n = 12) with different levels of CD117 antigen expression. Using flow cytometry, two cell populations differing in size were found. A majority (93%) of cells with high forward scatter revealed a phenotype positive for CD117 and CD34. Isolated cells revealed a high fraction of hematopoietic progenitors (16%). The technique presented allows for an alternative approach of stem cell enrichment and might be useful in autologous transplantation of cells with hematopoietic properties.

CELL ADHESION MOLECULES ON LARGE GRANULAR LYMPHOCYTES AND ENDOTHELIAL CELLS IN DECIDUA OF EARLY HUMAN PREGNANCY

Sirs: We read with great interest the article by Tortosa et al. (1993) concerning the expression of cell adhesion molecules (CAM) by endothelial cells of early human decidua. Using immunohistochemical techniques, these authors found strong expression of ICAM-1, P-selectin and HLA-DR on decidual endothelial cells, whereas staining of these cells for E-selectin, INCAM-110 and VCAM-1 was weak or absent. Tortosa et al. suggest that this differential expression of CAM by decidual endothelial cells may be responsible for the recruitment of the large and unusual population of lymphoid cells found in the decidua, which is composed mainly of CD56+ + large granular lymphocytes (LGL) with an NK cell phenotype (representing more than 70% of the intradecidual lymphoid cells) and T cells. However, decidual lymphocytes were not investigated for the expression of counter-receptors for the endothelial CAM in this study. In recent investigations of the distribution of CAM on decidual LGL and endothelial cells we found a similar pattern of immunoreactivity of decidual endothelial cells for ICAM-1, VCAM-1 and E-selectin (Marzusch et al. 1993; Ruck et al., in press). In addition to the immunohistochemical investigations, we also studied the expression of CAM on decidual and peripheral CD56 + lymphocytes by flow cytometry, which revealed that the e-chains of the integrin counter-receptors for both ICAM-1 and VCAM-1 - eL and e4, respectively (Springer 1990) - are found on the majority of decidual and peripheral LGL. As the expression of