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In this study, mononuclear cells (MNCs) from granulocyte colony-stimulating factor (G-CSF)-mobilized blood stem cell (BSC) harvests from 104 healthy donors were analyzed for their immunological functions and compared with MNCs from 28 steady-state nonmobilized donors. The relationships between donor characteristics (age, gender, weight, and HLA type) and immune functions of the harvests were also analyzed. There was a significant (P <.01) decrease in natural killer and lymphokine-activated killer (LAK) cell-mediated cytotoxicity for G-CSF-mobilized effector cells compared with nonmobilized cells. Similarly, there was a significant (P <.005) decrease in both T-cell and B-cell mitogen response in G-CSF-mobilized cells compared with nonmobilized cells. There was dose-dependent inhibition of LAK cell-mediated cytotoxicity, but this effect was not seen with other immune function assays. Changes in immune function did not appear to be determined by frequency of cellular phenotypes or expression of effector function genes seen in a reverse-transcription polymerase chain reaction. There was a significant relationship between expression of certain HLA alleles (A1, A3, A24, B44, B62, DR15, DR17; all P <.01) and increased immune function, such as cytotoxicity and/or mitogen response. A decrease in immune function with the HLA-DR13 expression was also observed (P <.01). Since the G-CSF increases the number of MNCs, the increase in effector cells might compensate for decreased immune functions of these cells in vivo when transplanted into patients. These results suggest a decreased immune function in G-CSF-mobilized BSC harvests and warrant further studies to correlate these data with clinical outcome.

Bone marrow transplantation (BMT) for severe combined immunodeficiency (SCID) with human leukocyte antigen (HLA)-identical sibling donors but no pretransplantation cytoreduction results in T-lymphocyte engraftment and correction of immune dysfunction but not in full hematopoietic engraftment. A case of a 17-month-old girl with adenosine deaminase (ADA) deficiency SCID in whom full hematopoietic engraftment developed after BMT from her HLA-identical sister is reported. No myeloablative or immunosuppressive therapy or graft-versus-host disease (GVHD) prophylaxis was given. Mild acute and chronic GVHD developed, her B- and T-cell functions became reconstituted, and she is well almost 11 years after BMT. After BMT, repeated studies demonstrated: (1) Loss of a recipient-specific chromosomal marker in peripheral blood leukocytes (PBLs) and bone marrow, (2) conversion of recipient red blood cell antigens to donor type, (3) conversion of recipient T-cell, B-cell, and granulocyte lineages to donor origin by DNA analysis, and (4) increased ADA activity and metabolic correction in red blood cells and PBLs.

A considerable number of patients with malignancies who are treated with high-dose therapy and hematopoietic stem cell transplantation subsequently relapse. Analyses of peripheral blood stem cell (PBSC) harvests obtained from 49 cancer patients showed that the PBSC harvest contained precursors for antitumor effector cells. Ex vivo manipulation of these harvests to maximize the antitumor effector cell activity may provide a new therapeutic approach to decrease or eliminate any minimal residual disease that remains after high-dose therapy. Characterization of PBSC from consecutive collections determined the collections best suited for ex vivo augmentation of antitumor cytotoxic effector cells. We report the results of a functional and phenotypical characterization of PBSC obtained from six consecutive collections from 18 cancer patients receiving granulocyte-macrophage colony-stimulating factor (GM-CSF) for hematopoietic stem/progenitor cell mobilization. The PBSC were evaluated for their cytotoxicity using the 51Cr-release assay. The frequency and subsets of lymphocytes were determined using flow cytometry with appropriate specific marker antibodies and differential cell counts. The content of hematopoietic progenitor cells in each collection was determined using a colony-forming unit granulocyte-macrophage (CFU-GM) culture assay. The frequency of cytotoxic effector cells including lymphokine-activated killer (LAK) cell precursors and lymphocytes was significantly greater (P < .05) in the early collections, whereas the later collections contained significantly (P < .05) more CFU-GM progenitor cells and fewer cytotoxic effector cells. Thus, our results show that PBSC obtained from advanced cancer patients do contain considerable levels of precursor cells for the generation of LAK cell populations. These results suggest that cells from the earlier collections are best suited for ex vivo manipulation to augment the antitumor effects.

The coagulant activity of tissue factor (TF) in the membranes of cultured cells is increased after physical disruption of the cells, and a similar increase can be elicited by treatment of cell cultures with calcium ionophore and calcium. We observed that the supernatants of cultures treated with calcium and ionophore 4-Br-A23187 contained more TF activity than those of control cultures. Phase contrast microscopy showed that cultures treated with ionophore and calcium contained rounded cells, membrane vesicles, and cell fragments. Laser-activated fluorescence microscopy of cells stained for tissue factor antigen showed that 4-Br-A23187, in the presence of 5 mmol/L calcium, caused progressive changes in cell morphology. Treatment of cultures with thrombin receptor agonist peptide caused a transient increase in cytoplasmic calcium, but had no short-term effect on TF activity or cell morphology. These combined results show that 4-Br-A23187, with extracellular calcium, increases TF activity concomitant with dramatic changes in cell morphology and the plasma membrane. The effect of increased cytoplasmic calcium on TF expression may therefore be similar in mechanism to other models of cell injury and may be caused by the effects of a sustained increase of cytosolic calcium on cellular elements that influence membrane stability and the distribution of TF per se.

OMA-AML-1 was established from a patient with acute myelomonocytic (M4) leukemia at fifth relapse when blasts were greater than 85% CD34+, CD15-. Leukemic cells were established in suspension culture and independently grown as subcutaneous tumors in SCID mice. Cells growing in suspension culture underwent differentiation by phenotypic and morphologic criteria. In contrast, cells grown as subcutaneous solid tumors in SCID mice maintained progenitor cell characteristics with high-density CD34 expression and lack of morphologic differentiation. A tendency toward differentiation to CD15+, CD34- cells in vitro and self-renewal of CD34+, CD15- cells in vivo was consistently demonstrated regardless of whether cells were initially grown in vitro or in vivo. The cell line maintains both a CD34+, CD15- progentitor cell pool and a non-overlapping, CD15+, CD34- differentiating cell compartment after more than 1 year in continuous culture. Cell cycle analysis and cloning experiments were consistent with terminal differentiation occurring in the CD15+, CD34- population. The cell line shows concentration-dependent proliferative responses to interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6, but not to granulocyte CSF (G-CSF). OMA-AML-1 appears to mimic several features of normal myeloid hematopoiesis and should prove useful for the study of normal and malignant myeloid differentiation.

In the hematopoietic system, the B-cell associated antigen CD24 is expressed at high density on B cells, B-cell precursors, and B-cell malignancies as well as at low density on peripheral blood polymorphonuclear leukocytes. The 42-Kd sialoglycoprotein has not been previously demonstrated to be expressed on T cells, thymocytes, or T-cell malignancies. We identified three patients with mycosis fungoides/Sézary syndrome that showed low density expression of the CD24-related epitope recognized by antibody BA-1 on circulating T cells. All three patients had Sézary cells by morphologic assessment and clonal T-cell populations in the peripheral blood by gene rearrangement studies. In two of these patients, indirect immunofluorescence with a panel of six anti-CD24 monoclonal antibodies demonstrated reactivity for two of six antibodies in one case and only one of six antibodies in the other. The biologic significance of CD24-related epitope expression on circulating T cells in mycosis fungoides/Sézary syndrome is unclear. However, these findings suggest that differential, low density expression of CD24-related epitopes (BA-1+, OKB2-) may be a useful phenotypic marker for identifying circulating Sézary cells.