Generation of antigen-presenting cells for soluble protein antigens ex vivo from peripheral blood CD34+ hematopoietic progenitor cells in cancer patients

Peripheral blood CD34⁺ hematopoietic progenitor cells (PBPC) mediate hematopoietic reconstitution in cancer patients after autologous transplantation and can be expanded ex vivo in the presence of colony-stimulating factors. This study shows that functionally active antigen-presenting cells (APC) for soluble proteins are generated and expanded in these PBPC cultures. CD34⁺ cells were cultured ex vivo in medium containing stem cell factor, interleukin-1β (IL-1β), IL-3, IL-6, and erythropoietin (EPO). The cells from these cultures developed into very potent APC of tetanus toxid and purified derivative of tuberculin for autologous T cells in vitro. The antigen-presenting capacity of these cells was maintained for at least 38 days of culture. These APC resembled immature cells of the myelomonocytic cell lineage by surface marker, immunocytochemistry and ultrastructural analysis. Such APC might be able to present antigens from certain tumors to the immune system.     

Maintenance of transplantation potential in ex vivo expanded CD34(+)- selected human peripheral blood progenitor cells

CD34(+)-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase I/II clinical trial using CD34(+)-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+ cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34(+)-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34(+)-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+ fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 x 10(4) LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo-expanded CD34+ PBPCs might be able to allow long-term reconstitution of hematopoiesis.     

Erythropoietin for the treatment of anemia of malignancy associated with neoplastic bone marrow infiltration

This clinical trial was performed to study the effects of intravenously (IV) administered recombinant human (rh) erythropoietin (EPO) at escalating doses (150, 300, and 450 U/kg, administered as an IV bolus injection, twice weekly, for 6, 4, and 4 weeks, respectively) in five patients with low-grade non-Hodgkin's lymphoma (Ig NHL) and bone marrow involvement and one patient with multiple myeloma (MM). All patients were anemic due to underlying disease. None of the patients had a history of bleeding, hemolysis, renal insufficiency, or other disorders causing anemia in addition to bone marrow infiltrating malignancy. Endogenous EPO serum levels were significantly increased in all patients (74 to 202 mU/mL). Five patients (one MM, four small-cell lymphocytic [SCLC] NHL) showed a dramatic increase of hemoglobin (Hb), hematocrit (Hk) and RBC count becoming obvious on the second EPO dose level. Initial ferritin serum values, which were high mostly due to polytransfusion, were significantly reduced in responding patients. Erythropoiesis of one patient with extensive follicular mixed (fm) NHL did not respond to EPO treatment. Platelet (PLT) count increase (greater than 75% above starting levels) during and following EPO therapy was observed in one patient with MM. Adverse events due to EPO therapy have not been recorded. These findings point out a previously unrecognized capacity of EPO given at pharmacologic doses to stimulate erythropoiesis in patients with anemia due to bone marrow infiltration by neoplastic lymphocytes in spite of enhanced endogenous EPO expression.     

Human Interleukin 2 Gene is Located on Chromosome 4

The interleukin-2 (IL2) gene is assigned to human chromosome 4. A synthetic oligonucleotide representing bases 285 to 324 of the IL2 cDNA clone described by Taniguchi et al. [Nature (London) 302:305-310, 1983] was used as hybridization probe in Southern blots. Eco RI digests of DNA derived from 11 somatic mouse-human cell hybrid clones were used. The IL2 gene was localized to human chromosome 4 based on the observed combinations of segregating chromosomes and bands. Under the conditions of stringency utilized, a single 3.6 kilobase Eco RI fragment hybridized to the oligonucleotide. Molecular weight standards were provided by rehybridizing the blots with an actin cDNA clone, pAct 1, which identified actin gene Eco RI fragments of defined size.     

Subpopulations of human T lymphocytes. VI. Analysis of cell markers in acute lymphoblastic leukemia with special reference to Fc receptor expression on E-rosette-forming blasts

A variety of surface markers, terminal deoxynucleotidyl transferase (TdT) activity, morphologic appearance, and cytochemical composition were studied in a group of 16 patients (13 children, 3 adults) with acute lymphoblastic leukemia (ALL). In 9 children, no surface markers were detected on lymphoblasts (null-type ALL). Leukemic blasts of 4 children formed E-rosettes. These E-rosette-forming blasts from 3 adult patients with ALL, were studied for the presence of Fc receptors. Of the leukemic blasts from these 7 patients, 2--76% expressed receptors for IgG Fc. Only 3 of 7 patients showed 9--42% receptors for IgM Fc. In addition, complement receptors were investigated in 6 of those 7 patients with T-cell ALL. Complement receptors were detected on 9--70% of the E rosette forming blasts from all 6 patients. TdT activity was elevated in T-cell ALL and in children with null-type ALL. The heterogeneity of Fc receptor expression on leukemic blasts in these patients demonstrates a malignant proliferation of T cells in different stages of differentiation or maturation. This observation might be helpful in subclassifying T-cell leukemias with regard to prognosis and the response to therapy.     

Hematologic effects of recombinant human granulocyte colony-stimulating factor in patients with malignancy

The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on hematologic parameters was evaluated in a phase I clinical study in 18 patients with advanced malignancy. G-CSF was administered once daily as a 30-minute infusion for 14 days; three patients each were treated at increasing dose levels of 1, 3, 10, 30, and 60 micrograms kg-1 day-1. A transient decrease in neutrophil and monocyte counts was observed immediately after the G-CSF infusion, followed by a dose-dependent increase of up to 15-fold. G-CSF-induced neutrophils exhibited an increased O2- radical production, and serum levels of enzymes related to granulocyte turnover, including lysozyme and elastase, were markedly elevated during therapy. A dose-dependent depression of platelet counts occurred in the second third of the treatment course, followed by a spontaneous recovery despite continuing therapy. G-CSF was well-tolerated; minor to moderate bone pain was the most common side effect. The primary course of the malignant diseases studied was not significantly altered. G-CSF appears to be an appropriate means to selectively increase the number of functionally competent polymorphonuclear phagocytes.     

Apparent decrease and elimination of BCR/ABL mRNA-expressing residual cells in patients with chronic myelogenous leukemia after allogeneic bone marrow transplantation

Summary A modified two-step polymerase chain reaction (PCR) was used for the amplification of BCR/ABL mRNA in 16 patients with Philadelphia chromosomepositive (Ph+) chronic myelogenous leukemia (CML) following allogeneic bone marrow transplantation (BMT). At different intervals after BMT, patient cells were assessed for the presence of BCR/ABL mRNA by two subsequent rounds of PCR amplification; this procedure increased the sensitivity for the detection of one Ph+ cell in 10^4–5 to one cell in 10^5–6. Eight of 16 patients were negative by two-step PCR 1–39 months after BMT, suggesting an elimination of Ph-positive cells or a decrease below the threshold of detection. Although five patients showed negative results by the one-step PCR only, they were tested positive when nested primers were used, indicating a substantial decrease in the amount of BCR/ABL target mRNA compared with earlier pre- or post-transplant analyses. One patient who was still PCR positive 27 months after BMT became negative 12 months later. Persistence of BCR/ABL mRNA-expressing cells correlated with subsequent clinical relapse only when the transplantation was performed during blast crisis. All patients who underwent transplantation in chronic phase, including those with BCR rearrangement by PCR, are in clinical and hematological remission between 24 and 95 months after BMT. We conclude that aggressive chemotherapy combined with total body irradiation is unable to completely eradicate the malignant clone in all CML patients, and it might be speculated that other mechanisms (e.g., graft versus host reaction [GVHD] or graft versus leukemia effect [GVL]) may effectively eliminate residual leukemic cells.The studies were supported by grant Do 176/5-1 from theDeutsche Forschungsgemeinschaft