Defective cytokine production following autologous stem cell transplantation for solid tumors and hematologic malignancies regardless of bone marrow or peripheral origin and lack of evidence for a role for interleukin-10 in delayed immune reconstitution.

A substantial body of evidence accumulated in recent years indicates a protracted delay in immune reconstitution following autologous stem cell transplantation. In order to investigate the cellular basis of this phenomenon, peripheral blood mononuclear cells were studied from recipients of autologous stem cell transplantation for solid tumors and hematological malignancies. On stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate, transplant-derived peripheral blood mononuclear cells demonstrate statistically significant depressed production of interleukin 3 (IL-3), IL-4, granulocyte-macrophage-colony-stimulating factor, and gamma-interferon as compared to normal controls, during the first 6 months following engraftment, which recover to normal levels 6 months or more posttransplant. When the overall group of transplant recipients is compared to the control group, there is a statistically significant lower production of IL-2. In addition, no differences were observed regardless of the source of the engrafted stem cells, whether from bone marrow alone (autologous bone marrow transplantation), from peripheral blood stem cells alone, or from a combination of autologous bone marrow transplantation and peripheral blood stem cells. The defect persisted past 6 months postengraftment. Transplant-derived peripheral blood mononuclear cells were stimulated with combinations of either phytohemagglutinin plus the calcium ionophore A23187, thereby circumventing the requirement for accessory cell function, or with phorbol 12-myristate 13-acetate plus anti-CD28 monoclonal antibody, mimicking the CD28-B7 cell surface-ligand interaction capable of triggering and stabilizing IL-2 gene transcription. In both situations, decreased production of IL-2 as compared to controls was observed in individuals within 6 months of transplantation. Quantitative polymerase chain reaction indicates that decreased transcription of IL-2 mRNA following transplantation is not due solely to a decrease in the absolute numbers of CD4+ T-cells but is secondary to reduced numbers of transcript copies per cell. Production of IL-10 was found to be decreased regardless of whether the autologous graft was of bone marrow or peripheral blood origin. These findings are consistent with the conclusion that: (a) multiple dysregulations exist in the production of cytokines important in immune homeostasis; (b) a defect occurs at or prior to the level of transcription of IL-2 mRNA; (c) IL-10 does not play a direct role in the pathogenesis of posttransplantation immunosuppression; and (d) there is no evidence that peripheral blood stem cells may be superior to bone marrow-derived stem cells in accelerating immune reconstitution.

Transcription of genes encoding granulocyte-macrophage colony-stimulating factor, interleukin 3, and interleukin 6 receptors and lack of proliferative response to exogenous cytokines in nonhematopoietic human malignant cell lines.

Studies in recent years have suggested that human tumor cell lines are capable of responding in vitro to hematopoietic growth factors. In the present study, we investigate the transcription of the alpha and beta subunits of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the alpha and beta subunits of interleukin 3 (IL-3) receptor, and the single subunit of interleukin 6 (IL-6) receptor and its associated gp130 transduction protein by PCR amplification of reverse-transcribed cellular mRNA in 34 malignant cell lines derived from a variety of histological cell types. mRNA for only a single subunit polypeptide was found in a significant minority of cell lines (23%), while in 20% both the alpha and beta subunits of either the GM-CSF receptor or the IL-3 receptor were detected among a number of different histological cell types. Transcription of the gene encoding the IL-6 receptor was found in 38% of cell lines, and all lines transcribed the gp130 transduction protein, consistent with previous observations on the ubiquity of that polypeptide. In order to test the in vitro effect of exogenously added growth factors on those malignant cell lines transcribing complete cytokine receptor, either GM-CSF, IL-3, or IL-6 was added in therapeutic concentrations (20-500 ng/ml) and cellular proliferation was measured by incorporation of [3H]thymidine. No stimulation was seen at either 3 and 6 days of culture. Production of cytokine by these cell lines was investigated at the level of transcription and by assay of peptide product. None transcribed mRNA for either GM-CSF or IL-3, while 5 of 6 (STD, DOZ, ADE, Hep-2, and Detroit) expressed IL-6 mRNA. Of these latter, 2 cell lines (ADE and Hep-2) produced IL-6 as determined by bioassay, while none produced GM-CSF or IL-3 by enzyme-linked immunosorbent assay. This suggests that in the case of GM-CSF and IL-3, failure to proliferate on addition of cytokine is not due to the prior presence of endogenous production. In contrast, at least a subset of malignant cell lines may involve a closed IL-6 autocrine loop saturating cell surface sites. These findings suggest that the ability to transcribe the genes encoding cytokine receptor is by itself insufficient to render cells cytokine responsive and that malignant cells may lack the cellular machinery for cytokine-induced proliferation. This in turn suggests that therapeutic administration of either GM-CSF, IL-3, or IL-6 may involve no additional risk of tumor regrowth in vivo.

Immunological and pharmacological removal of small cell lung cancer cells from bone marrow autografts.

Autologous bone marrow transplantation is used in small cell lung cancer (SCLC) to reverse the hematological toxicity induced by high dose therapy even though the presence of cancerous cells in the graft is potentially dangerous by reinfusion of the disease along with the hematopoietic stem cells. The present studies were undertaken to examine the effectiveness of anti-SCLC rat monoclonal antibodies LCA1 and LC66 plus human complement combined with a derivative of cyclophosphamide (Asta-Z 7557) for the elimination of cancerous clonogenic cells from the graft. In a series of assays conducted with three SCLC cell lines, used alone or mixed with normal bone marrow cells, the addition of Asta-Z 7557 to two cycles of treatment with monoclonal antibodies plus complement results in a 4- to 5-logarithmic reduction of the clonogenic SCLC cells detectable by limiting dilution analysis. This was superior to either treatment used alone. When normal bone marrow was submitted to the same treatment, a median (range) of 44% (15-77%) of the colony-forming unit, granulocyte-macrophage was recovered. These results suggest that the association of immunological (LCA1 and LC66 plus human complement) and pharmacological (Asta-Z 7557) removal methods is effective for purging metastatic clonogenic cells from bone marrow of SCLC patients and could be considered before autologous bone marrow transplantation.

Recombinant human interleukin-2 restores in vitro T-cell colony formation by peripheral blood mononuclear cells after autologous bone marrow transplantation.

Immune reconstitution after autologous bone marrow transplantation (ABMT) was studied in peripheral blood by phytohemagglutinin stimulated T-cell colony formation (CFU-TL) and by surface phenotype analysis of T-lymphocytes with monoclonal antibodies. Twenty-six patients (15 small-cell lung cancer, 5 lymphoma, 3 acute myeloid leukemia [AML], 2 germ cell cancer, and 1 melanoma) were conditioned with high-dose multiple drug combinations (plus total body irradiation in AML patients). No maintenance chemotherapy was given following treatment. Despite a rapid return to normal values of peripheral T3+, T11+ lymphocytes, the T4/T8 ratio remained below 1.0 up to 24 months after transplant, regardless of infection by cytomegalovirus (CMV). A high percentage (26% +/- 3%) of lymphocyte cells with immature phenotype (T8+, Ia+) was found during the first 6 months after transplant. Out of 84 cultures, performed in 26 patients, no growth was observed in 47 instances (22 patients) up to 28 months after grafting. Growth occurred in 37 cultures (11 patients, from 1 to 51 months after transplant) although it never reached the colony numbers of normal controls. Recombinant human interleukin-2 (rIL-2) added to lymphocyte culture induced proliferation in 8 (4 CMV-positive and 4 CMV-negative patients) out of 12 instances of no growth. In cases of depressed CFU-TL (20 cultures), rIL-2 induced a 48% and 92% increase in six CMV-positive patients and nine CMV-negative patients, respectively. These observations show that after ABMT and regardless of CMV status, defects in CFU-TL can be partially corrected by rIL-2.