Peripheral blood mononuclear cells from autologous hematopoietic stem cell transplantation recipients produce and respond to IL-12.

Autologous hematopoietic stem cell transplantation effectively results in restoration of hematopoiesis, but induces an often prolonged period of T cell dysfunction including redistribution of T cell subsets and defective T cell proliferation. Because IL-2 production is markedly decreased following autologous stem cell engraftment, and because IL-12 has direct stimulatory effects on TH1 cells, a major source of IL-2, we investigated the production and responsiveness of IL-12 of peripheral blood mononuclear cells (PBMC) of autologous stem cell recipients in the first 6 months following engraftment. When stimulated with S. aureus Cowan I (SAC), PBMC from autologous hematopoietic transplant recipients in the early months following engraftment show no decrease in production of IL-12 as compared to control PBMC. Furthermore, transplant-derived PBMC appear to be functionally responsive to exogenously provided IL-12 as indicated by several criteria. In vitro proliferation of total PBMC and of isolated CD4+ T cells from transplanted recipients to PHA (1 microgram/ml) + IL-12 (20 U/ml) was comparable to controls, excluding the possibility that only NK or CD8+ cells respond to IL-12. Culture of both control and transplant-derived PBMC in PHA + IL-12 (20 U/ml) or IL-2 (100 U/ml) + IL-12 (20 U/ml) combinations yielded comparable production of IFN-gamma, one of the major biological effects of IL-12 in vivo, as well as equal production of TNF-alpha, a costimulatory factor of IL-12-mediated induction of IFN-gamma by NK cells. Taken together, this in vitro evidence suggests that following autologous transplantation, PBMC do not appear to have either decreased production of endogenous IL-12 or defective functional responsiveness to exogenously provided IL-12.

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.

Blunted rise in intracellular calcium in CD4+ T cells in response to mitogen following autologous bone marrow transplantation.

Following autologous bone marrow transplantation (ABMT), both impaired T cell activation and defective production of the principal T cell growth factor, interleukin-2 (IL-2), has been observed. These processes are dependent on a rise of intracellular calcium ([Ca2+]i), a step which follows binding of T cell receptor (TCR) and transduction of signal via the generation of cytoplasmic second messengers. In order to better understand the nature of defective cellular immunity in ABMT, in the present study we investigated the rise of [Ca2+]i in T cells of recipients of ABMT. By concomitant labelling lymphocytes with anti-CD4 antibody and addition of fluo-3 as fluorescent calcium indicator, we have selected for the T cell subset which is the principal source of IL-2. Short-term (less than 1 year post-transplantation) recipients of ABMT show a statistically significant blunted rise in [Ca2+]i in response to concanavalin A as compared to normal controls not accounted for solely by a decreased percentage of CD4+ cells in these patients. The [Ca2+]i response of CD4+ cells from long-term (greater than 1 year post-transplant) recipients was lower than that of the normal group although not to a statistically significant level. These findings suggest that following ABMT is a defect in the early stages of T cell activation involving either T cell receptor binding or early signal transduction ultimately resulting in depressed transcription of IL-2 mRNA. These defects are analogous to findings in both allogeneic transplantation where factors of histoincompatibility and graft-versus-host disease (GVHD) come into play, as well as in the defective T cell activation of the normal ageing process.

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.

Concentration of bone marrow progenitor cells by separation on a Percoll gradient using the Haemonetics model 30.

To perform an optimal ex vivo bone marrow purge, it is necessary to concentrate the bone marrow progenitor cells and to eliminate both the red blood cells and the polymorphonuclear leucocytes. To achieve this goal which cannot be accomplished by using the Haemonetics model 30 alone, we used the Haemonetics model 30 and a density gradient together in a two-step procedure. In the first step we obtained the buffy coat from original bone marrow grafts and in the second we reintroduced these buffy coats into the Haemonetics bowl followed by Percoll, adjusted to 1.079 g/ml, at 5 ml/min in order to recover the light density mononuclear cells. After this second step, the mean volume of the marrow and the RBC and nucleated cell contaminations were reduced to 9%, 0.96% and 16% of their original values the unseparated bone marrow, respectively. This was far better than the values obtained after the first step (volume: 19%; RBC: 10%; nucleated cells: 54%). The CFU-GM recoveries after the first and second steps were 71% and 70% of the original samples, respectively. The entire procedure lasted between 75 and 150 min. At this time, 17 of the 24 patients whose bone marrow was separated using Percoll gradient in the Haemonetics bowl have been grafted. Thirteen of these 17 patients had an evaluable haematological recovery which was complete and rapid for all but one patient with acute myeloid leukaemia. These results demonstrate that the introduction of a density gradient into the Haemonetics model 30 bowl is possible and effective. The reduction in total volume and cell number permits ex vivo purging, without decreasing the grafting capability.

Detection of small cell lung cancer bone marrow metastases by immunofluorescence.

It is well known that small cell lung cancer (SCLC) has a high propensity to metastasize to the bone marrow and that such involvement has a prognostic significance. A more accurate detection of these bone marrow metastases is thus mandatory. In this study, we analysed the results of the detection of these metastases using an indirect immunofluorescence test. For this purpose, 3 anti-SCLC rat monoclonal antibodies (MoAbs) specific for 3 different antigens (LCA1, LCA2, LCA3) have been utilized to examine 59 bone marrow samples from patients at time of diagnosis and 20 samples from chemotherapy treated patients. Eight patients had bone marrow clearly involved by morphological analysis. They all had fluorescent cells recognized at immunodetection with at least 2 MoAbs positive for 7 out of the 8 samples. Fifteen samples, negative by morphological analysis, were proven positive by immunofluorescence. In 12 cases, involvement was detected only by 1 MoAb (6 anti-LCA1, 2 anti-LCA2, 4 anti-LCA3). A correlation was found between the number of samples proven positive by morphological analysis and the number of positive MoAbs for these samples (p less than 0.005). Among the bone marrow samples provided by the 32 limited disease patients, LCA positive cells were detected in 9 (28%) compared to 14 out of the 27 (52%) samples from extensive disease patients (p less than 0.05). We concluded that the indirect immunofluorescence with a panel of MoAbs increases the rate of detection of bone marrow SCLC metastases.