A comparison of ex vivo expanded DCs derived from cord blood and mobilized adult peripheral blood plastic-adherent mononuclear cells: decreased alloreactivity of cord blood DCs.

BACKGROUND: Cord blood (CB) has been used as an alternative source of transplantable allogeneic stem cells for a variety of malignant and non-malignant diseases. However, we have demonstrated delayed recovery of T- and B-cell function, and T-cell subsets post unrelated CB transplantation (UCBT), and deficiencies of CB mononuclear cells (MNC) in producing cytokines, including G-CSF, GM-CSF, M-CSF, IL-12, and IL-15. In this study we have investigated the ex vivo generation of DC from CB versus mobilized adult peripheral blood (APB) for later use as adoptive cellular immunotherapy. METHODS: CB and APB-adherent MNC were cultured in serum-free media with GM-CSF IL-4, FLT-3 ligand, tumor growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) for 7 days. Morphology, phenotype, immunohistochemistry, clonogenic activity, and alloreactivity in MLR were evaluated. RESULTS: CB and APB monocyte-derived ex vivo expanded DC expressed similar DC markers CD83 (31.27+ 11.7% versus 34.0+ 5.2%, CB versus APB), CD1a (23.4+ 4.2% versus 27.6+ 6.3%), and CD80 (21.97+ 12.01% versus 27.7+ 5.95). Immunohistochemistry showed that cells with DC morphology expressed CDla but not CD14. Neither FLT-3 ligand nor TGF-fl enhanced DC expansion. Addition of 10% autologous plasma to CB cultures promoted greater cell survival and a 150% increase in CDla + /CD80+ cell recovery. CB DC were 62% as effective stimulators of adult allogeneic T-cels as APB DC (p < .05) in allogeneic MLR. DISCUSSION: While phenotypically similar, CB and APB DC have differential potency in allogeneic MLR, which may account for the difference in GvHD and infection incidence and severity between UCBT and allogeneic stem cell transplantation, and may require a different approach for adoptive cellular immunotherapy. The mechanism(s) associated with these differences require further elucidation.

Feasibility study of IL-11 and granulocyte colony-stimulating factor after myelosuppressive chemotherapy to mobilize peripheral blood stem cells from heavily pretreated patients.

PURPOSE: Pediatric patients with solid tumors treated with prolonged dose-intensive chemoradiotherapy are poor mobilizers of peripheral blood stem cells (PBSC). We have conducted a pilot study to mobilize PBSC in eight pediatric patients with relapsed solid tumors using ifosfamide, carboplatin, and etoposide (ICE) followed-up by IL-11 plus granulocyte colony-stimulating factor (G-CSF). PATIENTS AND METHODS: Patients received ifosfamide 1.8 g/m2 per day for 5 days, carboplatin 400 mg/m2 per day for 2 days, and etoposide 100 mg/m2 per day for 5 days. After completion of ICE chemotherapy, patients received daily subcutaneous injections of G-CSF (5 microg/kg per day) and IL-11 (50-100 microg/kg per day) until peripheral stem cell apheresis. RESULTS: The median age was 11 years. Diagnosis included three relapsed Hodgkin disease, three relapsed central nervous system tumors, one relapsed Wilms tumor, and one relapsed rhabdomyosarcoma. The median number of apheresis procedures required to obtain 5 x 10(6) CD34+ cells/kg was one. The mean +/- standard error of mean (SEM) total CD34+ cells collected was 14.0+/-2.7 x 10(6)/kg. The mean +/- SEM total CD34+/CD41+ cells collected was 4.6+/-1.9 x 10(6)/kg. Seven of the eight patients have subsequently undergone myeloablative chemotherapy with autologous PBSC transplantation and have reconstituted hematopoiesis with a median time to neutrophil recovery of 10 days and platelet recovery of 15.5 days. CONCLUSIONS: We conclude that the regimen of ICE/IL-11 plus G-CSF is successful in mobilizing large numbers of CD34+ PBSC cells with a limited number (one) of apheresis collections in patients that have previously been heavily pretreated with chemotherapy/radiotherapy.

Mobilization, collection, and processing of autologous peripheral blood stem cells: development of a clinical process with associated costs.

We surveyed five academic medical centers to develop a clinical process for patients undergoing cytokine mobilization and leukapheresis prior to autologous peripheral blood stem cell transplantation. Costs were obtained from three centers and applied to each component of the pathway. Costs were divided into three categories: (1) pre-apheresis evaluation; (2) process of apheresis; (3) post-apheresis and peripheral blood stem cells processing. All centers participated in the development of the leukapheresis pathway. Because charges vary greatly among institutions, costs were determined from three of the institutions and a mean was calculated for each of the components of the process. Pre-apheresis costs consisted of central line placement, blood work, and the price of cytokine (rhG-CSF). Costs associated with apheresis included professional fees (for physicians and nurses), leukapheresis with stem cell cryopreservation, storage, sterility testing, analysis of circulating CD34+ cell counts, and 1 day of cytokine therapy. The post-apheresis process included thawing with sterility testing along with CD34+ cell number analysis and the performance of clonogenic assays. Total costs were as follows: (1) pre-apheresis, $2711; (2) apheresis, $2990; and, (3) post-apheresis/stem cell processing, $754. This survey from five academic medical centers provides the average costs associated with three main components of the apheresis procedure. Because many patients require multiple aphereses, interventions to achieve target CD34+ cell collections in as few collections as possible would result in significant cost reduction.

Immunotherapy with interleukin-2 and alpha-interferon after IL-2-activated hematopoietic stem cell transplantation for breast cancer.

We previously demonstrated findings suggestive of autologous GVHD in patients receiving IL-2-activated peripheral blood stem cells (PBSC) with IL-2 after transplantation. A pilot study was designed to test tolerability, feasibility and frequency of autologous GVHD and engraftment using IL-2 and alpha-IFN post-transplantation. After cyclophosphamide (6 g/m2) and carboplatin (1800 mg/m2), patients with high-risk stage II or III breast cancer received chemotherapy and rhG-CSF mobilized autologous PBSC that had been cultured in IL-2 for 24 h. Subcutaneous administration of IL-2 began on day 0 at 6 x 10(5) IU/m2/day for 5 of 7 days each week and continued for 4 weeks. Once engraftment occurred, alpha-IFN was initiated at a dose of 1 x 10(6)/m2/day subcutaneously for 30 days. Thirty-four consecutive patients with stage II (n=20), IIIA (n=6) and IIIB (n=8) disease were treated. All patients were without evidence of disease at the time of transplantation. The average time required for the ANC to reach 500/mm3 was 10 days (range: 8-11 days) and for platelets to reach 20000/mm3 was 10.7 days (range: 6-21 days). Forty-seven percent of patients (n=16) completed the full course of immunotherapy; the remaining patients received attenuated doses due to patient's request (n=6), development of temperature >38 degrees C (n=3), development of neutropenia (n=3), serious infection (n=1) and miscellaneous reasons (n=5). Four patients experienced transient moderate toxicities (level 3) including elevated liver function tests, nausea, rash and capillary leak syndrome. Pathological findings suggestive of skin GVHD developed in 43% of patients (12/28 patients) when skin biopsies were evaluated in a blinded fashion. At 13 months post-transplant (median; range: 5-24 months), 28 patients (82%) remain disease-free. These results demonstrate the feasibility and toxicity of this regimen along with pathological findings compatible with autologous GVHD of the skin.

Differential effects of IL-2 incubation on hematopoietic potential of autologous bone marrow and mobilized PBSC from patients with hematologic malignancies.

Culturing of hematopoietic progenitor cells for 24 h with IL-2 generates cytotoxic effector cells that mediate in vitro and possibly in vivo antitumor activity. We examined the effect of IL-2 incubation on progenitor cells from 24 patients with hematologic malignancies using paired autologous bone marrow (ABM) and PBSC to determine differences in hematopoietic potential. Cells were cryopreserved and stored in liquid nitrogen until conditioning therapy was completed. After thawing, cells were incubated with IL-2 for 24 h at 37 degrees C. Paired samples of ABM and PBSC from the same patient were analyzed for nucleated and mononuclear cell number, CD34 antigen expression, and colony-forming unit (CFU) activity before and after IL-2 incubation. There was a significant decrease in the average number of mononuclear cells (MNC) (x10(8)/kg) (<0.001) and CD34+ cells (x10(6)/kg) (0.006) from both ABM and PBSC after 24 h IL-2 culture (ABM MNC: 0.6+/-0.1 vs. 0.4+/-0.0, p = <0.001; PBSC MNC: 4.4+/-0.5 vs. 3.7+/-0.4, p = 0.03; ABM CD34+: 2.4+/-0.5 vs. 1.3+/-0.3, p = <0.001; PBSC CD34+: 6.6+/-1.8 vs. 5.0+/-1.2, p = 0.05). However, whereas ABM CFU/10(5) MNC plated (269.3+/-47.2 vs. 385.6+/-70.6) were significantly increased (p = 0.005), there was no change in PBSC CFU (271.0+/-47.2 vs. 257.3+/-48.5). The mean plating efficiency (%) of ABM CD34+ cells was markedly increased after IL-2 incubation (10.1+/-3.3 vs. 19.0+/-7.2, p = 0.04), although it was lower than that of PBSC CD34+ cells, which did not change significantly in culture (29.4+/-5.5 vs. 36.0+/-6.5). Additional work is in progress to determine the cause and significance of the enhanced plating efficiency of the ABM progenitor cells.

A pilot study evaluating interleukin-2-activated hematopoietic stem cell transplantation for hematologic malignancies.

Cytotoxic effector cells are generated when autologous hematopoietic cells (HSC) are cultured with IL-2 for 24 h. Infusion of these cells followed by IL-2 administration may moderate a graft-versus tumor (GvT) effect in vivo. Sixteen patients--7 with non-Hodgkin's lymphoma (NHL), 2 with AML, 4 with multiple myeloma (MM), and 3 with Hodgkin's disease (HD)--received busulfan (4 mg/kg/day for 4 days) and cyclophosphamide (60 mg/kg/day for 2 days) or cyclophosphamide/TBI (1320 cGy). Autologous HSC were activated by culturing with IL-2 for 24 h before reinfusion. Subcutaneous administration of IL-2 began after engraftment at 1.8 x 10(6) IU/m2/day for 1-4 weeks. Neutrophil engraftment occurred on day 13.1 (median) (range 9-45 days), and platelet engraftment occurred on day 19.3 (median) (range 7-54 days) for 15 patients, with delayed engraftment observed in 3 patients. One patient experienced a fatal cardiac arrhythmia. Five patients developed transient skin rashes with histologic evaluation demonstrating findings consistent with GvHD. At 17 months (median) (range 7-23 months), 9 patients are alive, with 6 patients remaining disease free. This pilot trial demonstrates mild to moderate toxicities and a possible delay in platelet engraftment. Further trials will determine the optimal dose and duration of IL-2 therapy and possible impact of this therapy on survival. Laboratory investigations are evaluating the presence of autologous GvHD and a possible GvT effect.

Interleukin-2-activated hematopoietic stem cell transplantation for breast cancer: investigation of dose level with clinical correlates.

Incubating hematopoietic stem cells with IL-2 in vitro for 24 h generates cytotoxic T cells. When infused into patients, these cells may stimulate a graft-versus-tumor (GVT) effect. This clinical trial was designed to assess the ability of IL-2 activated peripheral blood stem cells (PBSC) to reconstitute hematopoiesis, to investigate dose levels and dose-limiting toxicities of IL-2, and to evaluate clinical results and preliminary laboratory effects using a combination of IL-2-activated autologous PBSC followed by IL-2 after transplantation. Sixty-one women with stage II-IV breast cancer were treated. After the administration of carboplatin (200 mg/m2/day for 3 days) and cyclophosphamide (2 g/m2/day for 3 days), patients received autologous PBSC that were cultured in IL-2 for 24 h followed by parenteral administration of IL-2 beginning the day of transplantation. Three escalating doses of IL-2 were evaluated with increasing duration up to 4 weeks. Of the 57 patients receiving IL-2 after tranplantation, 19 patients (33.3%) were unable to complete the planned course of IL-2 therapy due to persistent fevers (n = 9), diarrhea (n = 2), pulmonary capillary leak syndrome (n = 3), development of a rash (n = 1), atrial fibrillation (n = 1), or patient's request (n = 3). One death occurred during hospitalization. Engraftment of neutrophils occurred on day 11.5 (mean; range 8-21 days) and platelets on day 11.7 (mean; range 7-33 days). The maximal tolerated dose of IL-2 was 6 x 10(5) IU/m2/day for 4 weeks. Disease-free survival rates for all stages were comparable to current reports in the literature. Preliminary laboratory evaluations include FACScan analysis of the IL-2 activated PBSC demonstrating an increased percentage of CD3+, CD25+, HLA-DR+ T cells. Phenotypically similar cells were present in peripheral blood samples of patients when tested 15 days after transplantation. This study demonstrates successful engraftment with IL-2-activated PBSC after high-dose chemotherapy for women with stage II-IV breast cancer. The regimen is feasible and, although toxicities are common, they are manageable and correlate with increasing dose and duration of IL-2.

Hematopoietic potential of IL-2-cultured peripheral blood stem cells from breast cancer patients.

Patients receiving autologous transplants for various malignancies generally experience an increased incidence of relapse compared with patients receiving unmanipulated allogeneic transplants. We initiated a protocol for IL-2 activation of peripheral blood stem cells (PBSC) for induction of in vitro and in vivo autologous graft-versus-tumor (GVT) activity in patients with breast cancer. In this study we analyzed the effects of 24 h of IL-2 incubation on the hematopoietic potential of PBSC from these patients. Cells collected by leukapheresis were first cryopreserved and stored in liquid nitrogen, then thawed rapidly and incubated with IL-2 in a serum-free system for 24 h, with samples analyzed before and after incubation. Although there was a significant drop in mononuclear cells (MNC) (from 4.5 to 3.7 x 10(8)/kg) and CD34+ cells (from 12.3 to 7.5 x 10(6)/kg) after 24 h in culture, there was no significant change in colony-forming units (CFU) (from 12.5 to 11.5 x 10(5)/kg). Time to engraftment (neutrophils: < 0.5 x 10(9)/l; platelets: > 20 x 10(9)/l) was comparable to a cohort of similar patients receiving non-cultured PBSC transplants. These results indicate that mobilized frozen/thawed PBSC which have been cultured in IL-2 for 24 h retain adequate potential for hematopoietic reconsistution in this group of patients.

Interleukin-2 activation of chemotherapy and growth factor-mobilized peripheral blood stem cells for generation of cytotoxic effectors.

Based on our previous studies demonstrating marked anti-tumor activity of interleukin-2 (IL-2)-activated bone marrow in vitro and in vivo, we studied the generation of anti-tumor cytotoxic effectors from chemotherapy- and growth factor-mobilized PBSC from 12 patients with different solid tumors. As chemotherapy and growth factor priming could lead to important qualitative and quantitative alterations of lymphoid cells, we also looked at the distribution of lymphoid cells contained in primed PBSC. In addition, different variables were defined for successful application of the technique to clinical protocols. The cells were placed in culture at varying cell densities in either serum-containing or serum-free culture medium, supplemented with IL-2, 100 or 1000 Cetus units/ml at 37 degrees C for 24 or 72 h, in flasks or in culture bags. Anti-tumor cytotoxicity was tested against A375 (melanoma), K562 (CML) and Daudi (Burkitt's lymphoma) cell lines in standard 4 h 51Cr release assay. Marked cytotoxicity was seen against all cell lines tested (A375: 32.7% +/- 5.2; K562: 52.8% +/- 4.8; Daudi: 50.5% +/- 7.2). Cytotoxicity was comparable in serum-containing and serum-free culture conditions and in tissue culture flasks and bags. Cell density up to 10 x 10(6)/ml was not associated with any significant decline in cytotoxicity. IL-2 activation of PBSC after thawing led to the generation of cytotoxicity comparable to that obtained with fresh PBSC. On flow cytometric analysis, the proportion of CD8+ T cells and NK cells (CD56+) was found to be higher in primed PBSC than in control peripheral blood mononuclear cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The impact of harvest center on quality of marrows collected from unrelated donors.

The total number and distribution of nucleated cells in harvested bone marrow are potentially important determinants of patient outcome following bone marrow transplantation. In order to assess whether marrows collected from predominantly unrelated donors at Georgetown University Medical Center (GUMC) were different in cellular content from marrows collected at harvest centers outside of GUMC, we compared the nucleated cell counts and mononuclear cell subset distribution (CD34, CD3, CD4, CD8, CD19 antigen-positive cell content) of 10 consecutive marrows harvested at GUMC to 10 unrelated donor marrows from outside harvest centers. Significantly higher nucleated cell counts and CD34 antigen-positive cell content and significantly lower CD3 and CD4 antigen-positive T-cell numbers were demonstrated among the marrows harvested at GUMC. These results confirmed significant variability in marrow collection practices between GUMC and 10 different outside harvest centers and suggest that strict adherence to a specific collection procedure, involving small volume marrow aspirations and multiple puncture sites, results in a product with a high number of early hematopoietic progenitor cells and minimal contamination by peripheral blood. These data further suggest the need for careful monitoring of individual unrelated donor marrow collection centers' practices to optimize the quality of the harvested marrow.

Use of a licensed electrolyte solution as an alternative to tissue culture medium for bone marrow collection.

Bone marrow for transplantation is traditionally collected into tissue culture medium with heparin. A licensed electrolyte solution (Plasma-Lyte A [PLA]) was used as a substitute for tissue culture medium in the harvesting of 28 bone marrows, 17 autologous and 11 allogeneic, which were subsequently transplanted. Data that were analyzed from the 25 evaluable patients consisted of the numbers of cells and colony-forming units in the transplanted marrow as well as the time to neutrophil and platelet engraftment. These results were compared with those in the 30 (26 evaluable) preceding transplanted marrows that were collected into a tissue culture medium (RPMI-1640 [RPMI]). The autologous marrow transplant patients in both the PLA and RPMI groups reached a neutrophil count of > or = 0.5 x 10(9) per L a mean of 19 days following transplantation. The patients who underwent transplantation with allogeneic bone marrow collected in RPMI achieved > or = 0.5 x 10(9) per L of neutrophils an average of 20 days following transplantation, while those who received marrow collected in PLA achieved engraftment of neutrophils to that level in a mean of 21 days. Because in vitro and in vivo results with RPMI and PLA are similar in this study, further work using a licensed solution for clinical bone marrow transplantation is indicated.

A novel approach to immunomodulation of frozen human bone marrow with interleukin-2 for clinical application.

Interleukin-2 (IL-2) activation of fresh or frozen bone marrow (BM) in vitro generates killer cells with potent anti-tumor effect both in vitro and in vivo. The IL-2-activated BM (ABM) retains the capacity to reconstitute the hematopoietic system in an autologous bone marrow transplantation (ABMT) setting. The killer cells lose their cytotoxicity if the ABM undergoes the procedures of freezing and thawing. Therefore, for clinical application, the ABM has to be generated after thawing a frozen stock of BM before ABMT. The thawed BM cells are fragile and may undergo lysis, resulting in clump formation and cell loss. The frozen autograft also contains components of cryoprotectant mixture whose effects on the generation of ABM have not been defined. The present studies have been carried out to optimize a technique of handling the frozen BM for immunomodulation with IL-2 for 24 h at 37 degrees C prior to ABMT, with minimal loss of cells. IL-2-activation of BM was carried out in bags containing serum free medium which were designed to permit gaseous exchange. Addition of deoxyribonuclease (DNAse) (100 micrograms/ml of BM concentrate) immediately after thawing and the presence of heparin (20 units/ml) in the medium completely abrogated immediate or delayed clumping of cells. The presence of DNAse and/or heparin during in vitro culture did not affect the cell viability, cytotoxicity against tumor cells or the progenitor cell activity of the ABM; all these functions were well maintained even when BM was placed in culture immediately after thawing (without washing). There was no microbial contamination.(ABSTRACT TRUNCATED AT 250 WORDS)

Automated processing of human bone marrow can result in a population of mononuclear cells capable of achieving engraftment following transplantation.

A concentrate of mononuclear bone marrow cells is often desired for ex vivo treatment with pharmacologic agents, monoclonal antibodies, cytokines, and other agents prior to transplantation. A method has been developed for automated separation of mononuclear cells from large volumes of harvested bone marrow. A programmable instrument originally designed for clinical ex vivo cell separation and the plasma-pheresis of patients and blood donors was adapted to permit rapid preparation, in a closed sterile system, of a bone marrow product enriched with mononuclear cells. A mean (+/- SEM) of 53 +/- 30 percent of the original mononuclear cells was recovered in a volume of 125 +/- 42 mL containing 82 +/- 12 percent mononuclear cells. This technique removed 95 +/- 9 percent of the red cells in the original marrow. No density gradient materials or sedimenting agents were employed in this process. Of 36 marrows processed by this technique, 19 autologous (6 of which were purged with 4-hydroperoxycyclophosphamide) and 7 allogeneic marrows have been transplanted, with all evaluable patients achieving a neutrophil count of 0.5 x 10(9) per L in a mean (+/- SEM) of 21 +/- 6 days.

Bone marrow processing for transplantation.

As indications for BMT increase, so do variations in bone marrow processing and manipulation techniques. Many centers have their own unique methods of mononuclear cell purification, concentration and storage. This is particularly evident in the processing of bone marrow for autologous BMT to allow dose intensification as salvage therapy for malignant disease. Unique procedures have been developed to maximize yields, concentrate mononuclear cells necessary for engraftment, and reduce the likelihood of GVH disease. Graft rejection and disease relapse still remain a problem in some of these "manipulated" marrows. Newer procedures may allow titration of the optimum numbers of immune reconstituting cells; however, at this time, these techniques are not precise and the balance between preventing GVH disease at the expense of graft failure or relapse may still jeopardize disease-free survival. Innovative purging techniques that include pharmacologic and immunologic methods, continue to evolve, necessitating standards for bone marrow processing that are flexible yet practical. Quality control and viability assays are essential to verify the biologic proliferative potential of progenitor cells capable of marrow reconstitution. Although no standards are yet established, all centers should have criteria to monitor the quality of the processed marrow. Blood banks and transfusion services are well versed in regulations governing processing, labeling, storage, and quality control of blood components. Bone marrow is the ultimate blood component, and it stands to reason that methods outlined in this article be integrated into transfusion medicine.

Processing and storage of human bone marrow: a survey of current practices in North America.

We conducted a two-step survey to question 110 transplant centers in the United States and Canada regarding marrow processing and storage policies and procedures. Approximately 65% of the centers surveyed responded to the questionnaires. Major differences with respect to patient diagnoses, amount of marrow harvested, purging method applied, freezing procedure, storage bag, cell concentrations, storage duration, interval until transplantation, cell counting, viability determination and so forth were reported. Among those centers responding 13% stored not only autologous but also allogeneic marrow. There was no consensus regarding patient consent for duration of storage, coverage of cost for cryopreservation or utilization of stored marrow after a patient's death. Additional studies will be necessary to correlate in vitro methods of marrow storage with clinical transplantation results, and to determine the cost/benefit ratio of this approach to various diagnoses. This should provide the basis for the establishment of standards and should facilitate the approach to various ethical questions.