Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells.

CD4(+)CD25(+) regulatory T (T(reg)) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking T(reg) cells develop severe autoimmune disease, and depletion of T(reg) cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of T(reg) cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2-responsive T(reg) cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain T(reg) cells, the question arises as to the effects of IL-2 therapy on them. We monitored T(reg) cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4(+)CD25(hi) cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the T(reg) cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent T(reg) cells in normal hosts, and IL-2-induced T(reg) cell expansion was further augmented by lymphopenia. On a per-cell basis, T(reg) cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to T(reg) cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4(+)CD25(+) T(reg) cell homeostasis.

Multicenter study on in vitro characterization of dendritic cells.

BACKGROUND: There is growing interest in the use of in vitro-expanded dendritic cells (DC) in cancer immunotherapy as cellular-based vaccines. However, the methods used for in vitro preparation vary widely between institutions. Therefore, a strong need exists for standardization, characterization and quality control (QC) of such vaccines. A first prospective multicenter pilot study was performed to investigate basic QC parameters of frozen/thawed DC. The study design was focused on comparison of test results for cell counts, immunophenotyping and cell viability. METHODS: CD14+ monocytes were isolated from three healthy volunteers. The cells were expanded in vitro, matured and cryopreserved using a standardized protocol in one laboratory. The aliquots of cryopreserved DC and a panel of reagents were shipped to eight laboratories worldwide. The objective was to compare the results of non-functional QC assays between sites by testing identical DC vaccines and using a pre-defined test protocol. RESULTS: Measurements of nucleated cell (NC) content of thawed DC vaccines with different types of hematology analyzers (HA) gave similar results for the majority of sites. Immunophenotyping using identical clones of monoclonal antibodies for the detection of surface antigens (i.e. CD1a, CD14, CD16, CD83, CD86 and HLA-DR) provided mostly comparable results between laboratories with an acceptable level of variation. In contrast, highly different results between study sites were generated for measuring the viability of thawed DC by flow cytometry using 7-amino-actinomycin D (7-AAD) dye exclusion. DISCUSSION: In characterizing frozen/thawed DC vaccines, NC counts generated by HA yielded similar results between different laboratories. Furthermore, immunophenotyping of DC vaccines can be standardized between centers, i.e. by using identical reagents. Because of highly variable results between laboratories, 7-AAD viability testing of thawed DC needs to be studied further to identify potential causes for the observed variability.

A multicenter comparison study between the Endosafe PTS rapid-release testing system and traditional methods for detecting endotoxin in cell-therapy products.

BACKGROUND: Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Health's Department of Transfusion Medicine, USA. METHODS: Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing. RESULTS: Valid paired results, based on each center's LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases. DISCUSSION: The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.

In vivo distribution of adoptively transferred indium-111-labeled tumor infiltrating lymphocytes and peripheral blood lymphocytes in patients with metastatic melanoma.

Patients with metastatic melanoma undergoing therapy with cyclophosphamide (CPM), tumor-infiltrating lymphocytes (TIL), and interleukin-2 (IL-2) were studied for the ability of their 111In-labeled TIL or peripheral blood lymphocytes (PBL) to localize in sites of tumor using gamma camera imaging and biopsies. Nineteen infusions of radiolabeled TIL were given to 18 patients, while five patients received radiolabeled autologous PBL during TIL therapy. Clear tumor localization was seen on 13 of 18 nuclear scan series performed on 111In-TIL recipients, while tumor was imaged in only one of four scan sequences on patients given 111In-PBL. Nineteen paired biopsies of tumor and normal skin were completed on 10 patients receiving 111In-TIL, while eight biopsies were done on three PBL patients receiving 111In-PBL. The mean percentage of total injectate activity localizing per gram of tumor tissue was 0.0049% in the TIL group and 0.0010% in the PBL group (P2 = .0004). The mean of the tumor to normal skin ratios of the 111In-TIL group was three times that for 111In-PBL (P2 = .0072). One patient was studied by nuclear scanning on three consecutive treatment courses of CPM, TIL, and IL-2. He initially demonstrated clear tumor localization by 111In-TIL at several sites, then faint localization with 111In-PBL at a single site, and subsequently positive tumor imaging on repeat 111In-TIL infusion at multiple sites. These results confirm and expand our initial data demonstrating that human TIL transferred with CPM pretreatment and followed by IL-2 preferentially localize to tumor sites and indicate that this localization is greater for TIL than PBL.

Tumor localization of adoptively transferred indium-111 labeled tumor infiltrating lymphocytes in patients with metastatic melanoma.

Lymphoid cells infiltrating into human tumors can be expanded in vitro in medium containing interleukin-2 (IL-2). Adoptive transfer of these tumor-infiltrating lymphocytes (TIL) mediates potent antitumor effects in murine tumor models. Clinical trials to evaluate the efficacy of these cells in patients with advanced cancer are underway. We have investigated whether infused TIL labeled with indium 111 (111In) oxine can traffic and localize to metastatic deposits of tumor. Six patients with metastatic malignant melanoma who had multiple sites of subcutaneous, nodal, and/or visceral disease were the subjects of the study. The patients received cyclophosphamide 36 hours before receiving the intravenous (IV) infusion of TIL followed by IL-2 IV every eight hours. The distribution and localization of the TIL were evaluated using serial whole body gamma camera imaging, serial blood and urine samplings, and serial biopsies of tumor and normal tissue. 111In-labeled TIL localized to lung, liver, and spleen within two hours after the infusion of activity. Activity in the lung diminished within 24 hours. As early as 24 hours after injection of 111In-labeled TIL, localization of TIL to sites of metastatic deposits was demonstrated in all six patients using either imaging studies or biopsy specimens or both. 111In activity in tumor tissue biopsies ranged from three to 40 times greater than activity in normal tissue. A progressive increase in the radioactive counts at sites of tumor deposit was seen. This study shows that labeled TIL can localize preferentially to tumor, and provides information concerning the possible mechanism of the therapeutic effects of TIL.

Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.

The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study. Hematopoietic stem cells (HSCs) were collected by both peripheral blood apheresis and bone marrow harvest after mobilization with a single dose of cyclophosphamide (4 g/m2) and daily filgrastim therapy (10 microg/kg/day). After enrichment for CD34+ cells, one-third of each collection was incubated ex vivo for 72 h with a replication-incompetent retrovirus containing the MDR1 gene (G1MD) in the presence of stem-cell factor, interleukin 3, and interleukin 6. The remaining CD34+ cells were stored without further manipulation. All of the CD34+ cells were reinfused for hematopoietic rescue after conditioning chemotherapy with ifosfamide, carboplatin, and etoposide regimen. After hematopoietic recovery, patients received six cycles of paclitaxel (175 mg/m2 every 3 weeks). Bone marrow and serial peripheral blood samples were obtained and tested for the presence of the MDR1 transgene using a PCR assay. Six patients were enrolled in the study and four patients received infusion of genetically altered cells. The ex vivo transduction efficiency, estimated by the PCR assay, ranged from 0.1 to 0.5%. Three of the four patients demonstrated engraftment of cells containing the MDR1 transgene. The estimated percentage of granulocytes containing the MDR1 transgene ranged from a maximum of 9% of circulating nucleated cells down to the limit of detection of 0.01%. One patient remained positive for the MDR1 transgene throughout all six cycles of paclitaxel therapy, whereas the other 2 patients showed a decrease in the number of cells containing the transgene to undetectable levels. Despite the low level of engraftment of MDR1-marked cells, a correlation was observed between the relative number of granulocytes containing the MDR1 transgene and the granulocyte nadir after paclitaxel therapy. No adverse reactions to the genetic manipulation procedures were detected. Therefore, engraftment of human HSCs transduced with the MDR1 gene can be achieved. However, the overall transduction efficiency and stable engraftment of gene-modified HSCs must be improved before MDR1 gene therapy and in vivo selection with anticancer drugs can be reliably used to protect cancer patients from drug-related myelosuppression.

Engraftment of hematopoietic progenitor cells transduced with the Fanconi anemia group C gene (FANCC).

Fanconi anemia (FA) is an autosomal recessive disorder that leads to aplastic anemia. Mutations in the FANCC gene account for 10-15% of cases. FA cells are abnormally sensitive to DNA-damaging agents such as mitomycin C (MMC). Transfection of normal FANCC into mutant cells corrects this hypersensitivity and improves their viability in vitro. Four FA patients, representing the three major FANCC mutation subgroups, were entered into a clinical trial of gene transduction aimed at correction of the hematopoietic defect. Three patients received three or four cycles of gene transfer, each consisting of one or two infusions of autologous hematopoietic progenitor cells that had been transduced ex vivo with a retroviral vector carrying the normal FANCC gene. Prior to infusion, the FANCC transgene was demonstrated in transduced CD34-enriched progenitor cells. After infusion, FANCC was also present transiently in peripheral blood (PB) and bone marrow (BM) cells. Function of the normal FANCC transgene was suggested by a marked increase in hematopoietic colonies measured by in vitro cultures, including colonies grown in the presence of MMC, after successive gene therapy cycles in all patients. Transient improvement in BM cellularity coincided with this expansion of hematopoietic progenitors. A fourth patient, who received a single infusion of transduced CD34-enriched BM cells, was given radiation therapy for a concurrent gynecologic malignancy. The FANCC transgene was detected in her PB and BM cells only after recovery from radiation-induced aplasia, suggesting that FANCC gene transduction confers a selective engraftment advantage. These experiments highlight both the potential and difficulties in applying gene therapy to FA.

In vivo traffic of indium-111-oxine labeled human lymphocytes collected by automated apheresis.

The in vivo traffic patterns of autologous lymphocytes were studied in five normal human volunteers using lymphocytes obtained by automated apheresis, separated on Ficoll-Hypaque gradients, and labeled ex vivo with 111In-oxine. Final lymphocyte infusions contained 1.8-3.1 X 10(9) cells and 270-390 microCi (9.99-14.43 MBq) 111In, or 11-17 microCi (0.41-0.63 MBq) per 10(8) lymphocytes. Gamma imaging showed transient lung uptake and significant retention of radioactivity in the liver and spleen. Progressive uptake of activity in normal, nonpalpable axillary and inguinal lymph nodes was seen from 24 to 96 hr. Accumulation of radioactivity also was demonstrated at the forearm skin test site, as well as in its associated epitrochlear and axillary lymph nodes, in a subject who had been tested for delayed hypersensitivity with tetanus toxoid. Indium-111-oxine labeled human lymphocytes may provide a useful tool for future studies of normal and abnormal lymphocyte traffic.

Cyclosporine is required to prevent severe acute GVHD following T-cell-depleted peripheral blood stem cell transplantation.

Reduced immunosuppression may improve immune recovery and increase the graft-versus-leukemia effect after allogeneic stem cell transplantation. Furthermore, the requirement for post-transplant immunosuppression following extensive T-cell depletion remains unclear. We therefore evaluated the role of cyclosporine (CSA) in recipients of HLA-identical T-cell-depleted peripheral blood stem cell transplants (PBSCT), followed by donor lymphocyte infusions (DLIs) scheduled on days +45 and +100. Before day+45, successive cohorts of patients received decreasing amounts of CSA: standard-dose (SD) CSA, low-dose (LD) CSA, or no CSA until day+45. LD CSA was as effective as SD CSA in preventing acute graft-versus-host disease (GVHD). However, moderate-to-severe acute GVHD was significantly more frequent before the day +45 DLI in patients receiving no CSA (33.3 vs 12.7%, P=0.036, including the only four grade III-IV cases). As a result of higher rates of early acute GVHD, more patients in the 'no CSA' group failed to receive any DLI (30.7 vs 7.1%, P=0.01). Overall, there was no difference in the incidence of acute GVHD, as patients receiving CSA developed more GVHD after DLI. Similarly, no significant differences were found in chronic GVHD, transplant-related mortality, or survival. These results define a role for CSA in preventing GVHD at low T-cell doses following PBSCT.

T cell-depleted granulocyte colony-stimulating factor (G-CSF) modified allogenic bone marrow transplantation for hematological malignancy improves graft CD34+ cell content but is associated with delayed pancytopenia.

To increase the stem cell content of T cell-depleted bone marrow transplants (BMT), we treated 12 patients with hematological malignancies with BMT from HLA-identical sibling donors given G-CSF 10 microg/kg/day for 5 days before marrow harvest. After CD34+ cell selection, patients received a median of 1.7 (range, 0.82-3.1) x 10(6) CD34+ cells/kg and 2.3 (range, 0.25-4.0) x 10(5) CD3+ cells/kg. All patients had initial engraftment but four developed pancytopenia between days 55-130 post-BMT. In two patients, this required a second infusion of G-CSF-mobilized donor peripheral blood progenitor cells. We observed no delayed pancytopenia in a matched historical group of 24 patients receiving T cell-depleted BMT without prior G-CSF stimulation. Compared to this control group, G-CSF-stimulated marrow recipients showed a significant decline in neutrophil and monocyte counts after 8 weeks. However, outcome after BMT was otherwise comparable, with a similar incidence of acute graft-versus-host disease and transplant-related mortality. Disease-free survival was 63 vs 67% for controls matched for CD34+ cell dose (P = NS). These results indicate that G-CSF stimulation can increase the CD34+ cell content of T cell-depleted marrow but carries a risk of late graft failure.

T cell-depleted bone marrow transplantation and delayed T cell add-back to control acute GVHD and conserve a graft-versus-leukemia effect.

Thirty-eight patients with hematological malignancies, received T cell-depleted marrow transplants (BMT) and cyclosporine to prevent acute graft-versus-host disease (aGVHD), followed by delayed add-back of donor lymphocytes to prevent leukemia relapse. In 26 patients scheduled for donor T cell add-back of 2 x 10(6) cells/kg on day 30 and 5 x 10(7) cells/kg on day 45 (schedule 1), the overall probability of grade > or = II aGVHD developing was 31.5%, with a 15.5% probability of aGVHD occurring after T cell add-back. In 12 patients receiving 10(7) donor T cells/kg on day 30 (schedule 2), the probability of grade > or = II aGVHD was 100%. The incidence of grade III-IV aGVHD was higher in schedule 2 than in schedule 1 (P=0.02). Of 24 evaluable patients, 10 (46%) developed chronic GVHD which was limited in eight and extensive in two. Current disease-free survival for 18 patients at standard risk for relapse (chronic myeloid leukemia (CML) in chronic or accelerated phase, acute myeloid leukemia in remission) vs 20 patients with more advanced leukemia or multiple myeloma were respectively 72% vs 12% (P < 0.01) with a 29% vs 69% probability of relapse (P=0.08). In 12 CML patients surviving more than 3 months, PCR analysis of the BCR/ABL transcript showed that minimal residual disease after T cell add-back was transient except in two patients who developed hematological relapse. Results indicate that the risk of acute GVHD is low following substantial T cell doses, transfused 45 days after transplant, using cyclosporine prophylaxis. Furthermore a graft-versus-leukemia effect was conserved.

Listeria monocytogenes sepsis and small cell carcinoma of the rectum: an unusual presentation of the acquired immunodeficiency syndrome.

A 26-year-old male homosexual initially presented with Listeria monocytogenes sepsis and a small cell carcinoma of the rectum. His subsequent course included esophageal candidiasis, Pneumocystis carinii pneumonia, and severe T-lymphocyte abnormalities on immunologic testing, consistent with the acquired immunodeficiency syndrome (AIDS). This represents the first case of AIDS associated with this unusual tumor and Listeria infection.

Placental metastasis from maternal carcinoma of the lung.

A case of large cell undifferentiated carcinoma of the lung metastatic to the placenta and a review of the literature are presented. This is the 44th documented case of placental and/or fetal metastasis from maternal cancer in the past 113 years. Fifty percent of these cases are either malignant melanomas or hematopoietic malignancies. Evidence indicates that these 2 malignancies are more likely to metastasize to the products of conception than are other malignancies.

The automated analysis of rat sperm motility following subchronic epichlorohydrin administration: methodologic and statistical considerations.

The automated analysis of sperm motion endpoints is potentially useful in identifying male reproductive toxicants and ultimately in predicting fertility in humans. The present study was designed to evaluate the automated analysis of rat sperm motility characteristics following subchronic administration of epichlorohydrin. This type of validation is a prerequisite for inclusion of sperm motion measurements in the process of reproductive risk assessment. In the present studies videotapes were made of cauda epididymal spermatozoa from Long-Evans rats, both untreated and treated with epichlorohydrin. From analysis of videotapes of control epididymal spermatozoa, the relationship of various sperm motion endpoints and settings of the CellSoft computer-assisted sperm motion analysis system (Cryo Resources, Ltd., New York, NY) is described. Optimal settings of the system for analysis of rat spermatozoa are detailed. Employing data from both control and epichlorohydrin-treated animals, a statistical methodology is described that evaluates: (1) the distributions of CellSoft generated sperm motion endpoints, (2) the correlations between these endpoints, and (3) techniques for detection of dose-related effects.

Correlation of sperm motion parameters with fertility in rats treated subchronically with epichlorohydrin.

We investigated the relationship between fertility and sperm motin endpoints in rats treated subchronically with the male reproductive toxicant, epichlorohydrin (ECH). Male rats were given ECH orally for 23 days at dosages of 0, 6.25, 12.5, or 25 mg/kg/day. They were mated twice (at 19 and 22 days) to estimate fertility by (1) detection of fertilized ova (presence of sperm head and tail or two pronuclei) 18 hours after mating and by (2) counting implants on day 14 of gestation. Both indices showed dose-related reductions (P less than 0.001). Motion parameters of cauda epididymal sperm were assessed using the CellSoft computer-assisted sperm motion analysis (CASA) system after the rats were asphyxiated on day 25. Curvilinear velocity, straight-line velocity, linearity, and amplitude of lateral head displacement were reduced in a dose-related manner. The fertility indices, percent fertilized ova, and percent implantation on day 14 of gestation were correlated significantly (r = 0.68; P = 0.0001). The following motion parameters were also correlated significantly with fertility (P less than 0.0003; r1 = percent fertilized ova and r2 = percent implantation): linearity (r1 = 0.42; r2 = 0.40), amplitude of lateral head displacement (r1 = 0.54; r2 = 0.48), curvilinear velocity (r1 = 0.53; r2 = 0.50), straight-line velocity (r1 = 0.55; r2 = 0.50), and percent motile sperm (r1 = 0.42; r2 = 0.32). These results suggest a relationship between toxicant-induced reductions in sperm motion and fertility.

Sources of variation in the computer-assisted motion analysis of rat epididymal sperm.

Random and nonrandom factors associated with sample preparation and the automated analysis (CellSoft) of rat cauda epididymal sperm motion were studied. Random factors included inherent system variation at both the individual cell level and at the multiple cell level. Repeated analyses of identical tracks across grey level revealed a statistical interaction between grey settings and curvilinear velocity. However, in multiple track analyses, grey level was seen to be a factor only at higher settings. Nonrandom factors included time after sample preparation, dilution medium, and sample preparation procedures. Using a nicked preparation of the entire cauda epididymis from Long-Evans rats, the effects of time were studied on sperm suspended in 1) phosphate-buffered saline + 10 mg BSA/mL, 2) TEST yolk buffer, and 3) Medium 199. In PBS/BSA, the percent motile sperm estimate decreased (50% to 30%) over an hour, while the curvilinear velocity increased (127 to 142 microns/sec). Both sperm motion parameters were maintained in the TEST yolk buffer and in the Medium 199, although at lower values for the latter. Evaluation of the relative contribution of several factors, nested within sample, to the overall variance of three separate motion endpoints revealed that there was a large variation from field to field, negligible variation between overall CellSoft analyses of 200 cells or more, low variation at the preparation aliquot level, and moderate variation at the animal level. In planning experiments to test for effects on sperm motion endpoints, consideration of the relative contribution of the individual study factors to the overall variance of the parameter estimates will result in more sensitive experimental designs.

The presence-absence coliform test for monitoring drinking water quality.

The concern for improved monitoring of the sanitary quality of drinking water has prompted interest in alternative methods for the detection of total coliform bacteria. A simplified qualitative presence-absence test has been proposed as an alternate procedure for detecting coliform bacteria in potable water. In this paper data from four comparative studies were analyzed to compare the recovery of total coliform bacteria from drinking water using the presence-absence test, the multiple fermentation tube procedure, and the membrane filter technique. The four studies were of water samples taken from four different geographic areas of the United States: Hawaii, New England (Vermont and New Hampshire), Oregon, and Pennsylvania. Analysis of the results of these studies were compared, based upon the number of positive samples detected by each method. Combined recoveries showed the presence-absence test detected significantly higher numbers of samples with coliforms than either the fermentation tube or membrane filter methods, P less than 0.01. The fermentation tube procedure detected significantly more positive samples than the membrane filter technique, P less than 0.01. Based upon the analysis of the combined data base, it is clear that the presence-absence test is as sensitive as the current coliform methods for the examination of potable water. The presence-absence test offers a viable alternative to water utility companies that elect to use the frequency-of-occurrence approach for compliance monitoring.

Innovation meets quality: moving cellular therapies at the National Institutes for Health from bench to bedside.

The National Institutes of Health (NIH) Department of Transfusion Medicine has supported clinical investigation in cellular therapies over the past 20 years. This experience, which encompasses product development research, quality system design and product manufacturing for a wide range of early phase clinical trials, provides a firm basis for future work with novel stem cell therapies.

Colloid cyst of the third ventricle.

Colloid cyst of the third ventricle is a rare, histologically benign but potentially lethal tumor. Two cases reflecting the variability in symptoms, physical findings, and outcome seen with these rare tumors are presented. One patient with classic age of onset in the third to fifth decade of life, symptoms of escalating intermittent headaches, and signs of papilledema and altered level of consciousness had successful surgical removal of the cyst. Another patient with nonspecific symptoms well outside the usual age of onset and without papilledema succumbed to sudden, complete impaction and was not diagnosed until autopsy.