Octamer proteins inhibit IL-4 gene transcription in normal human CD4 T cells.

The balance of Th1 (eg, interleukin-2 (IL-2)) and Th2 (eg, IL-4) cytokines produced by CD4 T cells markedly influences the outcome of the adaptive immune response. Although octamer transcription factor proteins increase IL-2 transcription in T cells, their role in IL-4 gene transcription remains controversial. We have previously shown and now confirm that the proximal octamer binding site of the human IL-4 promoter, which separates the two most proximal NFAT binding sites, is bound prior to, but not after, activation in vivo. Since these two NFAT sites are essential for optimal IL-4 promoter activity, this suggested that prior engagement by octamer proteins might prevent adjacent NFAT binding and inhibit IL-4 gene transcription. In support of this hypothesis, here we show that NFAT proteins are unable to bind to a combined octamer/NFAT site unless the octamer proteins are competed away. Moreover, activity of an IL-4 reporter gene mutated in the proximal octamer binding site is increased compared to the wild-type promoter in human peripheral blood CD4 T cells. In addition, over-expression of either Oct-1 or Oct-2 decreased wild-type IL-4 promoter activity, while increasing IL-2 promoter activity. No decrease in promoter activity was seen when Oct-1 or Oct-2 was over-expressed with the octamer-mutant IL-4 reporter gene. Thus, octamer proteins are candidates to promote a Th1 rather than Th2 pattern of cytokine gene expression by activated CD4 T cells.

T cell priming enhances IL-4 gene expression by increasing nuclear factor of activated T cells.

The repetitive activation of T cells (priming) enhances the expression of many cytokines, such as IL-4, but not others, such as IL-2. Molecular mechanisms underlying selective expression of cytokines by T cells remain poorly understood. Here we show that priming of CD4 T cells selectively enhances IL-4 expression relative to IL-2 expression by a transcriptional mechanism involving nuclear factor of activated T cells (NFAT) proteins. As detected by in vivo footprinting, priming markedly increases the activation-dependent engagement of the P0 and P1 NFAT-binding elements of the IL-4 promoter. Moreover, each proximal P element is essential for optimal IL-4 promoter activity. Activated primed CD4 T cells contain more NFAT1 and support greater NFAT-directed transcription than unprimed CD4 T cells, while activator protein 1 binding and activator protein 1-mediated transcription by both cell types is similar. Increased expression of wild-type NFAT1 substantially increases IL-4 promoter activity in unprimed CD4 T cells, suggesting NFAT1 may be limiting for IL-4 gene expression in this cell type. Furthermore, a truncated form of NFAT1 acts as a dominant-negative, reducing IL-4 promoter activity in primed CD4 T cells and confirming the importance of endogenous NFAT to increased IL-4 gene expression by effector T cells. NFAT1 appears to be the major NFAT family member responsible for the initial increased expression of IL-4 by primed CD4 T cells.

Canine T cells transduced with a herpes simplex virus thymidine kinase gene: a model to study effects on engraftment and control of graft-versus-host disease.

BACKGROUND: Alloreactive donor T cells in marrow grafts mediate graft-versus-host disease (GVHD), but T-cell depletion has resulted in increased graft failure. Add-back of gene-modified alloreactive donor T cells could prevent graft rejection. After engraftment, in vivo depletion of those modified T cells with ganciclovir may control GVHD. METHODS: Canine recipient-specific donor cytotoxic T lymphocytes (CTL) were retrovirally transduced with the herpes simplex virus thymidine kinase gene. RESULTS: Gibbon ape leukemia virus-pseudotyped vector yielded primary CTL transduction efficiency of 22.9+/-9.9%. After selection and expansion, 96.7+/-0.8% of CTL expressed retrovirally transferred genes. Recipient-specific cytotoxic activity was maintained with 84.3% specific lysis. After ganciclovir treatment, herpes simplex virus thymidine kinase-transduced CTL proliferation was reduced 98.7+/-0.2% compared with controls. CONCLUSIONS: We have demonstrated efficient ex vivo transduction, expansion, maintenance of alloreactivity, and ganciclovir-mediated ablation of canine CTL, which will permit in vivo studies in the dog, a well-established model for GVHD and engraftment.

High-dose fractionated total-body irradiation, etoposide and cyclophosphamide for treatment of malignant lymphoma: comparison of autologous bone marrow and peripheral blood stem cells.

Consecutive patients with non-Hodgkin's lymphoma (NHL, n = 133) or Hodgkin's disease (HD, n = 20) were treated with 12.0 Gy of fractionated total body irradiation, etoposide 60 mg/kg, and CY 100 mg/kg followed by infusion of autologous hematopoietic stem cells. Seventy-nine patients received purged (n = 62) or unpurged BM (n = 17), and 74 received unpurged PBSCs alone (n = 56) or with BM (n = 18). The median day for achieving a sustained granulocyte count of 0.5 x 10(9)/I was 14 range (7-66) for BM recipients and 10 (7-30) for PBSC +/- BM recipients (P = 0.03). A platelet count of 20 x 10(9)/I was achieved at a median of day 24 (6-145) in BM recipients and day 11 (range, 7-56) in PBSC +/- BM recipients (P = 0.007). The median number of platelet units transfused was 86 (0-1432) for BM recipients and 30 (6-786) for PBSC +/- BM recipients (P = 0.001). The median number of hospital days was 36 (10-88) for BM recipients and 27 (14-76) for PBSC +/- BM recipients (P = 0.0001). The unadjusted Kaplan-Meier (KM) estimates of survival, event-free survival (EFS) and relapse at 2 years were 0.57, 0.45 and 0.43 for patients receiving BM and 0.55, 0.36 and 0.59 for patients receiving PBSC +/- BM. After adjusting for confounding variables, the estimated relative risk (RR) of death from any cause was 0.92 (P = 0.75), of relapse was 1.25 (P = 0.39), of non-relapse mortality was 0.71 (P = 0.42) and of mortality and/or relapse was 1.17 (P = 0.48) for patients receiving PBSC +/- BM as compared to BM. For 46 patients with NHL receiving unpurged PBSC alone, the unadjusted KM estimate of relapse was 0.61 compared with 0.48 for 52 comparable patients receiving purged BM, while the RR for relapse for patients receiving unpurged PBSCs was 1.37 (P = 0.33) after adjusting for other significant covariates. These data confirm previous observations that patients who receive PBSC +/- BM have faster engraftment, fewer transfusions and shorter hospital stays than patients who receive only BM. There were no statistically significant differences between the two groups in survival, relapse, death from causes other than relapse and event-free survival.

High-dose fractionated total-body irradiation, etoposide, and cyclophosphamide followed by autologous stem-cell support in patients with malignant lymphoma.

PURPOSE: To evaluate a high-dose treatment regimen of fractionated total-body irradiation (TBI), etoposide, and cyclophosphamide (Cy) followed by autologous stem-cell transplantation (ASCT) in patients with malignant lymphoma. PATIENTS AND METHODS: Fifty-three patients with non-Hodgkin's lymphoma (NHL; n = 43) or Hodgkin's disease (HD; n = 10) received 12.0 Gy of fractionated TBI, etoposide 60 mg/kg, and Cy 100 mg/kg followed by infusion of autologous hematopoietic stem cells. RESULTS: Thirty-one of 53 patients are alive a median of 643 (range, 177 to 1,144) days after transplant. The 2 year Kaplan-Meier (K-M) estimates of survival, event-free survival (EFS), and relapse for all 53 patients were 54%, 45%, and 43%, respectively. Sixteen of 24 patients with less advanced disease and 10 of 29 patients with more advanced disease survive free of disease for K-M estimates of EFS of 61% and 31%, respectively (P = .006). The K-M estimates of relapse were 34% for patients with less advanced disease and 53% (P = .05) for patients with more advanced disease. The K-M estimates of dying from causes other than relapse were 8% in patients with less versus 25% in patients with more advanced disease (P = .09). CONCLUSION: These data indicate that approximately 60% of patients transplanted early after failure of initial therapy for malignant lymphoma are projected to be disease-free more than 2 years after treatment with fractionated TBI, etoposide, and Cy and infusion of autologous hematopoietic stem cells. The transplant-related mortality rate is low and relapse is the main cause of treatment failure in patients with less advanced disease. For patients with more advanced disease, the K-M estimates of both transplant-related deaths (25%) and relapse (53%) remain major problems.

Phase I study of high-dose busulfan, melphalan and thiotepa with autologous stem cell support in patients with refractory malignancies.

The purpose of this study was to determine the maximal tolerated dose of thiotepa administered with busulfan 12 mg/kg and melphalan 100 mg/m2 followed by autologous stem cell transplantation in patients with refractory malignancies. Twenty-eight patients with refractory malignancies received high-dose busulfan 12 mg/kg, melphalan 100 mg/m2 and escalating doses of thiotepa 450-550 mg/m2 followed by infusion of cryopreserved autologous peripheral blood stem cells (n = 26) or marrow (n = 2). The maximum tolerated dose was determined to be busulfan 12 mg/kg, melphalan 100 mg/m2 and thiotepa 500 mg/m2. Two of three patients receiving thiotepa 550 mg/m2 experienced grade 3 colitis. Twenty patients were enrolled at the maximum tolerated dose and the incidence of grade 3-4 regimen-related toxicity and mortality was 10% and 5%, respectively. Ninety-five per cent of patients experienced grade 1-2 mucositis, 50% grade 1-2 gastrointestinal toxicity, 35% grade I hepatic toxicity and 20% experienced grade 1-2 skin toxicity. The median time to achieve a granulocyte count of 0.5 x 10(9)/I was 10 days (range 8-20 days) and platelet transfusion independence was 10 days (range 1-26 days). Five of ten patients with stage 4 refractory breast cancer achieved a complete and two a partial remission with a complete response rate of 50% and a overall response rate of 70%. In conclusion, busulfan, melphalan and thiotepa can be administered in high doses with tolerable mucositis as the major side-effect. This combination has significant activity in patients with breast cancer, and phase II studies in patients with breast cancer and other chemotherapy sensitive malignancies are warranted.

In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T cells.

The IL-2 gene is a T cell specific gene that is expressed early during the activation-specific T lymphocyte development program. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assays have defined DNA/protein interactions at the IL-2 promoter cis-elements in vitro. To determine if the trans-activators documented in T cell nuclear extracts actually bind the IL-2 promoter in vivo, ligation mediated PCR (LMPCR) genomic footprinting was performed on the IL-2 promoter in both activated and non-activated T cells and HL60 promyelocytes, which do not express the IL-2 gene. The in vivo footprints indicate that the IL-2 gene transcription start site and TATA sequence are protected in both activated and resting T cells, prior to the appearance of detectable IL-2 steady state message. The distal NF-AT and the NF kappa B sites are each footprinted and the Oct/OAP site contains hypersensitive residues in the unstimulated T lymphocytes. Additional residues are protected in each of these sites after T cell activation. The proximal NF-AT site (NF-IL-2B) and the AP-1 site at -150 are protected in activated Jurkat T lymphocytes, but these two sites are not protected in activated Jurkat lymphocytes stably transfected a gene construct containing multiple NFAT binding sites.

Induction of alloantigen-specific hyporesponsiveness in human T lymphocytes by blocking interaction of CD28 with its natural ligand B7/BB1.

The specificity of T lymphocyte activation is determined by engagement of the T cell receptor (TCR) by peptide/major histocompatibility complexes expressed on the antigen-presenting cell (APC). Lacking costimulation by accessory molecules on the APC, T cell proliferation does not occur and unresponsiveness to subsequent antigenic stimulus is induced. The B7/BB1 receptor on APCs binds CD28 and CTLA-4 on T cells, and provides a costimulus for T cell proliferation. Here, we show that prolonged, specific T cell hyporesponsiveness to antigenic restimulation is achieved by blocking the interaction between CD28 and B7/BB1 in human mixed leukocyte culture (MLC). Secondary T cell proliferative responses to specific alloantigen were inhibited by addition to the primary culture of monovalent Fab fragments of anti-CD28 monoclonal antibody (mAb) 9.3, which block interaction of CD28 with B7/BB1 without activating T cells. Hypo-responsiveness was also induced in MLC by CTLA4Ig, a chimeric immunoglobulin fusion protein incorporating the extracellular domain of CTLA-4 with high binding avidity for B7/BB1. Cells previously primed could also be made hyporesponsive, if exposed to alloantigen in the presence of CTLA4Ig. Maximal hyporesponsiveness was achieved in MLC after 2 d of incubation with CTLA4Ig, and was maintained for at least 27 d after removal of CTLA4Ig. Accumulation of interleukin 2 (IL-2) and interferon gamma but not IL-4 mRNA was blocked by CTLA4Ig in T cells stimulated by alloantigen. Antigen-specific responses could be restored by addition of exogenous IL-2 at the time of the secondary stimulation. Addition to primary cultures of the intact bivalent anti-CD28 mAb 9.3, or B7/BB1+ transfected CHO cells or exogenous IL-2, abrogated induction of hyporesponsiveness by CTLA4Ig. These data indicate that interaction of CD28 with B7/BB1 during TCR engagement with antigen is required to maintain T cell competence and that blocking such interaction can result in a state of T cell hyporesponsiveness.

The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region.

A conditional block to transcriptional elongation is an important mechanism for regulating c-myc gene expression. This elongation block within the first c-myc exon was defined originally in mammalian cells by nuclear run-on transcription analyses. Subsequent oocyte injection and in vitro transcription analyses suggested that sequences near the end of the first c-myc exon are sites of attenuation and/or premature termination. We report here that the mapping of single stranded DNA in vivo with potassium permanganate (KMnO4) and nuclear run-on transcription assays reveal that polymerase is paused near position +30 relative to the major c-myc transcription initiation site. Deletion of 350 bp, including the sites of 3'-end formation and intrinsic termination defined in oocyte injection and in vitro transcription assays does not affect-the pausing of polymerase in the promoter-proximal region. In addition, sequences upstream of +47 are sufficient to confer the promoter-proximal pausing of polymerases and to generate the polarity of transcription farther downstream. Thus, the promoter-proximal pausing of RNA polymerase II complexes accounts for the block to elongation within the c-myc gene in mammalian cells. We speculate that modification of polymerase complexes at the promoter-proximal pause site may determine whether polymerases can read through intrinsic sites of termination farther downstream.

Pneumocystis carinii pneumonia associated with profound lymphopenia and abnormal T-lymphocyte subset ratios during treatment for early-stage breast carcinoma.

Two women with Stage II breast carcinoma treated with lumpectomy followed by breast irradiation and adjuvant chemotherapy developed Pneumocystis carinii pneumonia while receiving cytotoxic chemotherapy. Neither woman had evidence of immunosuppression before therapy. They both had profound lymphopenia, reversed CD4/CD8 ratios, and normal peripheral blood total leukocyte counts at the time of their infections. Both women were seronegative for human immunodeficiency virus type 1 and had no risk factors for such an infection. The patients' CD4 lymphocyte counts increased after chemotherapy for breast carcinoma was discontinued. Thus, it appears that the therapy they received may have caused severe T-lymphocyte mediated immunosuppression.

Nuclear factors interacting with the mitogen-responsive regulatory region of the interleukin-2 gene.

We have investigated the interaction of nuclear factors with the regulatory region of the interleukin-2 (IL-2) growth factor gene, which is induced upon activation of T lymphocytes. The regulatory region of IL-2 has been defined previously on the basis of transient transfection experiments and DNase I hypersensitive site analyses. This region of about 275 base pairs contains both tissue-specific as well as activation-dependent elements. Here we demonstrate nuclear factors which bind in this region. Some of these interactions are cell type-specific and several depend on prior activation of cells. This suggests that multiple activation responsive elements reside in the IL-2 promoter region. The inducible DNA binding activity of one of the factors is dependent also on protein synthesis. We have established an in vitro transcription assay to assess functional requirements for these DNA binding proteins. The possible involvement of some of these factors in regulating other genes expressed in T cells is discussed.

Chronic recurrent Goodpasture's syndrome.

A distinctive case of Goodpasture's syndrome characterized by multiple recurrences occurring over 14 years was observed. Each recurrence followed cessation of prednisone and azathioprine therapy, and reinstitution of this therapy resulted in prompt remission. Initially, both glomerulonephritis and hemoptysis were prominent clinical features. In recent years, hemoptysis has been the predominant clinical problem. This and other cases suggest that Goodpasture's syndrome may comprise a wider disease spectrum than generally appreciated.