Primary activation of murine CD8 T cells via cross-linking of T3 cell surface structures: two signals regulate induction of interleukin 2 responsiveness

In the model system used here to study the minimal signal requirements for the activation of murine resting CD8 T cells, cross-linking of T cell receptor structures by antigen-presenting cells is substituted for by the use of anti-CD3 monoclonal antibodies immobilized in Sepharose beads. We show that cross-linking of CD3 structures, even in combination with CD8 structures, is necessary but insufficient to induce responsiveness to the growth-promoting effect of interleukin 2 (IL2), i.e. fails to induce expression of functional IL2 receptors. A macrophage cell line product termed IL2 receptor-inducing factor (RIF), but not IL1, IL3, IL4 or tumor necrosis factor, efficiently functions as costimulator. Once activated, growth of CD8 T cells is entirely driven by IL2. We conclude that two restriction points control the activation of resting CD8 T cells. While cross-linking of CD3 structures is essential as a first step, RIF is required as competence factor to induce IL2 responsiveness. We consider the possibility that the ability of antigen-presenting cells to produce RIF determines the immunogenicity of presented antigen towards antigen-reactive resting CD8 T cells.     

Chemoradioimmunotherapy with 5-fluorouracil, cisplatin and interferon-α in pancreatic and periampullary cancer: Results of a feasibility study

BackgroundRecent studies give rise to the hypothesis, that adjuvant chemoradioimmunotherapy with 5-fluorouracil (5-FU), cisplatin and interferon-α (IFN-α) might be a possible new treatment of pancreatic cancer in resected patients. We report the up-to-now experience at our institution.Patients and methodsEleven patients with histological diagnosis of localized carcinoma of the pancreas (n = 7) or periampullary (n = 4) were prospectively analyzed. Four patients were deemed unresectable because of local invasion of adjacent organs (neoadjuvant setting) and seven patients underwent curative resection (adjuvant setting). Eight patients were classified as T3 carcinomas and three T4 carcinomas. Fifty-five per cent (6/11) of the patients presented with positive lymph node involvement. One histological Grade I, six Grade II and three Grade III were detected. External conformal irradiation to a total dose of 50.4 Gy with 1.8 Gy per day was delivered. All patients received a concomitant chemotherapy with continuous 5-FU 200 mg/m² per day on 28 treatment days and intravenous bolus cisplatin 30 mg/m² per week (Day 2, 9, 16, 23, 30). A recombinant r-IFN-α was administered on three days weekly during Week one to five of the radiotherapy course as subcutanous injections with 3*3 Mio. I.U. weekly.ResultsThe four-year overall survival rate for all patients was 55%. In the neoadjuvant group, three of four patients died due to progressive disease; in the adjuvant group, combined chemoradioimmunotherapy lead to controlled disease in five of seven patients. The overall toxicity was well-managed.ConclusionOur data strengthens the hypothesis of concomitant chemoradioimmunotherapy with 5-FU, IFN-α and cisplatin as a possible new treatment of pancreatic cancer in resected patients.     

Can contrast enhanced MRI predict the response of Graves' ophthalmopathy to orbital radiotherapy?

The purpose of this study was to try to determine by means of contrast-enhanced MRI, a subset of patients with Graves' ophthalmopathy who will not respond to orbital radiotherapy. 54 patients with Graves' ophthalmopathy were treated with orbital radiotherapy (10 x 2 Gy) and symptom relief was recorded. MRI examinations prior to radiotherapy were retrospectively evaluated for enlargement, contrast enhancement and fibrotic changes in extraocular muscles and surrounding soft tissue. Imaging data were correlated with clinical features and response. Symptom relief was observed in 61% of patients but this could not be predicted by any of the MRI signs investigated. However, there is a trend for a better treatment reponse in patients who show contrast enhancement of extraocular muscles prior to orbital radiotherapy (p=0.08). MRI could not adequately predict the efficacy of orbital radiotherapy in this group of patients. Clinical assessment of disease activity is still the most reliable method.     

Role of DNA-dependent protein kinase in the process of radiation-induced aberration formation

PURPOSE: To further investigate the role of DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutation (ATM) in the formation of chromosome aberrations, we analysed radiation-induced aberrations in M059J cells, complemented with the PRKDC (DNA-PK) gene by introducing a fragment of human chromosome 8 containing a copy of the human PRKDC gene. One hybrid cell line (M059J Fus1) displayed kinase activity and was radioresistant; the other hybrid cell line (M059J Fus9) showed no kinase activity and was radiosensitive. Both Fus1 and Fus9 cells have only a low ATM activity. Wortmannin, an inhibitor of DNA-PKCS and ATM, was added before irradiation in order to study the effect of DNA-PKCS--and ATM--inhibition on the formation of chromosome aberrations. Furthermore, aberration formation was studied in a lymphoblastoid ATM-deficient cell line AT-1 and in an ATM-proficient control. MATERIALS AND METHODS: Confluent cells were irradiated with 200 kV X-rays. Dicentrics, excess acentric fragments, chromatid/ isochromatid breaks and chromatid exchanges were scored in the absence and presence of wortmannin. RESULTS: In M059J-Fus1 cells and normal lymphoblastoid cells, only chromosome-type aberrations were observed independently of the presence of wortmannin. In DNA-PK-deficient Fus9 cells and in ATM-deficient AT-1 cells, an increasing proportion (30-80%) of cells containing chromatid-type aberrations was observed. This proportion increased with irradiation dose and with wortmannin addition. The aberration yields observed in the complemented M059J-Fus1 cell line were much lower than the corresponding yields observed in the deficient M059J and AT-1 cell lines. However, the low yields observed in the DNA-PK-proficient 'wild-type' cell line M059K were not completely restored. CONCLUSIONS: Since in M059J-Fus1 cells the radioresistant phenotype with respect to chromosome-type aberration formation was restored by the complementation of PRKDC, ATM expression determines the chromosomal radiosensitivity of M059J cells only to a minor extent. The increasing presence of chromatid-type aberrations in cells irradiated in G0/G1 phase as observed either in DNA-PK- or ATM-deficient cells definitely requires the lack of either kinase. Thus, the aberration spectrum observed is determined by the genetic profile of the respective cells and aberration class amplitudes can be modulated by the inhibition of either kinase.     

Role of DNA-PK in the process of aberration formation as studied in irradiated human glioblastoma cell lines M059K and M059J

PURPOSE: The participation of various DNA double-strand break repair mechanisms in the formation of chromosome aberrations is not yet fully understood. To investigate particularly the role of non-homologous end-joining, we analysed the formation of radiation-induced aberrations in a DNA-protein kinase (PK(CS))-proficient cell line M059K and in a deficient cell line M059J. MATERIALS AND METHODS: Plateau-phase M059K and M059J cells were irradiated with low doses of X-rays. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations and total visible complex exchanges (complex aberrations) for chromosomes 4, 7 and 11 using the FISH method. M059K cells were also analysed in the presence of 50 micro m wortmannin, a DNA-PK inhibitor. RESULTS: DNA-PK-deficient cells showed a higher yield of simple stable and unstable and of complex aberrations in comparison with DNA-PK-proficient cells. The largest differences were observed for excess acentric fragments and for complex aberrations. DNA-PK inhibition by wortmannin in M059-K cells resulted in increased aberration yield in the same qualitative and quantitative manner as in M059-J cells. CONCLUSIONS: The results obtained with DNA-PK-deficient M059J cells and with DNA-PK-proficient M059K cells treated with wortmannin, an inhibitor of DNA-PK and ATM, suggest that the elimination of DNA-PK-dependent non-homologous end-joining can recruit a slow, error-prone repair process, which is DNA-PK independent and favours the increased formation of chromosome aberrations. The nature of this pathway and the way of its participation in aberration formation need further elucidation.     

Radiotherapy for stages I and IIA/B testicular seminoma

Radiotherapy is generally accepted as a standard treatment for early-stage testicular seminoma. Relapse rates of 2% to 5% in clinical stage I and 10% to 20% in stage IIA/B (according to the Royal Marsden classification) can be achieved. Disease-specific survival reaches 100%. With such excellent cure rates, treatment-related side effects gain particular importance. Therefore, a prospective multicenter trial was initiated for radiotherapy of testicular seminoma with limited treatment portals and low total doses of irradiation. In clinical stage I, 483 patients were treated with 26 Gy to the para-aortic region only. In stage IIA, 42 patients and, in stage IIB, 18 patients received irradiation to the para-aortic and high iliac lymph nodes with 30 and 36 Gy, respectively. With a median time to follow-up of 55 months for stage I and 55.5 months for stage IIA/B, there were 18 (3.7%) and 4 (6.7%) cases of relapse in both treatment groups. Disease-specific survival was 99.6% in stage I and 100% in stage IIA/B. Acute toxicity was dominated by moderate gastro-intestinal side effects. No major late toxicity has been observed to date. Limited volume pure para-aortic treatment for stage I and para-aortic/high iliac irradiation for stage IIA/B with 26, 30 and 36 Gy, respectively, yields excellent cure rates with only moderate acute toxicity and is therefore recommended as standard treatment.     

In vitro study of a paclitaxel-radiotherapy combination on a human epidermoid tumor cell line]

PURPOSE: Paclitaxel is an agent which stabilizes microtubules, and has been shown to block different cells in the G2/M phase of the cell cycle and thus to modulate their radioresponsiveness. We investigated the radiosensitizing potential of paclitaxel in human head and neck cancer cells. MATERIALS AND METHODS: ZMK-1 cells were incubated with paclitaxel for 3, 9, or 24 h before or during 24 h after irradiation. Paclitaxel concentrations of 70 nM, 7 nM, and 0.7 nM were chosen to obtain equivalent toxicity at the different incubation times: 3 h, 9 h, and 24 h, respectively. Radiation doses ranged from 0 to 8 Gy using 60Co source. Cell survival was measured by a standard clonogenic assay after a 9-day incubation. Flow cytometry was used to measure the capacity of paclitaxel to accumulate cells in the G2/M phase. RESULTS: Paclitaxel alone possessed cytotoxicity dependent on time and concentration. There was a total of 40% of cells accumulated in G2/M after 24-36 h. When combined with radiation, the 9 h preincubation resulted in a radiosensitization. The 3 h pre-incubation as well as the 24 h post-incubation resulted in an infra-additive effect. CONCLUSION: In our cells a radiosensitizing effect of paclitaxel could not be demonstrated unambiguously. The blockage of the cells in the G2/M phase is not the only mechanism to explain the potential radiosensitization of paclitaxel.     

Radiotherapy in mice with yttrium-90-labeled anti-Ly1 monoclonal antibody: therapy of the T cell lymphoma EL4

Yttrium-90 is a potent beta-emitting radionuclide with potential for therapy of lymphoma. A monoclonal antibody against Ly1, the murine homologue of human CD5, was labeled with 90Y and found to selectively bind to Ly1-positive, radiation-sensitive, EL4 mouse lymphoma cells. When tested in this aggressive model of T cell lymphoma, in vivo studies in C57BL/6 mice showed that a single 140-microCi i.p. dose of 90Y-anti-Ly1, given 1 day after i.v. injection of a lethal dose of EL4 cells, resulted in significant but transient improvement in survival. Protection was selective, since a 90Y-labeled irrelevant control antibody did not prolong survival. Biodistribution studies showed that protection was likely limited by inadequate localization of labeled antibody to tumor. Importantly, labeled anti-Ly1 specifically localized in the immunological tissue (spleen and thymus) and lowered the WBC count, perhaps limiting the tolerated dose. Myelosuppression, which is considered one of the major side effects associated with 90Y usage, was not a lethal complication, since WBC counts recovered in mice given a 140-microCi dose of 90Y-anti-Ly1 without EL4 cells and 100% of these animals survived. The maximum tolerated dose was less than 200 microCi. Despite the high localization of 90Y-anti-Ly1 in spleen, splenectomies of tumor-injected mice did not improve the antitumor efficacy of radiolabeled antibody. Further evidence for inadequate delivery of radionuclide to tumor was shown when external total-body irradiation was given to mice given injections of a lethal dose of EL4 tumor cells. Comparison of internal and external irradiation studies indicated that the partially protective effect of 140 microCi 90Y-Ly1 was equivalent to external radiation of only 100-200 cGy. Because this model reflects the current clinical limitations of radiolabeled antibodies for therapy, including partial antitumor efficacy, delivery of labeled anti-T cell antibodies to the immune system, and low maximum tolerated dose, the model may be useful for examining strategies which could increase the tolerated dose and therapeutic efficacy.