B. Davies

Generation of cytotoxic T cell responses directed to human leucocyte antigen Class I restricted epitopes from the Aspergillus f16 allergen

Invasive aspergillosis (IA) is a major cause of infection‐related mortality in patients with haematological malignancies, especially in recipients of haematopoietic stem cell transplants. We have prepared overlapping pentadecapeptides (11‐aa overlap with previous peptide) spanning the entire 427‐aa coding region of the Aspergillus allergen, Asp f16 shown previously in mice to induce Th1‐type cell responses in vivo and in humans to induce proliferative and cytotoxic CD4+ T cell responses. Mature dendritic cells (DC) pulsed with a complete pool of peptides were used to generate T cell lines. Two lines from HLA‐B*3501+ donors were found to be strongly cytotoxic to autologous Asp f16‐peptide pool‐ and Aspergillus culture extract‐pulsed targets after 4–5 weekly primings. Cytotoxic T lymphocyte (CTL) culture supernatant killed Aspergillus conidia, and cells directly killed Aspergillus hyphae. Cytotoxic activity and interferon (IFN)‐γ production were mediated exclusively by CD8+ T cells in response to pool‐pulsed targets. Interleukin (IL)‐4 production was not detected. CTL activity was restricted by HLA‐B*3501 and based on peptide prediction programmes was most probably directed to YFKYTAAAL (YFK), LPLCSAQTW (LPL) and GTRFPQTPM (GTR) in one donor, while only LPL was recognized by CTL from the second donor. Pool‐pulsed B*3503+ BLCL but not B*3502+ or B*3508+ BLCL presented peptide to donor no. 1. B*3503+ BLCL presented YFK and to a lesser extent GTR, but not peptide LPL. Our data show that in addition to our previously identified Class II restricted peptide response, DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing polyclonal, HLA‐Class I‐restricted, Aspergillus‐specific T cells that may be capable of conferring immunity to IA.

Generation of Th1 T cell responses directed to a HLA Class II restricted epitope from the Aspergillus f16 allergen

The Aspergillus allergen Asp f16 has been shown to confer protective Th1 T cell‐mediated immunity against infection with Aspergillus conidia in murine models. Here, we use overlapping (11‐aa overlap with preceding peptide) pentadecapeptides spanning the entire 427‐aa coding region of Asp f16 presented on autologous dendritic cells (DC) to evaluate the ability of this antigen to induce Th1 responses in humans. Proliferative responses were induced in five out of five donors, and one line with a high frequency of interferon (IFN)‐γ‐producing CD4+ T cells in response to the complete peptide pool was characterized. This line was cytotoxic to autologous pool‐pulsed and Aspergillus culture extract‐pulsed targets. Limitation of cytotoxicity to the CD4+ T cell subset was demonstrated by co‐expression of the degranulation marker CD107a in response to peptide pool‐pulsed targets. Cytotoxic T lymphocytes (CTL) killed Aspergillus hyphae and CTL culture supernatant killed Aspergillus conidia. By screening 21 smaller pools and individual peptides shared by positive pools we identified a single candidate sequence of TWSIDGAVVRT that elicited responses equal to the complete pool. The defined epitope was presented by human leucocyte antigen (HLA)‐DRB1‐0301. These data identify the first known Aspergillus‐specific T cell epitope and support the use of Asp f16 in clinical immunotherapy protocols to prime protective immune responses to prevent or treat Aspergillus infection in immunocompromised patients.

Stimulation by means of dendritic cells followed by Epstein-Barr virus-transformed B cells as antigen-presenting cells is more efficient than dendritic cells alone in inducing Aspergillus f16-specific cytotoxic T cell responses.

Adoptive immunotherapy with in vitro expanded antigen‐specific cytotoxic T lymphocytes (CTLs) may be an effective approach to prevent, or even treat, Aspergillus (Asp) infections. Such lines can be generated using monocyte‐derived dendritic cells (DC) as antigen‐presenting cells (APC) but requires a relatively high volume of starting blood. Here we describe a method that generates Asp‐specific CTL responses more efficiently using a protocol of antigen presented on DC followed by Epstein–Barr virus (EBV)‐transformed B lymphoblastoid cell lines (BLCL) as APC. Peripheral blood mononuclear cells were stimulated weekly (2–5×) with a complete pool of pentadecapeptides (PPC) spanning the coding region of Asp f16 pulsed onto autologous mature DC. Cultures were split and stimulated subsequently with either PPC‐DC or autologous PPC‐pulsed BLCL (PPC‐BLCL). Lines from the DC/BLCL arm demonstrated Asp f16‐specific cytotoxicity earlier and to a higher degree than lines generated with PPC‐DC alone. The DC/BLCL‐primed lines showed a higher frequency of Asp f16‐specific interferon (IFN)‐γ producing cells but an identical effector cell phenotype and peptide specificity compared to PPC‐DC‐only‐primed lines. Tumour necrosis factor (TNF)‐α, but not IL‐10, appeared to play a role in the effectiveness of BLCL as APC. These results demonstrate that BLCL serve as highly effective APC for the stimulation of Asp f16‐specific T cell responses and that a culture approach using initial priming with PPC‐DC followed by PPC‐BLCL may be a more effective method to generate Asp f16‐specific T cell lines and requires less starting blood than priming with PPC‐DC alone.

DRUG RESISTANCE FOLLOWING IRRADIATION OF RIF-1 TUMORS: INFLUENCE OF THE INTERVAL BETWEEN IRRADIATION AND DRUG TREATMENT

RIF-1 tumors contain a small number of cells (1 to 100 per 10(6) cells) that are resistant to 5-fluorouracil, methotrexate, or adriamycin. The frequency of drug-resistant cells among individual untreated tumors is highly variable. Radiation, delivered in vivo at doses of 3 to 12 Gy, increases the frequency of methotrexate- and 5-fluorouracil-resistant cells, but not the frequency of adriamycin-resistant cells. The magnitude of induction of 5-fluorouracil and methotrexate resistance shows a complex dependence on the radiation dose and on the interval between irradiation and assessment of drug resistance. For a dose of 3 Gy, induced 5-fluorouracil and methotrexate resistance is seen only after an interval of 5 to 7 days, whereas for a dose of 12 Gy, high levels of induced resistance are observed 1 to 3 days after irradiation. The maximum absolute risk for induction of resistance is 4 per 10(4) cells per Gy for methotrexate, and 3 per 10(6) cells per Gy for 5-fluorouracil. These results indicate that tumor hypoxia may play a role in the increased levels of drug resistance seen after irradiation, and that both genetic and environmental factors may influence radiation-induction of drug resistance. These studies provide essential data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be caused by radiation-induced drug resistance.

RADIATION INDUCTION OF DRUG RESISTANCE IN RIF-1: CORRELATION OF TUMOR AND CELL CULTURE RESULTS

The RIF-1 tumor line contains cells that are resistant to various anti-neoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), adriamycin (ADR), and etoposide (VP16). The frequency of these drug-resistant cells is increased after irradiation. The frequency of drug-resistant cells and the magnitude of radiation-induced drug resistance are different in cell culture than in tumors. The dose-response and expression time relationships for radiation induction of drug resistance observed in RIF-1 tumors are unusual. We hypothesize that at high radiation doses in vivo, we are selecting for cells that are both drug resistant and radiation resistant due to microenvironmental factors, whereas at low radiation doses in vivo and all radiation doses in vitro, we are observing true mutants. These studies indicate that there can be significant differences in drug-resistance frequencies between tumors and their cell lines of origin, and that radiation induction of drug resistance depends significantly on whether the induction is done in tumors or in cell culture. These results imply that theories about the induction of drug resistance that are based on cell culture studies may be inapplicable to the induction of drug resistance in tumors.