H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies.

H-2 class I-negative, HLA-A2.1-transgenic HHD mice were used for a comparative evaluation of the immunogenicity of HLA-A2.1-restricted human tumor-associated cytotoxic T lymphocyte (CTL) epitopes. A hierarchy was established among these peptides injected into mice in incomplete Freund's adjuvant which correlates globally with their capacity to bind and stabilize HLA-A2.1 molecules. Co-injection of a helper peptide enhanced most CTL responses. In contrast, classical HLA class I-transgenic mice which still express their own class I molecules did not, in most cases, develop HLA-A2.1-restricted CTL responses under the same experimental conditions. Different monoepitope immunization strategies of acceptable clinical usage were compared in HHD mice. Recombinant Ty-virus-like particles, or DNA encoding epitopes fused to the hepatitis B virus middle envelope protein gave the best results. Using this latter approach and a melanoma-based polyepitope construct, CTL responses against five distinct epitopes could be elicited simultaneously in a single animal. Thus, HHD mice provide a versatile animal model for preclinical evaluation of peptide-based cancer immunotherapy.

Cytotoxic T cell response against the chimeric p210 BCR-ABL protein in patients with chronic myelogenous leukemia.

Human chronic myelogenous leukemia (CML) is characterized by a translocation between chromosomes 9 and 22 that results in a BCR-ABL fusion gene coding for chimeric proteins. The junctional region of the BCR-ABLb3a2 molecule represents a potential leukemia-specific antigen which could be recognized by cytotoxic T lymphocytes (CTL). In fact, we identified a junctional nonapeptide (SSKALQRPV) which binds to HLA-A2.1 molecules. This peptide, as well as those binding to HLA-A3, -A11, and -B8 molecules (previously identified by others), elicits primary CTL responses in vitro from PBLs of both healthy donors and CML patients. Such CTL recognize HLA-matched, BCR-ABL-positive leukemic cells, implying efficient natural processing and presentation of these junctional peptides. Specific CTL were found at high frequency in 5 of 21 CML patients, suggesting that these epitopes are, to some extent, immunogenic in vivo during the course of the disease. These peptides could be useful for the development of specific immunotherapy in CML patients.

Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo.

The homeodomain of the Antennapedia molecule (AntpHD) spontaneously crosses cellular membranes and can be used to deliver up to 50 additional amino acids to the cytoplasm. We exploited this approach to deliver antigenic peptides to the MHC class I processing and presentation pathway. AntpHD-based fusion peptides expressing the 170-179 HLA-Cw3 CTL epitope (pCw3) were produced in bacteria. Incubation of these fusion peptides with H-2d target cells resulted in efficient delivery to the cytosol as indicated by protease resistance and confocal microscopy. Moreover, this introduction of an exogenous Ag resulted in sensitization of the cell to lysis by a CTL clone specific for the 170-179 HLA-Cw3-derived peptide. Sensitivity of the Ag processing to brefeldin A but not to chloroquine is consistent with the delivery of AntpHD fusion peptides to the conventional class I-associated processing pathway. Immunization of DBA/2 (H-2d) mice with AntpHD pCw3 fusion peptide in the presence of SDS primed H-2Kd-restricted HLA-Cw3-specific CTL. Similar results were obtained with AntpHD fusion peptides expressing the 147-156 influenza nucleoprotein peptide. The strategy outlined in this paper provides a new approach for introducing molecules into the MHC class I Ag-presenting pathway. This approach has clear relevance to the design of synthetic peptide-based vaccines.

HIV-1 infection of lung alveolar fibroblasts and macrophages in humans.

We have studied the infected cell populations in the lungs of four human immunodeficiency virus type 1 (HIV-1) seropositive patients suffering from lymphocytic alveolitis or lymphocytic interstitial pneumonitis. Adherent cells were obtained by bronchoalveolar lavage (BAL) and were analyzed by various technical approaches. The cells considered here were alveolar macrophages and fibroblasts, and could be clearly identified morphologically and by the expression of specific cell-surface markers using monoclonal antibodies. The presence of HIV-1 in both of these cell types was established by serological, virological, and molecular procedures. Our results show that alveolar macrophages and fibroblasts are naturally infected in the lungs of HIV+ patients. Both cell types express the CD4 receptor molecule, in contrast to skin fibroblasts which are negative. Alveolar macrophages and fibroblasts thus may act as eventual HIV-1 reservoirs in vivo, and are probably involved in the induction of inflammatory reactions because they are targets for CD8 cytotoxic T lymphocytes (CTL).

Multiple subsets of HIV-specific cytotoxic T lymphocytes in humans and in mice.

The human immunodeficiency virus type 1 (HIV-1) induces a strong cytotoxic T lymphocyte (CTL) response in humans following infection. HIV-specific CTL can be detected directly in the blood and lungs of infected patients, and can be expanded in vitro by stimulation with autologous HIV-infected lymphoblasts. Furthermore, CTL specific for HIV envelope glycoprotein gp160 have been obtained in mice by immunization with recombinant vaccinia virus (VV) that carry the HIV env gene. In this study, we show that mice also produce strong CTL responses to gag and nef proteins following immunization with VV recombinants, thus providing a convenient model system to study T lymphocyte immunity to defined HIV antigens. To determine the specificity of circulating HIV-immune CTL in humans, a panel of doubly transfected mouse P815 tumor cells was produced which express the human HLA-A2 or HLA-A3 transplantation antigen gene and one HIV-1 gene (env, gag or nef). Using these cells as targets to CTL, we show that HIV-infected humans carry co-existing CTL sub-populations of different specificities. Each subpopulation appears to vary in intensity among different individuals. Surprisingly, CTL specific for regulatory, non-structural nef protein appear to be a major constituent of the human immune response to HIV.

Immune resistance to Trypanosoma cruzi: synergy of specific antibodies and recombinant interferon gamma in vivo.

The protective effects of interferon gamma (IFN-gamma) against infection by Trypanosoma cruzi were studied in vitro and in vivo in a murine model of infection. The possible synergy between IFN-gamma and trypomastigote-specific antibodies in the rejection of the parasite was also considered. Our results in vitro indicate that IFN-gamma activates macrophages to reject the parasite and this mechanism may lead to a decrease in parasitaemia in vivo. Finally, IFN production in vivo after infection by T. cruzi was compared among mice from different genetic backgrounds. Reduced parasitaemia and extended survival correlated with the early production of circulating IFN-gamma and anti-trypomastigote antibodies after infection. The appearance of an unusual type of circulating IFN in response to infection by T. cruzi was also detected; this IFN was resistant to neutralization by antibodies to IFN-alpha/beta and to IFN-gamma.

Selective suppression of tumour-immune cytolytic T lymphocytes in mice with chronic Trypanosoma cruzi infections.

Trypanosoma cruzi is the causative agent of Chagas' disease in man, often leading to suppression of T lymphocyte functions; the present study thus considered the effects of infection by T. cruzi on T-dependent immune responses in a murine model, namely, the immune resistance to a syngeneic tumour and a delayed-type hypersensitivity reaction to sheep red blood cells (SRBC). In BALB.B mice infected with T. cruzi, the graft of syngeneic Gross murine leukaemia virus-induced tumour cells leads to an increased incidence of progressive subcutaneous tumours and development of lymphatic leukaemia. This decreased resistance to tumours correlates with a suppression of the generation of tumour-specific cytolytic T lymphocytes (CTL) from the pre-CTL stage. In contrast, clonal expansion and circulation of T cells detected through their ability to locally transfer a delayed-type hypersensitivity (DTH) reaction to SRBC remained normal in T. cruzi-infected mice. However, at the site of the DTH reaction, a decreased availability of phagocytes was observed in T. cruzi-infected mice.

Antigenic polymorphism of Trypanosoma cruzi: clonal analysis of trypomastigote surface antigens.

The surface membrane antigens of infectious Trypanosoma cruzi trypomastigotes were studied at the levels of the strain and of individual trypomastigote clones. Blood trypomastigotes from three T. cruzi strains, Y, CL and Tehuantepec ("Teh"), were grown in vitro by weekly infection of J774 mouse macrophage tumor cells. Each T. cruzi strain was subsequently cloned by infection of J774 cells at limited trypomastigote dilution, and antisera were produced in mice against a selection of trypomastigote clones. Criss-cross panel analyses indicated the existence of a large degree of polymorphism among trypomastigote surface antigens. Various trypomastigote surface antigens were cross-reactive, appeared to be highly conserved, and were common to the three strains considered and to all the clones derived from each strain. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that numerous trypomastigote antigenic proteins were precipitated by mouse antisera generated against cloned trypomastigotes. Some of these proteins were commonly distributed, while others were polymorphic. Finally, a state of cross-reactive immunity could be induced in C3H/He mice by infection with a cloned T. cruzi trypomastigote population. Immune mice resisted subsequent infections with lethal doses of wild-type bloodstream trypomastigotes from any one of the three T. cruzi strains.