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The cloning of the genes encoding cancer antigens has opened new possibilities for the treatment of patients with cancer. In this study, immunodominant peptides from the gp100 melanoma-associated antigen were identified, and a synthetic peptide, designed to increase binding to HLA-A2 molecules, was used as a cancer vaccine to treat patients with metastatic melanoma. On the basis of immunologic assays, 91% of patients could be successfully immunized with this synthetic peptide, and 13 of 31 patients (42%) receiving the peptide vaccine plus IL-2 had objective cancer responses, and four additional patients had mixed or minor responses. Synthetic peptide vaccines based on the genes encoding cancer antigens hold promise for the development of novel cancer immunotherapies.

Most chemically induced tumors of mice express unique antigens that can be recognized by cytotoxic T lymphocytes (CTL) and thereby mediate tumor rejection. The number of different antigens expressed by a single tumor and their interplay during immunization and rejection are largely unexplored. We used CTL clones specific to individual tumor antigens to examine the number and distribution of CTL antigens expressed by cell lines derived from 3-methylcholanthrene-induced sarcomas of (C57BL/6J X SPRET/Ei)F1 mice. Each tumor cell line expressed one or more antigens that were unique, that is, not detected on cell lines from independent sarcomas. Immunoselection against an immunodominant antigen produced both major histocompatibility complex class I antigen and unique tumor antigen loss variants. Immunization of mice with antigen-negative immunoselected variants resulted in CTL that recognized additional antigens that were also expressed by the progenitor tumor. Some CTL recognized additional unique tumor antigen(s); other CTL recognized a shared antigen expressed not only by the immunizing cell line, but also by independent sarcoma cell lines and untransformed myoblastoid cell lines. CTL that recognized the shared antigen were also recovered from mice immunized in vivo with an untransformed myoblastoid cell line. These findings support a model of immunodominance among chemically induced tumor antigens in which shared antigens are masked by unique immunodominant antigens.

The common class I alleles (e.g., Kd and Db) within the H2g7 major histocompatibility complex (MHC) clearly contribute to autoimmune IDDM in NOD mice, but the mechanism by which this occurs has been controversial. One laboratory has reported that the peptide transporter encoded by the Tap1 gene within H2g7 is defective, and this contributes to IDDM by impairing MHC class I-mediated antigen presentation. If true, defective MHC class I-mediated antigen presentation should segregate with the H2g7 haplotype. NOD mice, related congenic stocks, and other control strains were used to test this hypothesis. H2g7-positive strains did not differ from those expressing other MHC haplotypes in ability to present MHC class I-restricted H3aa or H3ab minor histocompatibility (H) antigens to cytotoxic T-lymphocytes (CTL). The H2g7 haplotype was found to have a reduced capacity to mediate MHC class I-restricted presentation of the H47a minor H antigen. However, MHC class I-restricted presentation of H47a was found to be Tap independent. NOD mice and control strains also did not differ in ability to activate adenovirus-specific MHC class I restricted CTL. Thus, the H2g7 haplotype is not characterized by a Tap gene defect that only impairs the inductive phase of the immune response. In addition, MHC class I-restricted presentation of either minor H or adenoviral antigens was equivalent in male and female NOD mice. Therefore, while the class I alleles of the H2g7 haplotype exert diabetogenic functions in NOD mice, this is not elicited through a Tap gene defect. The absence of female-specific Tap gene defects also indicates this cannot account for the reduced male incidence of IDDM in some NOD mouse colonies.

Well-characterized murine mutations are powerful analytical tools for the genetic analysis of tumorigenesis. We crossed the multiple intestinal neoplasia (Min) allele of adenomatous polyposis coli (Apc), which produces a profound pre-disposition to intestinal neoplasia, with the severe combined immunodeficiency (scid) mutation, which causes defective double-strand DNA repair and severe immunodeficiency, on the common C57BL/6J genetic background to assay for any combined effect on intestinal tumorigenesis. Several phenotypic traits were exacerbated in an apparently additive manner in the double mutant mice, including reduced immunoglobulin levels, reduced body weight and increased morbidity. However, quantitation and histological evaluation of polyp phenotype indicated that these mutations did not interact to affect either polyp frequency or progression. Thus, neither genome instability nor lack of immunosurveillance conferred by scid contributes to intestinal polyps in this model.

Loss of heterozygosity (LOH) occurs commonly in some human tumors and is thought to reflect the selective pressure exerted by tumor suppressor genes. The purpose of this study was to examine the genomic location and relative frequency of LOH of chemically induced tumors in an established mouse model for chemical carcinogenesis. Twenty-four tumors were induced by subcutaneous injection of 3-methylcholanthrene into inter-subspecific hybrid (C57BL/6J x SPRET/Ei)F1 mice. Tumors were diagnosed as poorly differentiated, spindle cell sarcomas. DNA isolated from cell cultures derived from the sarcomas was analysed for LOH using a multi-locus genome scanning technique, motif-primed PCR, followed by conventional simple sequence length polymorphism (SSLP) PCR markers. Ninety-one motif-primed PCR products and seven SSLP markers were evaluated, covering segments of 39 out of 41 chromosomes. LOH was extremely rare; average fractional allelic loss per tumor was less than 0.01 suggesting a remarkable genetic stability of the tumors and that genetic events leading to transformation are only rarely revealed as LOH. However, some regions exhibiting LOH were identified encompassing both known and hypothesized tumor suppressor loci such as Trp53. These data indicate that motif-primed PCR is an efficient method of scanning the genome of interspecific sarcomas for rare mutational events generating LOH during tumor induction. Cell lines derived from the sarcomas promise to be useful for identifying genes whose inactivation causes increased malignancy during tumor progression.

Adoptive immunotherapy (AIT) involving transfer of tumor-sensitized T lymphocytes in combination with cyclophosphamide (CY)-injection results in the eradication of the C57BL/6J (B6) rhabdomyosarcoma, 76-9 and is associated with the accumulation of a large number of tumor-infiltrating lymphocytes (TIL). Using immune spleen cells (ISC) from B6 and congenic B6. PL. Thy-1a mice, it was shown that most (> or = 97%) of the TIL were donor-derived. This in situ increase in donor-derived T cells was confirmed by using positively-selected Thy- 1.1+ and Thy- 1.2+ TIL for AIT after isolating them from regressing tumors and expanding them in rIL-2. The extent of CD8+ TIL expansion in vivo correlated with the numbers of TIL adoptively transferred and this in turn determined the degree of anti-tumor effects. It was evident, however, that these in vitro-expanded TIL expressing mRNA for TNF alpha and IFN gamma were qualitatively different and therapeutically less efficacious than the T cells associated with ISC or with freshly-isolated TIL. Unlike freshly isolated TIL that expressed specific cytotoxicity towards the 76-9 targets in vitro, IL-2 expanded TIL killed 76-9 cells and unrelated tumor targets to the same extent. A cytotoxic CD8+ T cell line derived from ISC and selected for activity against the 76-9 tumor cells showed no therapeutic efficacy. The data suggest that, in this tumor model, expansion of CD8+ T cells in vitro selects against anti-tumor efficacy.

Minor histocompatibility (H) loci encode alloantigens that are recognized by cytotoxic T (Tc) lymphocytes. A (C57BL/10 x 129)F1-derived transformed lymphocyte cell line was immunoselected in vitro with cloned Tc cells that were specific for H-3aa, a Chromosome 2-encoded minor H antigen. This cell line is heterozygous at H-3a (former symbol, Cd-1) and other loci. Three groups of antigen-loss variants were identified. One group contained mutations affecting only the antigen-encoding gene. Another group probably arose through a single homologous interchromosomal exchange, resulting in extensive regions of loss of heterozygosity (LOH). The third group of variants contained an interstitial LOH, one of which was shown to be a significant deletion. Several deletion boundaries were identified, one of which ordered the closely linked H-3a and beta 2-microglobulin (B2m) genes. We suggest that Tc immunoselection against minor H antigens is a promising approach for targeting negative selection to specified chromosomal regions and can provide high-resolution genetic map information.

The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation of epidermal root hairs and the approximately concurrent stimulation of cell dedifferentiation and cell division in the root inner cortex. These early steps have been studied previously by analysis of R. meliloti mutants. Bacterial strains mutated in nodABC, for example, fail to stimulate either root hair curling or cell division events in the plant host, whereas exopolysaccharide (exo) mutants of R. meliloti stimulate host cell division but the resulting nodules are uninfected. As a further approach to understanding early symbiotic interactions, we have investigated the phenotype of a non-nodulating alfalfa mutant, MnNC-1008 (NN) (referred to as MN-1008). Nodulating and non-nodulating plants were inoculated with wild-type R. meliloti and scored for root hair curling and cell divisions. MN-1008 was found to be defective in both responses. Mutant plants inoculated with Exo- bacteria also showed no cell division response. Therefore, the genetic function mutated in MN-1008 is required for both root hair curling and cell division, as is true for the R. meliloti nodABC genes. These observations support the model that the distinct cellular processes of root hair curling and cell division are triggered by related mechanisms or components, or are causally linked.

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1-10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4'-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21-24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a "hybrid" structure intermediate between a nodule and a lateral root.