Vaccination with Designed Neopeptides Induces Intratumoral, Cross-reactive CD4+ T-cell Responses in Glioblastoma.

PURPOSE: The low mutational load of some cancers is considered one reason for the difficulty to develop effective tumor vaccines. To overcome this problem, we developed a strategy to design neopeptides through single amino acid mutations to enhance their immunogenicity. EXPERIMENTAL DESIGN: Exome and RNA sequencing as well as in silico HLA-binding predictions to autologous HLA molecules were used to identify candidate neopeptides. Subsequently, in silico HLA-anchor placements were used to deduce putative T-cell receptor (TCR) contacts of peptides. Single amino acids of TCR contacting residues were then mutated by amino acid replacements. Overall, 175 peptides were synthesized and sets of 25 each containing both peptides designed to bind to HLA class I and II molecules applied in the vaccination. Upon development of a tumor recurrence, the tumor-infiltrating lymphocytes (TIL) were characterized in detail both at the bulk and clonal level. RESULTS: The immune response of peripheral blood T cells to vaccine peptides, including natural peptides and designed neopeptides, gradually increased with repetitive vaccination, but remained low. In contrast, at the time of tumor recurrence, CD8+ TILs and CD4+ TILs responded to 45% and 100%, respectively, of the vaccine peptides. Furthermore, TIL-derived CD4+ T-cell clones showed strong responses and tumor cell lysis not only against the designed neopeptide but also against the unmutated natural peptides of the tumor. CONCLUSIONS: Turning tumor self-peptides into foreign antigens by introduction of designed mutations is a promising strategy to induce strong intratumoral CD4+ T-cell responses in a cold tumor like glioblastoma.

TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development.

TRAIL is a promising anticancer agent due to its ability to selectively induce apoptosis in established tumor cell lines but not nontransformed cells. Herein, we demonstrate a role for the apoptosis-inducing TRAIL receptor (TRAIL-R) as a metastasis suppressor. Although mouse models employing tumor transplantation have shown that TRAIL can reduce tumor growth, autochthonous tumor models have generated conflicting results with respect to the physiological role of the TRAIL system during tumorigenesis. We used a multistage model of squamous cell carcinoma to examine the role of TRAIL-R throughout all steps of tumor development. DMBA/TPA-treated TRAIL-R-deficient mice showed neither an increase in number or growth rate of benign papillomas nor an increase in the rate of progression to squamous cell carcinoma. However, metastasis to lymph nodes was significantly enhanced, indicating a role for TRAIL-R specifically in the suppression of metastasis. We also found that adherent TRAIL-R-expressing skin carcinoma cells were TRAIL resistant in vitro but were sensitized to TRAIL upon detachment by inactivation of the ERK signaling pathway. As detachment from the primary tumor is an obligatory step in metastasis, this provides a possible mechanism by which TRAIL-R could inhibit metastasis. Hence, treatment of cancer patients with agonists of the apoptosis-inducing receptors for TRAIL may prove useful in reducing the incidence of metastasis.

The epithelial glucocorticoid receptor is required for the normal timing of cell proliferation during mammary lobuloalveolar development but is dispensable for milk production.

Glucocorticoids have been shown to influence mammary gland function in vivo and to stimulate milk protein gene expression in vitro. Here, we describe the generation and analysis of a mouse model to study glucocorticoid receptor (GR, NR3C1) function in mammary epithelial cells. Using the Cre-loxP system, mutant mice were obtained in which the GR gene is specifically deleted in epithelial cells during lobuloalveolar development, leading to a complete loss of epithelial GR at the onset of lactation. Mice harboring the mammary-epithelial-specific GR mutation are able to nurse their litters until weaning. During pregnancy, however, GR deficiency delays lobuloalveolar development, leading to an incomplete epithelial penetration of the mammary fat pad that persists throughout lactation. We identified a reduced cell proliferation during lobuloalveolar development as reason for this delay. This reduction is compensated for by increased epithelial proliferation after parturition in the mutant glands. During lactation, GR-deficient mammary epithelium is capable of milk production and secretion. The expression of two milk proteins, namely whey acidic protein and beta-casein, during lactation was not critically affected in the absence of GR. We conclude that GR function is not essential for alveolar differentiation and milk production, but influences cell proliferation during lobuloalveolar development.

Evaluation of steroid receptor function by gene targeting in mice.

Corticosteroid hormones regulate a variety of developmental, physiological and pathological processes via their cognate receptors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). Using modern genetic technologies, including bacterial artificial chromosome-based transgenesis and conditional gene targeting, we have generated a panel of tissue-specific and function-selective mutations of the two corticosteroid hormone receptors in the mouse. These mouse models have allowed us to gain new insights into corticosteroid hormone signaling in vivo. By investigating a hepatocyte-specific GR mutation, it has been possible to define a novel biological action of GR, namely to function as a coactivator for Stat5-mediated gene transcription in the control of body growth. The investigation of brain-specific mutations have not only allowed us to better understand hypothalamo-pituitary-adrenal (HPA) axis regulation by glucocorticoids, but also to analyse corticosteroid action in various aspects of brain function like anxiety-related or addiction-related behaviour, and learning and memory. A function-selective mutation in the GR has allowed us to dissect different pathways in the gene expression regulation by this receptor, namely to separate DNA response element-binding dependent gene activation from response element-independent gene regulation via interference with other transcription factors. These different transcriptional activities of GR play an important role in glucocorticoid-mediated immunosuppression.

Targeting mammary epithelial cells using a bacterial artificial chromosome.

We describe the generation of transgenic mouse lines expressing Cre recombinase in epithelial cells of the lactating mammary gland. As an expression vector, we used a P1-derived bacterial artificial chromosome (PAC) which harbors the gene for the secretory milk protein, whey acidic protein (Wap). Using homologous recombination in E. coli, the PAC was modified to carry the improved coding sequence of Cre recombinase (iCre). Transgenic lines carrying the WAPiCre PAC express Cre recombinase efficiently in the majority of mammary epithelial cells upon lactation. Of only four transgenic lines produced, three express Cre recombinase to a high efficiency. LoxP-flanked DNA sequences are recombined in virtually all epithelial cells of WAPiCre transgenic mice at lactation day 3.