De novo identification of CD4+ T cell epitopes.

CD4+ T cells recognize peptide antigens presented on class II major histocompatibility complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor sequences and lack of antigen discovery approaches for MHC-II make profiling the specificities of CD4+ T cells challenging. We have expanded our platform of signaling and antigen-presenting bifunctional receptors to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ T cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, allowing a readable output. Furthermore, the SABR-II design is modular in signaling and deployment to T cells and B cells. Here, we demonstrate that SABR-IIs libraries presenting endogenous and non-contiguous epitopes can be used for antigen discovery in the context of type 1 diabetes. SABR-II libraries provide a rapid, flexible, scalable and versatile approach for de novo identification of CD4+ T cell ligands from single-cell RNA sequencing data using experimental and computational approaches.

p53 Promotes Cytokine Expression in Melanoma to Regulate Drug Resistance and Migration.

The transcription factor p53 is frequently lost during tumor development in solid tumors; however, most melanomas retain a wild type p53 protein. The presence of wild type p53 in melanoma has fueled speculation that p53 may play a neutral or pro-tumorigenic role in this disease. Here we show that p53 is functional in human melanoma cell lines, and that loss of p53 results in a general reduction in basal NF-kB regulated cytokine production. The reduced cytokine expression triggered by p53 loss is broad and includes key inflammatory chemokines, such as CXCL1, CXCL8, and the IL6 class cytokine LIF, resulting in a reduced ability to induce chemotactic-dependent migration of tumor cells and immune cells and increased sensitivity to BRAF inhibition. Taken together, this result indicates that wild type p53 regulates cytokine expression and induces cytokine-dependent phenotype on melanoma.

Identification of universal and cell-type specific p53 DNA binding.

BACKGROUND: The tumor suppressor p53 is a major regulator of the DNA damage response and has been suggested to selectively bind and activate cell-type specific gene expression programs. However recent studies and meta-analyses of genomic data propose largely uniform, and condition independent p53 binding and thus question the selective and cell-type dependent function of p53. RESULTS: To systematically assess the cell-type specificity of p53, we measured its association with DNA in 12 p53 wild-type cancer cell lines, from a range of epithelial linages, in response to ionizing radiation. We found that the majority of bound sites were occupied across all cell lines, however we also identified a subset of binding sites that were specific to one or a few cell lines. Unlike the shared p53-bound genome, which was not dependent on chromatin accessibility, the association of p53 with these atypical binding sites was well explained by chromatin accessibility and could be modulated by forcing cell state changes such as the epithelial-to-mesenchymal transition. CONCLUSIONS: Our study reconciles previous conflicting views in the p53 field, by demonstrating that although the majority of p53 DNA binding is conserved across cell types, there is a small set of cell line specific binding sites that depend on cell state.

Optimized systemic dosing with CpG DNA enhances dendritic cell-mediated rejection of a poorly immunogenic mammary tumor in BALB/c mice.

To model a clinical trial of dendritic cell (DC) therapy of a poorly immunogenic mammary tumor, we treated BALB/c mice bearing an established TS/A mammary tumor with lysate-pulsed DCs and CpG DNA. We observed that the dose of CpG DNA required to activate DCs in vitro was insufficient to mediate tumor rejection in vivo. We therefore undertook in vivo studies to identify an optimized dose of CpG DNA for tumor therapy, defined as the lowest and least frequently administered dose of CpG DNA that mediated complete tumor rejection. We show that one priming dose of 15 nanomoles and one booster dose of 10 nanomoles of CpG DNA given 7 days apart, respectively, with lysate-loaded DCs were sufficient to mediate complete tumor rejection in vivo. This dose of CpG DNA was 42-fold higher than that required to activate DCs in vitro but was not associated with any toxicity in mice. Also, the cured mice rejected a subsequent challenge with fresh TS/A tumor, and both CD4(+) and CD8(+) T cells were required for tumor rejection. We conclude that effective DC-based therapy of a poorly immunogenic TS/A tumor is enhanced by optimized dosing of CpG DNA. Our data have important implications for DC-based clinical trials of breast cancer immunotherapy.