Programmed Death-1 receptor (PD-1) is an inhibitory receptor on hematopoietic cells that can negatively regulate immune responses, particularly responses to tumors, which often upregulate PD-1 ligands. PD-1/PD-1 ligand blocking antibodies can reverse the inhibition and show significant therapeutic promise in treating renal cell carcinoma (RCC), lung cancer and melanoma. While PD-1 expression on tumor infiltrating lymphocytes has been associated with poor outcome in RCC, we sought to define immune cell biomarkers, including PD-1, on peripheral blood mononuclear cells (PBMC) that could predict disease progression of RCC patients before and after nephrectomy. We analyzed expression of numerous immune cell markers on fresh PBMC from 90 RCC patients preoperatively and 25 age-matched healthy controls by 10-color flow cytometry. Postoperative blood samples were also analyzed from 23 members of the RCC patient cohort. The most striking phenotypic immune biomarker in RCC patients was a significant increase in PD-1 expression on certain PBMC in a subset of patients. Increased PD-1 expression on CD14bright myelomonocytic cells, effector T cells, and NK cells correlated to disease stage, and expression was significantly reduced on all cell types soon after surgical resection of the primary tumor. The results indicate that PD-1 expression on fresh peripheral blood leukocytes may provide a useful indicator of RCC disease progression. Furthermore, measuring PD-1 levels in peripheral blood may assist in identifying patients likely to respond to PD-1 blocking antibodies, and these therapies may be most effective before and immediately after surgical resection of the primary tumor, when PD-1 expression is most prominent.

Toll-like receptors (TLRs) recognize pathogens and their components, thereby initiating immune responses to infectious organisms. TLR ligation leads to the activation of NF-κB and MAPKs through well-defined pathways, but it has remained unclear how TLR signaling activates PI3K, which provides an inhibitory pathway limiting TLR responses. Here, we show that the signaling adapter B-cell adaptor for PI3K (BCAP) links TLR signaling to PI3K activation. BCAP-deficient macrophages and mice are hyperresponsive to TLR agonists and have reduced PI3K activation. The ability of BCAP to inhibit TLR responses requires its capacity to bind PI3K. BCAP is constitutively phosphorylated and associated with the p85 subunit of PI3K in macrophages. This tyrosine-phosphorylated BCAP is transiently enriched in the membrane fraction in response to LPS treatment, suggesting a model whereby TLR signaling causes the phosphorylation of the small amount of BCAP that is associated with membranes in the resting state or the translocation of phosphorylated BCAP from the cytoplasm to the membrane. This accumulation of tyrosine-phosphorylated BCAP at the membrane with its associated PI3K would then allow for the catalysis of Ptd Ins P2 to Ptd Ins P3 and downstream PI3K-dependent signals. Therefore, BCAP is an essential activator of the PI3K pathway downstream of TLR signaling, providing a brake to limit potentially pathogenic excessive TLR responses.inflammatory response | innate immunity

We have developed 3D-tumoroids and tumor slice in vitro culture systems from surgical tumor specimens derived from patients with colorectal cancer (CRC) or lung cancer to evaluate immune cell populations infiltrating cultured tissues. The system incorporates patient's peripherally and tumor-derived immune cells into tumoroid in vitro cultures to evaluate the ability of the culture to mimic an immunosuppressive tumor microenvironment (ITM). This system enables analysis of tumor response to standard therapy within weeks of surgical resection. Here we show that tumoroid cultures from a CRC patient are highly sensitive to the thymidylate synthase inhibitor 5-fluorouracil (adrucil) but less sensitive to the combination of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (Lonsurf). Moreover, re-introduction of isolated immune cells derived from surrounding and infiltrating tumor tissue as well as CD45+ tumor infiltrating hematopoietic cells displayed prolonged (>10 days) survival in co-culture. Established tumor slice cultures were found to contain both an outer epithelial and inner stromal cell compartment mimicking tumor structure in vivo.Collectively, these data suggest that, 3D-tumoroid and slice culture assays may provide a feasible in vitro approach to assess efficacy of novel therapeutics in the context of heterogeneous tumor-associated cell types including immune and non-transformed stromal cells. In addition, delineating the impact of therapeutics on immune cells, and cell types involved in therapeutic resistance mechanisms may be possible in general or for patient-specific responses.

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Elotuzumab is a humanized therapeutic monoclonal antibody directed to the surface glycoprotein SLAMF7 (CS1, CRACC, CD319), which is highly expressed on multiple myeloma (MM) tumor cells. Improved clinical outcomes have been observed following treatment of MM patients with elotuzumab in combination with lenalidomide or bortezomib. Previous work showed that elotuzumab stimulates NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), via Fc-domain engagement with FcγRIIIa (CD16). SLAMF7 is also expressed on NK cells, where it can transmit stimulatory signals. We tested whether elotuzumab can directly activate NK cells via ligation with SLAMF7 on NK cells in addition to targeting ADCC through CD16. We show that elotuzumab strongly promoted degranulation and activation of NK cells in a CD16-dependent manner, and a non-fucosylated form of elotuzumab with higher affinity to CD16 exhibited enhanced potency. Using F(ab') or Fc-mutant forms of the antibody, the direct binding of elotuzumab to SLAMF7 alone could not stimulate measurable CD69 expression or degranulation of NK cells. However, the addition of soluble elotuzumab could costimulate calcium signaling responses triggered by multimeric engagement of NKp46 and NKG2D in a CD16-independent manner. Thus, while elotuzumab primarily stimulates NK cells through CD16, it can also transduce effective "trans"-costimulatory signals upon direct engagement with SLAMF7, since these responses did not require direct co-engagement with the activating receptors. Trans-costimulation by elotuzumab has potential to reduce activation thresholds of other NK cell receptors engaging with their ligands on myeloma target cell surfaces, thereby potentially further increasing NK cell responsiveness in patients.

DNA Photolyase is a flavoprotein that uses light to repair cyclobutylpyrimidine dimers in DNA. From considerations of the crystal structure of the protein, it has been hypothesized that the dimer lesion is flipped out of the DNA double helix into the substrate binding pocket. We have used a fluorescent adenine analog, 2-aminopurine (2-Ap), as a probe of local double helical structure upon binding of the substrate to the protein. Our results show that the local structure around the thymidine lesion changes dramatically upon binding to Photolyase. This is consistent with base flipping of the lesion into the protein binding cavity with concomitant destacking of the opposing complementary 2-Ap nucleotide.Cyclobutylpyrimidine dimers (CPDs) 1 are lesions formed in DNA between adjacent pyrimidines upon absorption of UV light. These lesions cause replicational errors and can lead to cell death or cancer if left unrepaired (1, 2). One repair protein, DNA photolyase, incorporates a non-covalently bound FAD and requires blue light for repair (3). There is ample evidence that repair of the CPD proceeds by electron transfer from a photoexcited fully reduced FADH Ϫ to the CPD, which subsequently monomerizes within 2 ns (3). Studies by Payne et al. (4) have demonstrated that the oxidized enzyme can bind CPD-containing DNA but cannot efficiently repair the CPD lesion. As we show below, this differential behavior is extremely useful in understanding the substrate binding mode of photolyase.The crystal structures of the Escherichia coli and Aspergillus nidulans holoenzymes were solved by Kim et al. (5) and Tamada and co-workers (6), respectively. These crystal structures revealed important structural elements that were both familiar and surprising. It was noted that the cavity has approximately the correct dimensions to enclose the CPD if the thymidines were folded in a coplanar geometry, leading to the prediction that photolyase would bind the CPD by "flipping out" the lesion from the double helical DNA (5). The FADH Ϫ cofactor was found to lie at the bottom of this cavity, consistent with the ability of the cofactor to resist oxidation by molecular O 2 . The surface of the protein above the cavity incorporates a strip of positively charged amino acid residues that are thought to help orient the negatively charged phosphate backbone of the damaged DNA strand.Unfortunately, no crystal structure of the enzyme-substrate complex is available, but a recent crystal structure of Thermus thermophilus complexed with thymine was published by Komori and co-workers (7), which shows the thymine residing in the putative substrate cavity. While thymine is not a substrate, it can be thought of as a partial product of the repair reaction. This is the strongest evidence regarding the substrate binding mode of photolyase for CPDs. However, it does not clarify whether one or both of the thymine bases of the CPD is bound in the cavity.Other experimental evidence for the mode of substrate binding is available. Most notable are the ethylation and ...

ONC201 is a first-in-class, orally active antitumor agent that upregulates cytotoxic TRAIL pathway signaling in cancer cells. ONC201 has demonstrated safety and preliminary efficacy in a first-in-human trial in which patients were dosed every 3 weeks. We hypothesized that dose intensification of ONC201 may impact antitumor efficacy. We discovered that ONC201 exerts dose- and schedule-dependent effects on tumor progression and cell death signaling in vivo. With dose intensification, we note a potent anti-metastasis effect and inhibition of cancer cell migration and invasion. Our preclinical results prompted a change in ONC201 dosing in all open clinical trials. We observed accumulation of activated NK+ and CD3+ cells within ONC201-treated tumors and that NK cell depletion inhibits ONC201 efficacy in vivo, including against TRAIL/ONC201-resistant Bax-/- tumors. Immunocompetent NCR1-GFP mice, in which NK cells express GFP, demonstrated GFP+ NK cell infiltration of syngeneic MC38 colorectal tumors. Activation of primary human NK cells and increased degranulation occurred in response to ONC201. Coculture experiments identified a role for TRAIL in human NK-mediated antitumor cytotoxicity. Preclinical results indicate the potential utility for ONC201 plus anti-PD-1 therapy. We observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients after ONC201 treatment. The results offer what we believe to be a unique pathway of immune stimulation for cancer therapy.

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