Successive crystal structure snapshots suggest the basis for MHC class i peptide loading and editing by tapasin

MHC-I epitope presentation to CD8 + T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptideloading complex, the peptide editor tapasin is key to the selection of MHC-I-bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles

Effects of 1-MCP and storage temperature on quince fruit quality

Quince (Cydonia oblonga) is an important underutilized Mediterranean fruit having many possible culinary uses but there is very little knowledge on its storage and quality and almost nothing about 1-MCP effects on quince fruit quality. Quince fruit ('Afrata Volou') were harvested at commercial maturity (slightly green skin color), treated or not with 600 nl L-1 1-MCP five days later and stored for up to 151 d at 2°C (no cover) or 10°C (in loosely closed PE bags), or in a storage room with gradual (within a month) drop of storage temperature from 20 to 2°C (RH 80-90%). Control fruit were kept at ambient conditions (temperature 4 to 17°C and RH 35-95%). Quality evaluation included skin color, flesh firmness, juice soluble solids content (SSC) and titratable acidity and flesh dry matter (DM). Quince fruit skin color became more yellow, fruit softened, acidity and DM decreased and SSC only slightly decreased with time in storage. Fruit stored at 2°C or treated with 1-MCP remained greener based on lower a∗, b∗ and chroma values and higher hue angle values compared to fruit stored at higher temperatures. In contrast, storage at 2°C or treatment with 1-MCP did not improve quince flesh firmness. As expected, fruit kept at ambient conditions were the softest and also developed flesh browning. Quince fruit stored at 2°C or ambient conditions had lower SSC and DM values than fruit stored at 10°C in PE bags. It seems that low storage temperature or ambient temperature increase sugar losses through respiration and storing quince at 10°C in PE bags without treatment with 1-MCP is recommended to keep quince fruit quality acceptable for up to 106 d

Adoptive nk cell therapy: A promising treatment prospect for metastatic melanoma

Adoptive cell therapy (ACT) represents a promising alternative approach for patients with treatment-resistant metastatic melanoma. Lately, tumor infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR)-T cell therapy have shown improved clinical outcome, compared to conventional chemotherapy or immunotherapy. Nevertheless, they are limited by immune escape of the tumor, cytokine release syndrome, and manufacturing challenges of autologous therapies. Conversely, the clinical use of Natural Killer (NK) cells has demonstrated a favorable clinical safety profile with minimal toxicities, providing an encouraging treatment alternative. Unlike T cells, NK cells are activated, amongst other mechanisms, by the downregulation of HLA class I molecules, thereby overcoming the hurdle of tumor immune escape. However, impairment of NK cell function has been observed in melanoma patients, resulting in deteriorated natural defense. To overcome this limitation, “activated” autologous or allogeneic NK cells have been infused into melanoma patients in early clinical trials, showing encouraging clinical benefit. Furthermore, as several NK cell-based therapeutics are being developed for different cancers, an emerging variety of approaches to increase migration and infiltration of adoptively transferred NK cells towards solid tumors is under preclinical investigation. These developments point to adoptive NK cell therapy as a highly promising treatment for metastatic melanoma in the future

Boosting Natural Killer Cell Therapies in Glioblastoma Multiforme Using Supramolecular Cationic Inhibitors of Heat Shock Protein 90

Allogeneic natural killer (aNK) cell adoptive therapy has the potential to dramatically impact clinical outcomes of glioblastoma multiforme (GBM). However, in order to exert therapeutic activity, NK cells require tumor expression of ligands for activating receptors, such as MHC Class I peptide A/B (MICA/B) and ULBPs. Here, we describe the use of a blood–brain barrier (BBB) permissive supramolecular cationic drug vehicle comprising an inhibitor of the chaperone heat shock protein 90 (Hsp90), which sustains a cytotoxic effect on GBM cells, boosts the expression of MICA/B and ULBPs on the residual population, and augments the activity of clinical-grade aNK cells (GTA002). First, we identify Hsp90 mRNA transcription and gain of function as significantly upregulated in GBM compared to other central nervous system tumors. Through a rational chemical design, we optimize a radicicol supramolecular prodrug containing cationic excipients, SCI-101, which displays &amp;gt;2-fold increase in relative BBB penetration compared to less cationic formulations in organoids, in vitro. Using 2D and 3D biological models, we confirm SCI-101 sustains GBM cytotoxicity 72 h after drug removal and induces cell surface MICA/B protein and ULBP mRNA up to 200% in residual tumor cells compared to the naked drug alone without augmenting the shedding of MICA/B, in vitro. Finally, we generate and test the sequential administration of SCI-101 with a clinical aNK cell therapy, GTA002, differentiated and expanded from healthy umbilical cord blood CD34+ hematopoietic stem cells. Using a longitudinal in vitro model, we demonstrate &amp;gt;350% relative cell killing is achieved in SCI-101–treated cell lines compared to vehicle controls. In summary, these data provide a first-of-its-kind BBB-penetrating, long-acting inhibitor of Hsp90 with monotherapy efficacy, which improves response to aNK cells and thus may rapidly alter the treatment paradigm for patients with GBM.</jats:p

Early TRAIL-engagement elicits potent multimodal targeting of melanoma by CD34+ progenitor cell-derived NK cells

Summary: Umbilical cord blood (UCB) CD34+ progenitor cell-derived natural killer (NK) cells exert efficient cytotoxicity against various melanoma cell lines. Of interest, the relative cytotoxic performance of individual UCB donors was consistent throughout the melanoma panel and correlated with IFNγ, TNF, perforin and granzyme B levels. Importantly, intrinsic perforin and Granzyme B load predicts NK cell cytotoxic capacity. Exploring the mode of action revealed involvement of the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46 and most importantly of TRAIL. Strikingly, combinatorial receptor blocking led to more pronounced inhibition of cytotoxicity (up to 95%) than individual receptor blocking, especially in combination with TRAIL-blocking, suggesting synergistic cytotoxic NK cell activity via engagement of multiple receptors which was also confirmed in a spheroid model. Importantly, lack of NK cell-related gene signature in metastatic melanomas correlates with poor survival highlighting the clinical significance of NK cell therapies as a promising treatment for high-risk melanoma patients

TGF-beta 1-induced EMT promotes targeted migration of breast cancer cells through the lymphatic system by the activation of CCR7/CCL21-mediated chemotaxis

Tumor cells frequently disseminate through the lymphatic system during metastatic spread of breast cancer and many other types of cancer. Yet it is not clear how tumor cells make their way into the lymphatic system and how they choose between lymphatic and blood vessels for migration. Here we report that mammary tumor cells undergoing epithelial-mesenchymal transition (EMT) in response to transforming growth factor-beta (TGF-beta 1) become activated for targeted migration through the lymphatic system, similar to dendritic cells (DCs) during inflammation. EMT cells preferentially migrated toward lymphatic vessels compared with blood vessels, both in vivo and in 3D cultures. A mechanism of this targeted migration was traced to the capacity of TGF-beta 1 to promote CCR7/CCL21-mediated crosstalk between tumor cells and lymphatic endothelial cells. On one hand, TGF-beta 1 promoted CCR7 expression in EMT cells through p38 MAP kinase-mediated activation of the JunB transcription factor. Blockade of CCR7, or treatment with a p38 MAP kinase inhibitor, reduced lymphatic dissemination of EMT cells in syngeneic mice. On the other hand, TGF-beta 1 promoted CCL21 expression in lymphatic endothelial cells. CCL21 acted in a paracrine fashion to mediate chemotactic migration of EMT cells toward lymphatic endothelial cells. The results identify TGF-beta 1-induced EMT as a mechanism, which activates tumor cells for targeted, DC-like migration through the lymphatic system. Furthermore, it suggests that p38 MAP kinase inhibition may be a useful strategy to inhibit EMT and lymphogenic spread of tumor cells

Reprogramming Tumor-Associated Macrophages by Antibody Targeting Inhibits Cancer Progression and Metastasis

Tumors are composed of multiple cell types besides the tumor cells themselves, including innate immune cells such as macrophages. Tumor-associated macrophages (TAMs) are a heterogeneous population of myeloid cells present in the tumor microenvironment (TME). Here, they contribute to immunosuppression, enabling the establishment and persistence of solid tumors as well as metastatic dissemination. We have found that the pattern recognition scavenger receptor MARCO defines a subtype of suppressive TAMs and is linked to clinical outcome. An anti-MARCO monoclonal antibody was developed, which induces anti-tumor activity in breast and colon carcinoma, as well as in melanoma models through reprogramming-TAM-populations to a pro-inflammatory phenotype and increasing tumor immunogenicity. This anti-tumor activity is dependent on the inhibitory Fc-receptor, Fc gamma RIIB, and also enhances the efficacy of checkpoint therapy. These results demonstrate that immunotherapies using antibodies designed to modify myeloid cells of the TME represent a promising mode of cancer treatment