Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15°relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.superresolution microscopy | focal adhesions | talin | mechanobiology | nanoscale architecture C ell adhesion to the ECM is a highly coordinated process that involves ECM-specific recognition by integrin transmembrane receptors, and their aggregation with numerous cytoplasmic proteins into dense supramolecular complexes called focal adhesions (FAs) (1). Actin stress fibers terminate at FAs where actomyosin contractility is transmitted to the ECM, generating traction (2-5). Mechanical tension impinging on each FA is implicated in key steps including the elongation, reinforcement, and maintenance of the FA structures (6). FA mechanotransduction is a major aspect of cellular microenvironment sensing with wide-ranging consequences in physiological and pathological processes (7-10). However, molecular-scale spatial parameters that specify FA nanoscale organization have been difficult to access experimentally. Nevertheless, these are essential to understand how mechanosensitivity arises within such complex molecular machines (11-15).Previously 3D superresolution fluorescence microscopy has unveiled the nanoscale organization of major FA components, whereby a core region of ∼30 nm interposes between the integrin and the actin cytoskeleton along the vertical (z) axis (16). The FA core consists of a membrane-proximal layer that contains signaling proteins such as FAK (focal adhesion kinase) and paxillin, an intermediate zone that contains force-transduction proteins s...

The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that the forced expression of the C-X-C chemokine receptor type 6 (CXCR6, whose ligand is highly expressed by human and murine pancreatic cancer cells and by tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell-adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained antitumoral activity and prolonged animal survival only when co-expressing CXCR6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.

Adoptive T cell therapy (ACT) is highly effective in the treatment of hematologic malignancies, but shows limited success in solid tumors. Inactivation of T cells in the tumor milieu is a major hurdle to a wider application of ACT. Cytotoxicity is the most relevant activity for tumor eradication. Here, we document that cytotoxic T cells (CTL) in lactic acidosis exhibited strongly reduced tumor cell killing, which could be compensated partly by increasing the CTL to tumor cell ratio. Lactic acid intervened at multiple steps of the killing process. Lactic acid repressed the number of CTL that performed lytic granule exocytosis (degranulation) in tumor cell co-culture, and, additionally impaired the quality of the response, as judged by the reduced intensity of degranulation and lower secretion of cytotoxins (perforin, granzyme B, granzyme A). CTL in lactic acid switched to a low bioenergetic profile with an inability to metabolize glucose efficiently. They responded to anti-CD3 stimulation poorly with less extracellular acidification rate (ECAR). This might explain their repressed granule exocytosis activity. Using live cell imaging, we show that CTL in lactic acid have reduced motility, resulting in lower field coverage. Many CTL in lactic acidosis did not make contact with tumor cells; however, those which made contact, adhered to the tumor cell much longer than a CTL in normal medium. Reduced motility together with prolonged contact duration hinders serial killing, a defining feature of killing potency, but also locally confines cytotoxic activity, which helps to reduce the risk of collateral organ damage. These activities define lactic acid as a major signaling molecule able to orchestrate the spatial distribution of CTL inside inflamed tissue, such as cancer, as well as moderating their functional response. Lactic acid intervention and strategies to improve T cell metabolic fitness hold promise to improve the clinical efficacy of T cell–based cancer immunotherapy.

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Introduction: The use of cellular immunotherapies has led to impressive complete and durable clinical responses in patients with certain types of hematological cancers. However, positive clinical results in solid tumor indications are still rare and many patients are in urgent need of alternative treatment options for several different indications. It has become clear that expression of inhibitory immune checkpoint molecules as well as harsh metabolic conditions in the tumor microenvironment (TME) are responsible for lack of activity of T cell immunotherapies in several settings, especially solid tumors. Here additional strategies are necessary to efficiently employ cellular immunotherapies. With the aim to further enhance the clinical efficacy of TCR-based immunotherapies under immunosuppressive conditions found in tumors, we analyzed the ability of PD1-41BB, a chimeric co-stimulatory receptor, to reverse the natural inhibitory PD-1/PD-L1 interaction into a supporting co-stimulatory signal in TCR-modified T cells encountering tumor cells. Methods: We evaluated the ability of the chimeric co-stimulatory receptor PD1-41BB to improve activity of TCR-modified T cells using 2-dimensional or 3-dimensional in vitro assays that model different immunosuppressive conditions found in tumors. Results: We demonstrate that chronic stimulation as well as several immunosuppressive factors of the TME, such as tumor cell expression of inhibitory immune checkpoint molecules or glucose restriction, impede the ability of TCR-transduced T cells to produce inflammatory cytokines and to efficiently lyse tumor cells. By using a chimeric co-stimulatory receptor consisting of the extracellular part of PD-1 and the co-stimulatory domain of 4-1BB we reversed the naturally occurring inhibitory PD-1/PD-L1 interaction to provide a co-stimulatory signal for improved T cell activity under immunosuppressive conditions or chronic stimulation. Addition of the chimeric co-stimulatory receptor PD1-41BB to TCR-modified T cells led to enhanced release of Interferon-γ, increased tumor cell killing, T cell proliferation and persistence in these T cell-tumor cell models. Conclusions: These preclinical studies support our approach to enhance the clinical efficacy of TCR-T therapies in PD-L1-positive malignancies by reversing naturally occurring inhibitory signals enabling counteraction of checkpoint-mediated dysfunction and metabolic insufficiency. The chimeric co-stimulatory PD1-41BB receptor has the potential to further enhance the clinical efficacy of TCR-modified T cells in patients with PD-L1-positive malignancies. Further preclinical in vitro and in vivo studies are ongoing to investigate the safety and efficacy of PD1-41BB in combination with multiple TCR candidates to explore its feasibility for the treatment of various cancers. Citation Format: Nadja Sailer, Melanie Salvermoser, Maria Gerget, Sarah Thome, Angelika J. Fischbeck, Svenja Ruehland, Luis F. Olguín-Contreras, Maja Buerdek, Christian Ellinger, Elfriede Noessner, Dolores J. Schendel, Patrik Kehler. The chimeric co-stimulatory receptor PD1-41BB enhances the function of T cell receptor (TCR)-modified T cells targeting solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3231.