EXTH-37. PAK4 INHIBITION REPROGRAMS VASCULAR MICROENVIRONMENT AND IMPROVES CAR T IMMUNOTHERAPY IN GLIOBLASTOMA

Malignant solid tumors are characterized by aberrant vascularity that inhibits T cell adhesion to vasculature and impedes T cell delivery into the tumors, contributing to glioblastoma (GBM) resistance to T cell-based immunotherapy such as adoptive chimeric antigen receptor (CAR)–T transfer. Vascular abnormality is driven by pro-angiogenic pathway activation and genetic reprogramming in tumor endothelial cells (ECs). Here, our kinome-wide functional screening of mesenchymal-like transcriptional activation in human GBM-derived ECs identifies p21-activated kinase 4 (PAK4) as a selective regulator of genetic programming and aberrant vascularization in tumor. Genetic ablation of PAK4 reprograms EC transcriptome and inhibits mesenchymal-like transformation. Interestingly, deficiency of PAK4 induces adhesion protein re-expression in tumor ECs, reduces vascular abnormalities in tumors, improves T cell infiltration into the tumors, and inhibits GBM growth in mice. Moreover, pharmacological PAK4 inhibition normalizes the tumor vascular microenvironment and sensitizes GBM to Egfrviii CAR–T cell immunotherapy. Finally, we reveal a MEF2D/ZEB1- and SLUG-mediated mechanism by which PAK4 reprograms the EC transcriptome and downregulates claudin-14 and VCAM-1 expression, enhancing vessel permeability and reducing T cell adhesion to the endothelium. Thus, targeting PAK4-mediated EC plasticity may offer exciting opportunities to recondition the vascular microenvironment and strengthen cancer immunotherapy.

Endothelial-Derived Extracellular Vesicles Induce Cerebrovascular Dysfunction in Inflammation

Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of naïve hCMEC/D3 cells with small EVs (sEVs) reduced the TEER and increased the shear-resistant T-cell adhesion. The levels of microRNA-155, VCAM1 and ICAM1 were increased in sEV-treated hCMEC/D3 cells. Blocking the expression of VCAM1, but not of ICAM1, prevented sEV-mediated T-cell adhesion to brain endothelia. These results suggest that sEVs derived from inflamed BECs promote cerebrovascular dysfunction. These findings may provide new insights into the mechanisms involving neuroinflammatory disorders.

Three-Dimensional Model of Sub-Plasmalemmal Ca2+ Microdomains Evoked by the Interplay Between ORAI1 and InsP3 Receptors

Ca2+ signaling plays an essential role in T cell activation, which is a key step to start an adaptive immune response. During the transition from a quiescent to a fully activated state, Ca2+ microdomains characterized by reduced spatial and temporal extents are observed in the junctions between the plasma membrane (PM) and the endoplasmic reticulum (ER). Such Ca2+ responses can also occur in response to T cell adhesion to other cells or extracellular matrix proteins in otherwise unstimulated T cells. These non-TCR/CD3-dependent Ca2+ microdomains rely on d-myo-inositol 1,4,5-trisphosphate (IP3) signaling and subsequent store operated Ca2+ entry (SOCE) via the ORAI/STIM system. The detailed molecular mechanism of adhesion-dependent Ca2+ microdomain formation remains to be fully elucidated. We used mathematical modeling to investigate the spatiotemporal characteristics of T cell Ca2+ microdomains and their molecular regulators. We developed a reaction-diffusion model using COMSOL Multiphysics to describe the evolution of cytosolic and ER Ca2+ concentrations in a three-dimensional ER-PM junction. Equations are based on a previously proposed realistic description of the junction, which is extended to take into account IP3 receptors (IP3R) that are located next to the junction. The first model only considered the ORAI channels and the SERCA pumps. Taking into account the existence of preformed clusters of ORAI1 and STIM2, ORAI1 slightly opens in conditions of a full ER. These simulated Ca2+ microdomains are too small as compared to those observed in unstimulated T cells. When considering the opening of the IP3Rs located near the junction, the local depletion of ER Ca2+ allows for larger Ca2+ fluxes through the ORAI1 channels and hence larger local Ca2+ concentrations. Computational results moreover show that Ca2+ diffusion in the ER has a major impact on the Ca2+ changes in the junction, by affecting the local Ca2+ gradients in the sub-PM ER. Besides pointing out the likely involvement of the spontaneous openings of IP3Rs in the activation of SOCE in conditions of T cell adhesion prior to full activation, the model provides a tool to investigate how Ca2+ microdomains extent and interact in response to T cell receptor activation.

Cooperative Stabilization of Close-Contact Zones Leads to Sensitivity and Selectivity in T-Cell Recognition

T cells are sensitive to 1 to 10 foreign-peptide-MHC complexes among a vast majority of self-peptide-MHC complexes, and discriminate selectively between peptide-MHC complexes that differ not much in their binding affinity to T-cell receptors (TCRs). Quantitative models that aim to explain this sensitivity and selectivity largely focus on single TCR/peptide-MHC complexes, but T cell adhesion involves a multitude of different complexes. In this article, we demonstrate in a three-dimensional computational model of T-cell adhesion that the cooperative stabilization of close-contact zones is sensitive to one to three foreign-peptide-MHC complexes and occurs at a rather sharp threshold affinity of these complexes, which implies selectivity. In these close-contact zones with lateral extensions of hundred to several hundred nanometers, few TCR/foreign-peptide-MHC complexes and many TCR/self-peptide-MHC complexes are segregated from LFA-1/ICAM-1 complexes that form at larger membrane separations. Previous high-resolution microscopy experiments indicate that the sensitivity and selectivity in the formation of closed-contact zones reported here are relevant for T-cell recognition, because the stabilization of close-contact zones by foreign, agonist peptide-MHC complexes precedes T-cell signaling and activation in the experiments.

Cooperative Stabilization of Close-Contact Zones Leads to Sensitivity and Selectivity in T-Cell Recognition

T cells are sensitive to 1 to 10 foreign-peptide-MHC complexes among a vast majority of self-peptide-MHC complexes, and discriminate selectively between peptide-MHC complexes that differ not much in their binding affinity to T-cell receptors (TCRs). Quantitative models that aim to explain this sensitivity and selectivity largely focus on single TCR/peptide-MHC complexes, but T cell adhesion involves a multitude of different complexes. In this article, we demonstrate in a three-dimensional computational model of T-cell adhesion that the cooperative stabilization of close-contact zones is sensitive to 1 to 3 foreign-peptide-MHC complexes and occurs at a rather sharp threshold affinity of these complexes, which implies selectivity. In these close-contact zones with lateral extensions of hundred to several hundred nanometers, few TCR/foreign-peptide-MHC complexes and many TCR/self-peptide-MHC complexes are segregated from LFA-1/ICAM-1 complexes that form at larger membrane separations. Previous high-resolution microscopy experiments indicate that the sensitivity and selectivity in the formation of closed-contact zones reported here is relevant for T-cell recognition, because the stabilization of close-contact zones by foreign, agonist peptide-MHC complexes precedes T-cell signaling and activation in the experiments.