PD1 inhibits PKCθ-dependent phosphorylation of cytoskeleton-related proteins and immune synapse formation.

The inhibitory surface receptor programmed cell death protein 1 (PD1) is a major target for antibody-based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with protein kinase Cθ (PKCθ) of a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a Src homology-2 domain-containing phosphatase-1/2 (SHP1/SHP2)-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. T-cell receptor induced phosphorylation of the PKCθ substrate and binding partner vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with the levels of the PD1 ligand, PDL1, in human lung carcinoma. Thus, PKCθ and its substrate vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway

PD-1 checkpoint inhibition enhances the antilymphoma activity of CD19-CAR-iNKT cells that retain their ability to prevent alloreactivity.

Relapse and graft-versus-host disease (GVHD) are the main causes of death after allogeneic hematopoietic cell transplantation (HCT). Preclinical murine models and clinical data suggest that invariant natural killer T (iNKT) cells prevent acute and chronic GVHD. In addition, iNKT cells are crucial for efficient immune responses against malignancies and contribute to reduced relapse rates after transplantation. Chimeric antigen receptors (CAR) redirect effector cells to cell surface antigens and enhance killing of target cells. With this study, we aimed to combine enhanced cytotoxicity of CD19-CAR-iNKT cells against lymphoma cells with their tolerogenic properties. iNKT cells were isolated from peripheral blood mononuclear cells and transduced with an anti-CD19-CAR retrovirus. After in vitro expansion, the functionality of CD19-CAR-iNKT cells was assessed by flow cytometry, image stream analysis and multiplex analysis in single-stimulation or repeated-stimulation assays. Moreover, the immunoregulatory properties of CD19-CAR-iNKT cells were analyzed in apoptosis assays and in mixed lymphocyte reactions. The effect of checkpoint inhibition through nivolumab was analyzed in these settings. In this study, we could show that the cytotoxicity of CD19-CAR-iNKT cells was mediated either through engagement of their CAR or their invariant T-cell receptor, which may circumvent loss of response through antigen escape. However, encounter of CD19-CAR-iNKT cells with their target induced a phenotype of exhaustion. Consequently, checkpoint inhibition increased cytokine release, cytotoxicity and survival of CD19-CAR-iNKT cells. Additionally, they showed robust suppression of alloreactive immune responses. In this work, we demonstrate that CAR-iNKT cells are a powerful cytotherapeutic option to prevent or treat relapse while potentially reducing the risk of GVHD after allogeneic HCT

Retromer and Its Role in Regulating Signaling at Endosomes.

The retromer complex is a key element of the endosomal protein sorting machinery being involved in trafficking of proteins from endosomes to the Golgi and also endosomes to the cell surface. There is now accumulating evidence that retromer also has a prominent role in regulating the activity of many diverse signaling proteins that traffic through endosomes and this activity has profound implications for the functioning of many different cell and tissue types from neuronal cells to cells of the immune system to specialized polarized epithelial cells of the retina. In this review, the protein composition of the retromer complex will be described along with many of the accessory factors that facilitate retromer-mediated endosomal protein sorting to detail how retromer activity contributes to the regulation of several distinct signaling pathways

Supplementary Material for: The Janus Face of a-Toxin: A Potent Mediator of Cytoprotection in Staphylococci-Infected Macrophages

<p>After phagocytosis by macrophages, <i>Staphylococcus aureus</i> evades killing in an a-toxin-dependent manner, and then prevents apoptosis of infected cells by upregulating expression of antiapoptotic genes like <i>MCL-1 (myeloid cell leukemia-1)</i>. Here, using purified a-toxin and a set of <i>hla</i>-deficient strains, we show that a-toxin is critical for the induction of <i>MCL-1 </i>expression and the cytoprotection of infected macrophages. Extracellular or intracellular treatment of macrophages with a-toxin alone did not induce cytoprotection conferred by increased Mcl-1, suggesting that the process is dependent on the production of a-toxin by intracellular bacteria. The increased expression of <i>MCL-1</i> in infected cells was associated with enhanced NFκB activation, and subsequent IL-6 secretion. This effect was only partially inhibited by blocking TLR2, which suggests the participation of intracellular receptors in the specific recognition of <i>S. aureus </i>strains secreting a-toxin. Thus, <i>S. aureus</i> recognition by intracellular receptors and/or activation of downstream pathways leading to Mcl-1 expression is facilitated by a-toxin released by intracellular bacteria which permeabilize phagosomes, ensuring pathogen access to the cytoplasmatic compartment. Given that the intracellular survival of <i>S. aureus</i> depends on a-toxin, we propose a novel role for this agent in the protection of the intracellular niche, and further dissemination of staphylococci by infected macrophages.</p