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Genetic engineering of immune cells has opened new avenues for improving their functionality but it remains a challenge to pinpoint which genes or combination of genes are the most beneficial to target. Here, we conduct High Multiplicity of Perturbations and Cellular Indexing of Transcriptomes and Epitopes (HMPCITE-seq) to find combinations of genes whose joint targeting improves antigen-presenting cell activity and enhances their ability to activate T cells. Specifically, we perform two genome-wide CRISPR screens in bone marrow dendritic cells and identify negative regulators of CD86, that participate in the co-stimulation programs, including Chd4, Stat5b, Egr2, Med12, and positive regulators of PD-L1, that participate in the co-inhibitory programs, including Sptlc2, Nckap1l, and Pi4kb. To identify the genetic interactions between top-ranked genes and find superior combinations to target, we perform high-order Perturb-Seq experiments and we show that targeting both Cebpb and Med12 results in a better phenotype compared to the single perturbations or other combinations of perturbations., (© 2023. Springer Nature Limited.)

The inhibitory receptor PD-1 is expressed on T cells to inhibit select functions when ligated. The complete signaling mechanism downstream of PD-1 has yet to be uncovered. Here, we discovered phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG) is phosphorylated following PD-1 ligation and associate this with inhibitory T cell function. Clinical cohort analysis correlates low PAG expression with increased survival from numerous tumor types. PAG knockdown in T cells prevents PD-1-mediated inhibition of cytokine secretion, cell adhesion, CD69 expression, and ERK 204/187 phosphorylation, and enhances phosphorylation of SRC 527 following PD-1 ligation. PAG overexpression rescues these effects. In vivo, PAG contributes greatly to the growth of two murine tumors, MC38 and B16, and limits T cell presence within the tumor. Moreover, PAG deletion sensitizes tumors to PD-1 blockade. Here PAG is established as a critical mediator of PD-1 signaling and as a potential target to enhance T cell activation in tumors.

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell-mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell-mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1-triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches., Competing Interests: Conflict of interest—A. M. received research support from NTB Pharma. The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Tocheva et al.)

Exosomes are cell-derived vesicles that have been implicated in the pathogenesis of many inflammatory diseases. In the immune system, it has been shown that T lymphocyte-derived exosomes are able to induce diverse cellular responses. There are several methods to isolate and to characterize exosomes, each with their own advantages and disadvantages. Here, we describe a centrifugation approach, combined with mass spectrometry characterization, as a means to study exosomes derived from primary human T lymphocytes. This method is sensitive and therefore can be applied when a limited amount of sample is available.

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP., Competing Interests: The authors declare no conflict of interest.

The adaptor protein CrkII regulates T cell adhesion by recruiting the guanine nucleotide exchange factor C3G, an activator of Rap1. Subsequently, Rap1 stimulates the integrin LFA-1, which leads to T cell adhesion and interaction with antigen-presenting cells (APCs). The adhesion of T cells to APCs is critical for their proper function and education. The interface between the T cell and the APC is known as the immunological synapse. It is characterized by the specific organization of proteins that can be divided into central supramolecular activation clusters (c-SMACs) and peripheral SMACs (p-SMACs). Through total internal reflection fluorescence (TIRF) microscopy and experiments with supported lipid bilayers, we determined that activated Rap1 was recruited to the immunological synapse and localized to the p-SMAC. C3G and the active (dephosphorylated) form of CrkII also localized to the same compartment. In contrast, inactive (phosphorylated) CrkII was confined to the c-SMAC. Activation of CrkII and its subsequent movement from the c-SMAC to the p-SMAC depended on the phosphatase SHP-1, which acted downstream of the T cell receptor. In the p-SMAC, CrkII recruited C3G, which led to Rap1 activation and LFA-1-mediated adhesion of T cells to APCs. Functionally, SHP-1 was necessary for both the adhesion and migration of T cells. Together, these data highlight a signaling pathway in which SHP-1 acts through CrkII to reshape the pattern of Rap1 activation in the immunological synapse., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.(1,2) These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

CD28 is a coreceptor expressed on T lymphocytes. Signaling downstream of CD28 promotes multiple T cell functions such as proliferation, survival, and cytokine secretion. Adhesion to APCs is another function of T cells; however, little is known with regard to the role of CD28 in this process. Our previous studies have shown that CD28 inhibits T cell adhesion, but the underlying mechanism that mediates this process remains unknown. In the present study we discovered that signaling downstream of CD28 resulted in inhibition of Rap1 activity and decreased LFA-1-mediated adhesion. We showed that this was regulated by the recruitment of calcium-promoted Ras inactivator (CAPRI), a GTPase-activating protein, to the plasma membrane downstream of CD28 signaling. CAPRI trafficking to the plasma membrane was secondary to calcium influx and was mediated by its C2A and C2B domains. We conclude that CD28 inhibits Rap1-mediated adhesion by recruiting the protein CAPRI to the plasma membrane., (Copyright © 2015 by The American Association of Immunologists, Inc.)

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits αL and β2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g. VLA-4 - fibronectin).

Background: Glatiramer acetate (GA, Copaxone®), a mixture of polymers comprising four amino acids, is approved for treatment of relapsing-remitting multiple sclerosis and clinically isolated syndrome. GA mediates its activity by induction of GA-specific T cells that shift the T cell balance from a dominant proinflammatory phenotype (Th1/Th17) to an anti-inflammatory phenotype (Th2/Treg)., Objective: To characterize the functional pathways by which GA acts on immune cells, the authors conducted gene expression profiling using glatiramoid-stimulated splenocytes., Methods: Mice were immunized with GA and harvested splenocytes were reactivated ex vivo with GA or a purported generic GA. Gene expression profiles and functional pathways were evaluated in reactivated splenocytes., Results: Overall, 1,474 genes were significantly upregulated or downregulated by GA. The main functional pathways induced by GA were: increased proliferation and activation of immune cells including T and B lymphocytes, stimulation of antigen presenting cells and differentiation of effector T lymphocytes. T-helper cell differentiation was the most significant canonical pathway associated with gene transcripts altered by GA. These expression patterns were not observed when splenocytes were activated with generic GA., Conclusion: GA-induced functional pathways coincide with known mechanisms of GA activity in MS patients and further support the unique therapeutic effect of this drug.