Faculty Bibliography

Programmed cell death 1 (PD-1) is a major coinhibitory receptor and a member of the immunological synapse (IS). To uncover proteins that regulate PD-1 recruitment to the IS, we searched for cytoskeleton-related proteins that also interact with PD-1 using affinity purification mass spectrometry. Among these proteins, EF hand domain family member D2 (EFHD2), a calcium binding adaptor protein, was functionally and mechanistically analyzed for its contribution to PD-1 signaling. EFHD2 was required for PD-1 to inhibit cytokine secretion, proliferation, and adhesion of human T cells. Interestingly, EFHD2 was also required for human T cell-mediated cytotoxicity and for mounting an antitumor immune response in a syngeneic murine tumor model. Mechanistically, EFHD2 contributed to IS stability, lytic vesicles trafficking, and granzyme B secretion. Altogether, EFHD2 is an important regulator of T cell cytotoxicity and further studies should evaluate its role in T cell-mediated inflammation.

Exosomes are cell derived vesicles that have been implicated in the pathogenesis of many inflammatory diseases. More specifically, it has been shown that T cell-derived exosomes can induce immunological responses; however, little is known about the mechanism and the molecular content of these vesicles. Here, we used a proteomic approach to characterize human T cell-derived exosomes. We found that specific proteins of the RAS signaling pathway were enriched in exosomes derived from activated T cells, and that these vesicles induced ERK phosphorylation in recipient immune cells. Our findings support a mechanistic role of exosomes in cellular activation, and further studies should consider exosomes as a biomarker for inflammatory diseases.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

PURPOSE OF REVIEW/OBJECTIVE:With the advent of cancer immunotherapy and immune checkpoint inhibitors, patients with malignancies can now achieve durable remissions for conditions previously described as terminal. However, immune-related adverse events (irAEs) associated with cancer immunotherapy have become an anticipated consequence of enhanced T cell activation. Through an extensive literature review, we assess the most recent clinical and basic research data concerning immune checkpoint blockade and describe the spectrum of associated irAEs as well as their management. RECENT FINDINGS/RESULTS:Anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies are widely used in the management of an array of tumors with incredible clinical remissions. However, irAEs cause significant morbidity and mortality and in some cases, result in withdrawal of cancer therapy and initiation of immunosuppression. While this is an exciting time in oncology, irAEs are a barrier to adequate care and therefore deserve close attention and improved capacity to predict and prevent toxicity. Rheumatologists should be familiar with these topics in the eventuality of patient evaluation and management.

Background/Purpose: T cells play a major role in the pathogenesis of Rheumatoid Arthritis (RA). These cells are regulated by signals provided via the T cell receptor (TCR) complex as well as by a set of co-receptors, which can propagate either stimulatory or inhibitory signals. CD28, a co-stimulatory receptor, and PD-1, a co-inhibitory receptor, are two essential T cell co-receptors whose ligands belong to the B7 family, but have opposing functions. Interestingly, both coreceptors play a role in the pathogenesis of RA and recent studies have shown that CD28 is targeted directly by PD-1. Accordingly, understanding the interplay between CD28 and PD-1 in the context of RA may provide novel approaches to better understand or treat autoimmunity. We hypothesize that PD-1 regulates CD28 function by dephosphorylating specific motifs in the tail of CD28 resulting in impaired downstream T cell function.
Method(s): Genetically modified T cell lines were used to study the contribution of different versions of CD28 to TCR signaling. Western blotting and cytokine levels were used to measure the role of CD28 on T cell function. The inhibitory effect of the PD-1 was examined by plating T cells on PD-1 ligand coated surfaces. Mass spectrometry was used to uncover additional proteins that regulate the interaction between PD-1 and CD28. To translate our finding to RA, blood and synovial fluid were collected from active RA patients (disease activity score (DAS)>5.1) to analyze expression of PD-1, CD28 and other signaling mediators. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were analyzed by flow cytometry.
Result(s): We discovered that signaling downstream of PD-1 dephosphorylates tyrosine 173 of CD28 and that this event was absolutely required for the ability of PD-1 to inhibit interleukin 2 (IL-2) secretion. Mass spectrometry results identified SAP, a hematopoietic-restricted adaptor protein found to be associated with autoimmunity, as a regulator of the functional interaction between PD-1 and CD28. SAP co-localized and physically interacted with CD28 to counter PD-1 mediated dephosphorylation. More specifically, SAP bound to tyrosine 173, but not to tyrosine 190 of the cytoplasmic tail CD28, and by doing so blocked the ability of PD-1 to dephosphorylate this site. Additionally, serine at position 171 of CD28 was required to stabilize the interaction between SAP and CD28. Finally, we also learned that SAP levels were elevated in synovial fluid and peripheral blood RA T cells, concordant with PD-1 levels and DAS.
Conclusion(s): Our results demonstrate that SAP binds to phosphorylated CD28 at tyrosine 173 to interfere with PD-1 activity. It has been suggested that RA T cells are in a state of dysfunction with limited ability to regulate IL-2 production. Our finding of elevated SAP levels in these cells provides a mechanistic explanation for these observations whereby SAP interferes with PD-1 signaling by shielding phosphorylated CD28 and leading to persistent activation in acute disease followed by dysfunction in chronic disease. Therefore, SAP has potential to be utilized as a biomarker for RA disease activity. Manipulation of the PD-1/CD28 axis may prove to be a promising therapeutic target in autoimmunity

The signaling lymphocytic activation molecule (SLAM) family is comprised of nine distinct receptors (SLAMF1 through SLAMF9) that are expressed on hematopoietic cells. All of these receptors, with the exception of SLAMF4, are homotypic by nature as downstream signaling occurs when hematopoietic cells that express the same SLAM receptor interact. The SLAM family receptor function is largely controlled via SLAM associated protein (SAP) family adaptors. The SAP family adaptors consist of SAP, Ewing sarcoma associated transcript (EAT)-2, and EAT-2-related transducer (ERT). These adaptors associate with the cytoplasmic domain of the SLAM family receptors through phosphorylated tyrosines. Defects in SLAM family members and SAP adaptors have been implicated in causing immune deficiencies. This is exemplified in patients with X-linked lymphoproliferative (XLP) disease, where SAP undergoes a loss of function mutation. Furthermore, evidence has been accumulating that SLAM family members are potential targets for inflammatory and autoimmune diseases. This review will discuss the structure and function of the SLAM family receptors and SAP family adaptors, their role in immune regulation, and potential approaches to target this family of receptors therapeutically.