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 co-receptors 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.

Methods: 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.

Results: 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: 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.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/regulation-of-autoimmune-t-cells-by-the-co-receptors-cd28-and-pd-1/