P-01.08 PVR-CD226 interaction regulates IL-10 production during human T cell differentiation

<h3>Background</h3> The co-inhibitory receptor <i>T cell immunoreceptor with Ig and ITIM domains</i> (TIGIT) and the co-stimulatory receptor CD226 can compete for binding their common ligands (poliovirus receptor (PVR, CD155) and nectin-2 (CD112)) regulating human T cell activation.¹ In addition, the ITIM-bearing receptor CD96 (Tactile) can also be expressed on the surface of T cells and bind PVR. We recently reported that an increase in PVR in an <i>ex vivo</i> bone marrow model reduced the number of cytotoxic T cells overcoming the effects of TIGIT blockade in multiple myeloma.² In this study, we aim to investigate the functional roles of PVR and nectin-2 in regulating human T cell differentiation. <h3>Materials and Methods</h3> Peripheral blood was obtained from healthy donors from the Blood and Tissue Bank (BST, Barcelona). Naive CD4⁺ T cells were isolated by negative selection (Stemcell) and sorted on a FACS Aria (BD Biosciences). Cells were cultured in 96-well plates in RPMI 1640 medium with 5% human AB serum, plate-bound anti-CD3 (clone UCHT1) at 5 µg/mL, soluble anti-CD28 (clone 28.2) at 1 µg/ml. Recombinant human IL-2 was used at 10 U/mL (Th0) or 100 U/mL plus TGFb at 25ng/mL (Treg conditions). After 7 days, cells were analyzed by spectral flow cytometry on a Cytek Aurora cytometer to quantify viability, proliferation (Ki67), surface expression of CD226, TIGIT, CD96 and intracellular staining of FoxP3 and IL-10. <h3>Results</h3> Presence of plate-bound PVR or nectin-2 at 2 µg/mL did not significantly modify cell viability nor frequency of proliferating cells compared to isotype control. As expected, Treg conditions significantly increased FoxP3 expression (paired T test p&lt;0.0001, n=6) which was accompanied by elevation of CD226 levels (p=0.0026). Surprisingly, Treg conditions led to reduction of TIGIT expression (p=0.0039) and CD96 (p=0.084). In addition, PVR ligation resulted in a higher frequency of IL10-producing cells in Th0 conditions (p=0.0193), most of them expressing high levels of CD226 (78.48% ± 2.324), CD96 (59.87% ± 4.032), and to a lesser extent, TIGIT (39.82% ± 4.043). Consistently, under these experimental conditions dual blockade of TIGIT and CD96 did not abrogate the increase in the percentage of IL-10 producing cells, suggesting that PVR binds CD226 expressing cells to upregulate IL-10 production. <h3>Conclusions</h3> PVR-CD226 interaction plays a critical role in IL-10 production during human T cell differentiation. <h3>References</h3> Lozano E, <i>et al</i>. The TIGIT/CD226 axis regulates human T cell function. <i>J. Immunol.</i> 2012:<b>188</b>(8):3869–3875. Lozano E, <i>et al</i>. Increased PVR expression on bone marrow macrophages may promote resistance to TIGIT blockade in multiple myeloma. <i>Clin Cancer Res</i> 2024;<b>30</b>:3944–55. E. Lozano: None. M. Mena: None. T. Diaz: None. L. Rosiñol: None.