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Prolonged exposure to antigens can lead to a state of reduced T cell responsiveness termed exhaustion. Restoring T cell functions from this state of reduced functionality by, for example, blockade of inhibitory immune checkpoints is now an established therapeutic modality with lasting impact on survival for some patients. Modelling human T cell exhaustion and its reversal in vitro can be challenging in medium or high-throughput assays. We have therefore sought to develop and characterize an in vitro model of T cell exhaustion using human T cells. Long-term (14-day) prolonged stimulation of human T cells in vitro led to both an up-regulation of established exhaustion markers PD-1, LAG-3 and TIM-3, and a lack of responsiveness to subsequent stimulation with human allogeneic monocyte-derived dendritic cells (DCs) as assessed by, for instance, IFNγ and TNFα production. Subsequent addition of blocking antibodies against PD-1 (pembrolizumab and nivolumab) led to a robust reversal of exhausted state by, for instance, restoration of cytokine secretion. The magnitude and nature of the response also correlated with the response of dissociated cells from lung and colon carcinomas. This response also showed greater sensitivity to PD-1 blockade compared to freshly isolated T cells or when using PBMCs stimulated with a pool of EBV peptides. By performing intracellular cytokine staining of human exhausted T cells stimulated with allogeneic dendritic cells, we further noted that PD-1 blockade using either pembrolizumab or nivolumab increased the percentage of cells producing IFNγ suggesting a greater responding pool of T cells. However, whilst the levels of the IFNγ in the supernatants of the exhausted T cell/allogeneic dendritic cell cultures was fully restored when compared to ‘fresh' T cell/allogeneic dendritic cell cultures, the percentage of IFNγ+ cells was not, nor was it fully restored by the inclusion of PMA/ionomycin. Taken together, this would suggest that PD-1 blockade induces responding cells to produce more IFNγ. We suggest that this chronic activation model of human T cell exhaustion with a robust assay window, and responsive to the clinically validated anti-PD-1 antibodies, nivolumab and pembrolizumab, may provide a platform for the discovery of new immuno-oncology therapeutics as well as the assessment of differences in mechanism of action between them. Citation Format: Omar S. Qureshi, Alexander Roberts, Lindsay Bentley, Tina Tang, Fay Stewart, Graham Wallace, Alison Cooper, Aaron Scott, David Thickett, Babu Naidu, Thomas Pinkney, Graham Taylor, Kristian Brock, Louise Healy, Zania Stamataki, Catherine Brady, S J. Curnow, John Gordon, Nicholas M. Barnes. A robust enhancement of cytokine production in a human chronic activation model of T cell exhaustion in vitro through blockade of PD-1/PDL-1 interactions using pembrolizumab or nivolumab; correlation with dissociated tumor immune cell responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3274.