Abstract
Redirecting CD8 T cell immunity with self/tumor-specific affinity-matured T cell receptors (TCRs) is a promising approach for clinical adoptive T cell therapy, with the aim to improve treatment efficacy. Despite numerous functional-based studies, little is known about the characteristics of TCR signaling (i.e., intensity, duration, and amplification) and the regulatory mechanisms underlying optimal therapeutic T cell responses. Using a panel of human SUP-T1 and primary CD8 T cells engineered with incremental affinity TCRs against the cancer-testis antigen NY-ESO-1, we found that upon activation, T cells with optimal-affinity TCRs generated intense and sustained proximal (CD3ζ, LCK) signals associated with distal (ERK1/2) amplification-gain and increased function. In contrast, in T cells with very high affinity TCRs, signal initiation was rapid and strong yet only transient, resulting in poor MAPK activation and low proliferation potential even at high antigen stimulation dose. Under resting conditions, the levels of surface TCR/CD3ε, CD8β, and CD28 expression and of CD3ζ phosphorylation were significantly reduced in those hyporesponsive cells, suggesting the presence of TCR affinity-related activation thresholds. We also show that SHP phosphatases were involved along the TCR affinity gradient, but displayed spatially distinct regulatory roles. While PTPN6/SHP-1 phosphatase activity controlled TCR signaling initiation and subsequent amplification by counteracting CD3ζ and ERK1/2 phosphorylation, PTPN11/SHP-2 augmented MAPK activation without affecting proximal TCR signaling. Together, our findings indicate that optimal TCR signaling can be finely tuned by TCR affinity-dependent SHP-1 and SHP-2 activity, and this may readily be determined at the TCR/CD3 complex level. We propose that these TCR affinity-associated regulations represent potential protective mechanisms preventing high affinity TCR-mediated autoimmune diseases.
Highlights
Adoptive transfer of tumor-reactive T cells has demonstrated that cytotoxic CD8 T cells are capable of mounting durable and protective immune responses against cancer
Using a panel of T cell receptor (TCR)-engineered A2/NY-ESO-1-specific SUP-T1 and primary CD8 T cells [3, 12], we first assessed the impact of TCR-peptide-major histocompatibility complex (pMHC) affinity on the distal pERK1/2 signaling node in terms of signal intensity and duration by quantitative flow cytometry-based intracellular staining and Reverse-Phase Protein Assays (RPPA)
Upon antigen-specific TCR triggering with pMHC multimers, ERK1/2 phosphorylation displayed an on-off activation pattern at the population level, from pERK1/2low to pERK1/2high (Figures 1A,D,E), consistent with the previously reported switch-like digital response of ERK1/2 activation [31]
Summary
Adoptive transfer of tumor-reactive T cells has demonstrated that cytotoxic CD8 T cells are capable of mounting durable and protective immune responses against cancer. Using a panel of engineered primary CD8 T cells expressing TCRs of incremental affinities for the NY-ESO-1 antigen, we found that T cells with supraphysiologically very high affinity TCRs lying above the optimal affinity window (KD between 5 and 1 μM) were functionally impaired [3, 11, 12] This impairment could be gradually restored by augmenting the dose of antigen stimulation [3]. Modeling-based analyses on therapeutic TCR-transduced NY-ESO-1-specific T cells further enabled to unravel a minimal signaling architecture combining antigen affinity as well as dose effects [5] These reports highlight that complex TCR affinity and antigen dose-associated regulatory mechanisms (positive and negative feedback loops) become integrated within the TCR signaling pathway following T cell activation
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