Abstract
BackgroundAffinity-optimized T cell receptor (TCR)-engineered lymphocytes targeting tumor antigens can mediate potent antitumor responses in cancer patients, but also bear substantial risks for off-target toxicities. Most preclinical studies have focused on T cell responses to antigen-specific stimulation. In contrast, little is known on the regulation of T cell responsiveness through continuous TCR triggering and consequent tonic signaling. Here, we addressed the question whether increasing the TCR affinity can lead to chronic interactions occurring directly between TCRs and MHC-(self) molecules, which may modulate the overall functional potency of tumor-redirected CD8 T cells. For this purpose, we developed two complementary human CD8 T cell models (i.e. HLA-A2 knock-in and knock-out) engineered with incremental-affinity TCRs to the HLA-A2/NY-ESO-1 tumor antigen.MethodsThe impact of HLA-A2 recognition, depending on TCR affinity, was assessed at the levels of the TCR/CD3 complex, regulatory receptors, and signaling, under steady-state conditions and in kinetic studies. The quality of CD8 T cell responses was further evaluated by gene expression and multiplex cytokine profiling, as well as real-time quantitative cell killing, combined with co-culture assays.ResultsWe found that HLA-A2 per se (in absence of cognate peptide) can trigger chronic activation followed by a tolerance-like state of tumor-redirected CD8 T cells with increased-affinity TCRs. HLA-A2pos but not HLA-A2neg T cells displayed an activation phenotype, associated with enhanced upregulation of c-CBL and multiple inhibitory receptors. T cell activation preceded TCR/CD3 downmodulation, impaired TCR signaling and functional hyporesponsiveness. This stepwise activation-to-hyporesponsive state was dependent on TCR affinity and already detectable at the upper end of the physiological affinity range (KD ≤ 1 μM). Similar findings were made when affinity-increased HLA-A2neg CD8 T cells were chronically exposed to HLA-A2pos-expressing target cells.ConclusionsOur observations indicate that sustained interactions between affinity-increased TCR and self-MHC can directly adjust the functional potential of T cells, even in the absence of antigen-specific stimulation. The observed tolerance-like state depends on TCR affinity and has therefore potential implications for the design of affinity-improved TCRs for adoptive T cell therapy, as several engineered TCRs currently used in clinical trials share similar affinity properties.
Highlights
Affinity-optimized T cell receptor (TCR)-engineered lymphocytes targeting tumor antigens can mediate potent antitumor responses in cancer patients, and bear substantial risks for off-target toxicities
Downregulation of basal TCR/CD3 complex depends on both TCR affinity and HLA-A2 expression Through structure-based rational predictions [18], we have established a panel of affinity-increased TCRs for the NY-ESO-1 tumor antigen presented in the context of HLA-A2 (Additional file 1: Table S1 [17];), and reported significant reduced levels of surface TCR/CD3ε in A2pos primary CD8 T cells redirected with increased affinity TCRs [14]
Following TCR transduction with affinityenhanced variants and under resting culture conditions, levels of surface TCRαβ and CD3ε as well as of the proximal phospho-CD3ζ signaling molecule were downmodulated in de novo A2-expressing J76 cells along the TCR affinity gradient, when compared to A2neg J76 cells (Fig. 1 b, Additional file 1: Figure S1D). This was not the case for the total levels of CD3ε (Additional file 1: Figure S1E) and the distal ERK1/2 signaling node (Fig. 1 b). These results demonstrate that basal downregulation of the TCR/CD3 complex and associated CD3ζ in redirected J76 CD8αβ T cells depends on both TCR affinity and A2 expression
Summary
Affinity-optimized T cell receptor (TCR)-engineered lymphocytes targeting tumor antigens can mediate potent antitumor responses in cancer patients, and bear substantial risks for off-target toxicities. We addressed the question whether increasing the TCR affinity can lead to chronic interactions occurring directly between TCRs and MHC-(self) molecules, which may modulate the overall functional potency of tumor-redirected CD8 T cells. For this purpose, we developed two complementary human CD8 T cell models (i.e. HLA-A2 knock-in and knock-out) engineered with incremental-affinity TCRs to the HLA-A2/NY-ESO-1 tumor antigen. T cells expressing TCRs with increased affinity, above the physiological range, or with prolonged halflives, display substantial functional defects. There is a clear need to promote better preclinical strategies, including the prediction of optimized T cell responsiveness and off-target toxicity related to enhanced TCR-pMHC affinity, to guarantee the safety of candidate TCRs for clinical testing
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