Magic™ Anti-CBL (Phospho Y731) monoclonal antibody, clone FQ084Z

Background Affinity-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. Methods The 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. Results We 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-A2(pos) but not HLA-A2(neg) 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 (K-D <= 1 mu M). Similar findings were made when affinity-increased HLA-A2(neg) CD8 T cells were chronically exposed to HLA-A2(pos)-expressing target cells. Conclusions Our 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.

RIG-I and MDA5 are cytoplasmic viral RNA sensors and are essential for antiviral innate immune responses, such as type I interferon production. Post-translational modification is critical for the activation and inactivation of RIG-I and MDA5. At least seven ubiquitin ligases have been reported to be involved in either K63- or K48-linked polyubiquitination of RIG-I and MDA5, and these ubiquitin ligases are further regulated by other factors. TRIM25 is an E3 ubiquitin ligase that delivers a K63-linked polyubiquitin moiety to the caspase activation and recruitment domains (CARDs) of RIG-I, thereby activating the antiviral innate immune response. Recent studies have shown that NDR2, ZCCHC3, and Lnczc3h7a promote TRIM25-mediated RIG-I activation. Riplet is another ubiquitin ligase that mediates the K63-linked polyubiquitination of the C-terminal domain (CTD) of RIG-I; however, it was also reported that Riplet delivers the K63-linked polyubiquitin moiety to the CARDs of RIG-I as well as to the CTD, thereby activating RIG-I. Further, there are several factors that attenuate the activation of RIG-I and MDA5. RNF125, TRIM40, and c-Cbl mediate K48-linked polyubiquitination and induce degradation of RIG-I and/or MDA5. USP21 and CYLD remove the K63-linked polyubiquitin chain from RIG-I, and NLRP12 inhibits polyubiquitin-mediated RIG-I activation. Although these new regulators have been reported, their distinctive roles and functional differences remain elusive, and in some cases, studies on the topic are contradictory to each other. In the present review, recent studies related to post-translational modifications of RIG-I and MDA5 are summarized, and several controversies and unanswered questions in this field are discussed.