Rational design of chimeric antigen receptors (CARs) with optimized anticancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen engagement triggers activation. We analyzed CAR-mediated antigen recognition via quantitative, single-molecule, live-cell imaging and found the sensitivity of CAR T cells toward antigen approximately 1,000-times reduced as compared to T cell antigen-receptor-mediated recognition of nominal peptide–major histocompatibility complexes. While CARs outperformed T cell antigen receptors with regard to antigen binding within the immunological synapse, proximal signaling was significantly attenuated due to inefficient recruitment of the tyrosine-protein kinase ZAP-70 to ligated CARs and its reduced concomitant activation and subsequent release. Our study exposes signaling deficiencies of state-of-the-art CAR designs, which presently limit the efficacy of CAR T cell therapies to target tumors with diminished antigen expression.

Rational design of chimeric antigen receptors (CARs) with optimized anti-cancer performance mandates detailed knowledge of how CARs engage tumor antigens and how antigen-engagement triggers activation. We analyzed CAR-mediated antigen recognition via quantitative single molecule live-cell imaging and found the sensitivity of CAR-T-cells towards antigen approximately 1000-times reduced when compared to T-cell antigen receptor (TCR)-mediated recognition of nominal peptide/MHC complexes. While CARs outperformed TCRs with regard to antigen binding within the immunological synapse, proximal signaling was significantly attenuated due to inefficient recruitment of the tyrosine-kinase ZAP70 to ligated CARs and its reduced concomitant activation and subsequent release. Our study exposes signaling deficiencies of state-of-the-art CAR-designs, which limit at present the efficacy of CAR-T-cell therapies to target tumors with diminished antigen expression.

The nonreceptor tyrosine kinase Syk, a central regulator of immune cell differentiation and activation, is a promising drug target for treatment of leukemia and allergic and inflammatory diseases. The clinical failure of Syk inhibitors underscores the importance of understanding the regulation of Syk function and activity. A series of previous studies emphasized the importance of three C-terminal tyrosines in Syk for kinase activity regulation, as docking sites for downstream effector molecules, and for Ca2+ mobilization. Here, we investigated the roles of these C-terminal tyrosines in the mouse. Surprisingly, expression of a triple tyrosine-to-phenylalanine human Syk mutant, SYK(Y3F), was not associated with discernible signaling defects either in reconstituted DT40 cells or in B or mast cells from mice expressing SYK(Y3F) instead of wild-type Syk. Remarkably, lymphocyte differentiation, calcium mobilization, and 2,4,6-trinitrophenyl (TNP)-specific immune responses were unperturbed in SYK(Y3F) mice. These results emphasize the capacity of immune cells to compensate for specific molecular defects, likely using redundant intermolecular interactions, and highlight the importance of in vivo analyses for understanding cellular signaling mechanisms.

The TNF receptor family member BAFFR is essential for providing mature B cells with pro-survival signals and has recently been claimed to transduce these, though not exclusively, via a Syk-dependent signaling hub that feeds into ERK/AKT activation. In this issue of The EMBO Journal, Hobeika et al (2015) describe a synergistic prosurvival scenario involving BAFFR and CD19, which remains functional under Syk null conditions and is able to maintain mature B-cell survival. The authors hence propose a BAFFR-/CD19-driven mechanism to act in parallel with homeostatic NF-κB/AKT activation in non-stimulated B cells.

Syk and Zap‐70 constitute a closely related nonreceptor protein tyrosine kinase family, of which both members are functionally indispensable for conferring their respective antigen receptors with enzymatic activity. In this study, we analyze the impact of altering BCR signaling output on B‐cell germinal center (GC) fate selection by constitutive, as well as inducible, monoallelic Syk kinase loss in the presence of a Zap‐70 knock‐in rescue allele. Cre‐mediated Syk deletion in Sykflox/Zap‐70 B cells lowers pErk, but not pAkt‐mediated signaling. Surprisingly, the use of a B‐cell‐specific constitutive mb1‐cre deleter mouse model showed that a small cohort of peripheral Sykflox/Zap‐70;mb1‐cre B cells efficiently circumvents deletion, which ultimately favors these Syk‐sufficient cells to contribute to the GC reaction. Using a developmentally unbiased Sykflox/Zap‐70;mb1‐creERT2 approach in combination with an inducible tdRFP allele, we further demonstrate that this monoallelic deletion escape is not fully explained by leakiness of Cre expression, but is possibly the result of differential Syk locus accessibility in maturing B cells. Altogether, this underscores the importance of proper Syk kinase function not only during central and peripheral selection processes, but also during GC formation and maintenance.

The spleen tyrosine kinase family members Syk and Zap‐70 are pivotal signal transducers downstream of antigen receptors and exhibit overlapping expression patterns at early lymphocytic developmental stages. To assess their differential kinase fitness in vivo, we generated mice, which carry a Zap‐70 cDNA knock‐in controlled by intrinsic Syk promoter elements that disrupts wild‐type Syk expression. Kinase replacement severely compromised Erk1/2‐mediated survival and proper selection of developing B cells at central and peripheral checkpoints, demonstrating critical dependence on BCR signalling quality. Furthermore, ITAM‐ and hemITAM‐mediated activation of platelets and neutrophils was completely blunted, while surprisingly FcγR‐mediated phagocytosis in macrophages was retained. The alteration in BCR signalling quality resulted in preferential development and survival of marginal zone B cells and prominent autoreactivity, causing the generation of anti‐insulin antibodies and age‐related glomerulonephritis. Development of concomitant fasting glucose intolerance in knock‐in mice highlights aberrant B cell selection as a potential risk factor for type 1 diabetes, and suggests altered BCR signalling as a mechanism to cause biased cellular and Ig repertoire selection, ultimately contributing to B cell‐mediated autoimmune predisposition. The related tyrosine kinases Syk and Zap‐70 are key signalling proteins downstream of antigen receptors. A knock‐in strategy reveals that the two kinases differentially affect BCR signalling, leading to aberrant B cell section and increased risk of autoimmune disease.