CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy

Abstract

Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions.

Keywords: T cells; gene therapy; therapy/immunotherapy.

Fig. 1

Immunoreceptor tyrosine-based activation motif (ITAM)-mutant chimeric antigen receptors retain a spectrum of functional activity in Jurkat E6·1 and JRT3-T3·5 cells –Jurkat E6·1 cells were transduced with retroviral constructs encoding the indicated chimeric antigen receptors (CARs) and sorted by flow cytometry to obtain enriched populations of CAR⁺ cells. CAR⁺ cells were lysed and subjected to sodium dodecyl sulphate-polyacrylamide gel electophoresis (SDS-PAGE). Transferred proteins were detected using an anti-FLAG monoclonal antibody (mAb). Reducing and non-reducing conditions are shown with molecular weight (KDa) markers (a). Transduced Jurkat E6·1 (b) or JRT3-T3·5 (c) cells were assessed for surface expression of the T cell activation marker CD69 after stimulation for 24 h with the indicated concentrations of immobilized carcinoembryonic antigen (CEA) protein. Significant increases in CD69 expression between 0 μg/ml and 1·0 μg/ml are shown as *P < 0·05, **P < 0·01 and ***P < 0·001 (Kruskal–Wallis test). For activation assays (b,c), arithmetic means of three independent experiments ± standard deviation are shown. NT: non-transduced.

Fig. 2

Residual chimeric antigen receptor cis- and trans-signalling mechanisms permit activity in CD3ζ-deficient MA5·8 cells – CD3ζ-deficient MA5·8 cells were transduced with the indicated chimeric antigen receptors (CARs) and enriched for expression. CD3ε surface expression was measured to evaluate CAR–T cell receptor (TCR) interactions (a). Transduced MA5·8 cells were assessed for surface expression of CD69 after stimulation for 24 h with the indicated concentrations of immobilized carcinoembryonic antigen (CEA) protein (b). Significant increases in CD69 expression between 0 μg/ml and 1·0 μg/ml are shown as *P < 0·05, **P < 0·01 and ***P < 0·001 (Kruskal–Wallis test). Arithmetic means of three independent experiments ± standard deviation are shown. NT: non-transduced.

Fig. 3

Immunoreceptor tyrosine-based activation motif (ITAM)-truncated chimeric antigen receptors retain functional capacity in Jurkat E6·1 cells – Jurkat E6·1 cells were transduced with the indicated full-length and truncated MFEζ variants and enriched for expression. Transduced cells were assessed for surface expression of CD69 after stimulation for 24 h with the indicated concentrations of immobilized carcinoembryonic antigen (CEA) protein (a) and EC₅₀ values were compared by one-way (anova) (b). *P < 0·05, **P < 0·01 and ***P < 0·001. Arithmetic means of three independent experiments ± standard deviation are shown. NT: non-transduced.

Fig. 4

Immunoreceptor tyrosine-based activation motif (ITAM)-truncated chimeric antigen receptors retain functional capacity in primary human T cells – T cells from three healthy donors were transduced with the indicated chimeric antigen receptors (CARs) and expanded in vitro. Transduced CD4⁺ and CD8⁺ cells, identified by FLAG expression, were assessed for CD107a mobilization and intracellular production of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-2 after stimulation for 6 h with the indicated concentrations of immobilized carcinoembryonic antigen (CEA) protein. Representative data from one donor are shown.

Fig. 5

Immunoreceptor tyrosine-based activation motif (ITAM)-truncated chimeric antigen receptors do not show significant variations in antigen sensitivity for most effector functions in CD4⁺ and CD8⁺ T cell lineages – T cells from three healthy donors were transduced with the indicated chimeric antigen receptors (CARs) and expanded in vitro. Transduced CD4⁺ and CD8⁺ cells, identified by FLAG expression, were assessed for CD107a mobilization and intracellular production of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-2 after stimulation for 6 h with the indicated concentrations of immobilized carcinoembryonic antigen (CEA) protein. EC₅₀ values were calculated using GraphPad Prism software and compared by one-way analysis of variance (anova). *P < 0·05, **P < 0·01 and ***P < 0·001 compared to three ITAM.

Fig. 6

Immunoreceptor tyrosine-based activation motif (ITAM)-truncated chimeric antigen receptors elicit polyfunctional responses in CD4⁺ and CD8⁺ T cell lineages – T cells from three healthy donors were transduced with the indicated chimeric antigen receptors (CARs) and expanded in vitro. Transduced CD4⁺ and CD8⁺ cells, identified by FLAG expression, were assessed for CD107a mobilization and intracellular production of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-2 after stimulation for 6 h with the indicated concentrations of immobilized carcinoembryonic antigen (CEA) protein. The functional profiles of CD8⁺ (a,b) and CD4⁺ (c,d) cells were analysed using spice software. Pie charts depict the number of functions as follows: grey, 0; blue, 1; green, 2; yellow, 3; red, 4. Arc graphs indicate the proportion of cells with each combination of effector functions for each CAR (from left to right: three ITAM, two ITAM, one ITAM and no ITAM) at a CEA concentration of 0·125 μg/ml for CD8⁺ (b) and CD4⁺ (d) cells.