CD4CAR T cells engage endogenous TCR to mediate antigen-independent cytotoxicity and distinct immune signatures.

Abstract

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy enables targeted tumor cell killing through synthetic receptors that recognize surface antigens independently of the native T cell receptor (TCR). Although CAR constructs bypass MHC restriction, CAR T cells retain their endogenous TCRs. The extent to which these intrinsic receptors contribute to T cell function—particularly in the context of antigen-negative tumor cells—remains poorly defined. This has important implications for CAR T cell specificity, therapeutic safety, and the risk of off-target toxicity. We investigated whether CD4CAR T cells engage their endogenous TCR to mediate cytotoxicity against CD4-negative tumor cells and characterized the underlying signaling pathways, distinguishing CAR versus TCR-driven responses. Methods: Third-generation CD4CAR T cells incorporating CD28, 4-1BB, and CD3ζ signaling domains were generated and tested for cytotoxic activity against CD4⁺ KARPAS-299 (on-target) and CD4⁻ tumor cell lines U-87 MG (glioblastoma) and SK-BR-3 (breast adenocarcinoma). Co-cultures were performed at E:T 1:1, 2:1, and 5:1 ratios for 24–48 hours. Target cell viability was assessed using 7-AAD staining. To evaluate the contribution of endogenous TCR signaling, CD4CAR T cells were treated with CTLA-4-Ig to block CD28-mediated co-stimulation. Total RNA was extracted for bulk RNA sequencing, followed by differential gene expression analysis and pathway enrichment using KEGG, Reactome, and Hallmark gene sets. Results: CD4CAR T cells exhibited robust cytotoxicity against CD4⁺ KARPAS cells, with >80% killing at E:T 5:1 (p = 0.003). Notably, moderate cytotoxicity (~15%) was observed against CD4⁻ U-87 MG and SK-BR-3 cells (p = 0.003), increasing with higher E:T ratios. Blockade of CD28-mediated co-stimulation with CTLA-4-Ig selectively reduced cytotoxicity against CD4⁻ tumor cells, with no impact on CD4⁺ target killing. This suggests that the endogenous TCR mediates off-target cytotoxicity, while on-target killing is driven primarily by CAR activation. Transcriptomic profiling revealed discrete transcriptional programs associated with CAR- versus TCR-mediated cytotoxicity. On-target killing induced TNFα–NF-κB signaling (p = 6.25×10⁻²⁰), granzyme B expression, and enrichment of p53/apoptosis and Wnt/β-catenin pathways. These responses were absent in CD4⁻ co-cultures and non-transduced (NT) T cells. In contrast, off-target killing was characterized by selective activation of TGF-β signaling and IL-6–JAK–STAT3 signaling (the latter restricted to U-87 MG; p = 1.18×10⁻⁶). Shared pathways across all cytotoxic conditions included IL-2–STAT5 signaling and IFN-γ response signatures. Granzyme B expression was elevated exclusively in CD4CAR T cells undergoing on-target killing. This supports a model in which granzyme B serves as a biomarker of full CAR engagement, whereas off-target killing occurs through weaker, sub-threshold TCR activation that fails to induce granzyme B expression. The IL-6–STAT3 axis, while commonly linked to CAR T cell activation, appeared more heterogeneous, being selectively upregulated during U-87 MG–mediated off-target responses but not SK-BR-3. TGF-β signaling was uniquely enriched in off-target co-cultures, suggesting immune regulatory responses elicited by incomplete T cell activation or tumor-derived suppressive cues. Multiplex cytokine analysis revealed distinct secretory profiles between on-target and off-target killing. CD4⁺ target engagement led to preferential secretion of IL-1α, IL-5, IL-12, IL-17RA, IL-17RD, and IL-18. Chemokines CXCL8, CCL1, CCL22, CXCL10, and CXCL13 were also enriched, suggesting antigen-specific recruitment and inflammatory activation. In contrast, IL-2 levels were elevated in both on- and off-target conditions, while IL2RA expression did not differ significantly. Conclusions: Our data demonstrate that CD4CAR T cells retain functional endogenous TCR signaling, capable of mediating off-target cytotoxicity through different molecular programs. On-target killing involves TNFα–NF-κB activation, granzyme B upregulation, and pro-apoptotic signaling, while off-target cytotoxicity involves TCR-driven pathways such as IL-6–STAT3 and TGF-β signaling. These findings offer mechanistic insights into off-target effects seen in clinical CAR T cell therapy and highlight the need for strategies to prevent TCR-mediated bystander cytotoxicity without reducing therapeutic efficacy.