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Background CAR T cell therapy has transformed the treatment landscape for hematologic malignancies, but its application in solid tumors—especially pediatric brain tumors—remains limited by multiple barriers, including antigen heterogeneity. In diffuse intrinsic pontine glioma (DIPG) and other pediatric high-grade gliomas, tumors frequently express a variety of antigens with variable affinities, reducing the effectiveness of conventional T cell recognition. Overcoming the intrinsic threshold required for TCR activation in response to low-affinity antigens could significantly improve immunotherapeutic efficacy.Methods We investigated the functional crosstalk between CAR and TCR signaling in a preclinical system. Human T cells co-expressing the same CAR and TCR were evaluated in real-time cytotoxicity assays using xCELLigence against U87 cells engineered to express IL13Rα2 or NLV or its APLs. In parallel, Jurkat triple-parameter reporter cells were engineered to express an IL13Rα2-targeting 41BB-based CAR and a TCR specific for the CMV-derived peptide NLVPMVATV (NLV). Using altered peptide ligands (APLs) of NLV—specifically NLVAMVATV (P4A, intermediate affinity) and NLVPMVAAV (T8A, low affinity)—we compared CAR-primed and TCR-primed Jurkat responses via CD69, NFκB, NFAT, and AP1 reporter activation.Results Under an E:T ratio of 1:1, CAR or TCR stimulation was applied using NLV or the low-affinity variant T8A for 2 days. CAR-primed TCR-CAR T cells exhibited superior cytolytic activity against T8A-expressing targets during the first two rounds of serial challenge, compared to TCR(NLV)-primed and TCR(T8A)-primed TCR-CAR T cells. Consistently, CAR-primed Jurkat cells showed significantly increased CD69 expression and enhanced activation of NFκB, NFAT, and AP1 reporters when stimulated with intermediate- and low-affinity APLs, relative to TCR-primed cells. These results support a model in which CAR engagement lowers the activation threshold of the TCR, allowing for improved recognition and killing of suboptimal antigens.Conclusions CAR priming sensitizes T cells to low-affinity tumor antigens, providing a potential strategy for improving immunotherapy in antigen-heterogeneous tumors like DIPG. This approach could enhance TCR responsiveness and expand the therapeutic reach of CAR T cell therapies.