Abstract Background: Chimeric antigen receptor (CAR) T-cell therapy has significantly altered the treatment landscape for various hematological malignancies, yet therapeutic responses remain inconsistent due to the significant heterogeneity of individual tumors. Currently, the evaluation of CAR-T therapy efficacy lacks simple and effective methods. CD58 serves as a ligand for the CD2 receptor on T cells, playing a critical role in the transmission of activation and proliferation signals to T cells. However, its expression is frequently deficient in various hematological cancers, which may represent a potential intrinsic mechanism underlying resistance to CAR T-cell therapy. Exploring the role of CD58 may yield valuable insights into the assessment of CAR-T therapy efficacy.

Methods: In this study, we analyzed single-cell RNA sequencing and bulk RNA-sequencing results from patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) to elucidate the effects of CD58 expression on T-cell function and tumor cell-intrinsic signaling pathways. In vitro co-culture experiments and flow cytometry were conducted to assess the effect of CD58 expression alterations in DLBCL cells on the killing function of CAR-T cells. Cellular imaging was utilized to develop a novel method based on CD58 expression, aimed at predicting CAR-T efficacy.

Results: Co-culture assay confirmed that CD58 expression on DLBCL cells markedly affected CAR-T cell cytotoxicity. Overexpression of CD58 in DLBCL cells significantly enhanced the killing effect of CD19 CAR-T cells compared with control groups, while CD58 low expression diminished the efficacy of CD19 CAR-T cells. CD58 expression levels may serve as a key indicator of CAR-T cell function.

To explore a simple and effective method for assessing CAR-T efficacy based on CD58 expression levels, we investigated the impact of CD58 expression on tumor cell intrinsic properties. RNA sequencing revealed that CD58 expression levels significantly influenced the metabolic pathways of DLBCL cells, particularly those related to nitric oxide (NO) synthesis. Meanwhile, we demonstrated that high CD58 expression activated the PI3K-AKT signaling pathway, leading to the upregulation of NOS3 expression and subsequently enhancing NO synthesis and metabolism.

NO has been shown to play a crucial role in various physiological and pathological processes, and several types of NO probes are available for in vivo imaging and disease detection. Fluorescent probes offer the advantage of rapid real-time imaging. Therefore, we employed the NO probe on DLBCL cells, and the cellular imaging results indicated that DLBCL cells with high CD58 expression exhibited significantly stronger fluorescent signals in response to the probe. The expression of CD58 in DLBCL cells may be observed and quantified with NO-specific fluorescent probes. We further correlated the fluorescence signal with the cytotoxic effect of CAR-T cells, revealing a significant positive correlation between CAR-T cell cytotoxicity and the fluorescence intensity of NO probe targeting DLBCL cells.

Conclusions: Our study comprehensively investigated the impact of altered CD58 expression in DLBCL on CAR-T cell cytotoxicity, and tumor cell-intrinsic signaling pathways. We demonstrated that high CD58 expression significantly enhanced the cytotoxic effect of CAR-T cells on tumor cells. Meanwhile, elevated CD58 expression promoted NO synthesis and metabolism by activating the PI3K-AKT pathway. The application of the NO probe cell imaging method demonstrated a positive correlation between fluorescence intensity and CD58 expression levels in DLBCL cells, thus establishing a link to the tumoricidal activity of CAR T cells. Our findings provide a NO probe cell imaging method based on CD58 expression in DLBCL cells as a novel and efficient tool for predicting CAR T-cell therapy efficacy.

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2025
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