Knockout IL4I1 affects macrophages to improve poor efficacy of CD19 CAR-T combined with PD-1 inhibitor in relapsed/refractory diffuse large B-cell lymphoma

Abstract

Chimeric antigen receptor (CAR) T-cell therapy plays a critical role in the treatment of B-cell hematologic malignancies. The combination of PD-1 inhibitors and CAR-T has shown encouraging results in treating patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). However, there are still cases where treatment is ineffective. This study aimed to investigate the role of IL4I1 in the poor efficacy of CD19 CAR-T combined with PD-1 inhibitors in R/R DLBCL and to explore potential mechanisms. Transcriptomic and metabolomic correlation analyses were performed on tumor tissue from DLBCL patients. We employed an in vitro co-culture system consisting of Pfeiffer cells, CD19 CAR-T and macrophages to investigate the underlying mechanisms. It was found that IL4I1 levels were significantly increased in the tumor tissues of R/R DLBCL patients compared to responders. Correlation analysis revealed a positive association between IL4I1 and tryptophan (Trp)-kynurenic acid (Kyn) related metabolites. In the in vitro co-culture model, the presence of IL4I1 inhibited the cytotoxicity of CAR-T cells. Depletion of IL4I1 disrupted the IDO-AHR-Kyn signaling pathway, thereby enhancing the effectiveness of PD-1 inhibitors in combination with CD19 CAR-T for DLBCL treatment. CAR-T-mediated cytotoxicity was significantly inhibited when IL4I1 was present in the in vitro co-culture model. These findings suggest that IL4I1 may be a contributing factor to poor prognosis in R/R DLBCL patients. IL4I1 expression enhances immunosuppression via the IDO-AHR-Kyn pathway, inhibiting the effectiveness of PD-1 inhibitors combined with CD19 CAR-T. Therefore, suppression of IL4I1 may represent a potential target for combination therapy in DLBCL.

Keywords: CD19 CAR-T; DLBCL; IL4I1; Metabolism; PD-1 inhibitor.

Fig. 1

IL4I1 as a Factor for Poor Prognosis in DLBCL Patients. The groups are divided into Group 1 and Group 2. Group 1: R/R patients after treatment with a PD-1 inhibitor in combination with CD19 CAR-T; Group 2: Patients with a response after treatment with a PD-1 inhibitor in combination with CD19 CAR-T. (A) The mRNA levels of IL4I1 in different groups. (B) Immunohistochemical analysis of IL4I1 in lymphoma tissue across different groups. (C) The volcano plot offers a visual representation of the differential metabolic pathways distribution between the two groups. (D) The heatmap illustrates several metabolic pathways between the two groups. (E) The volcano plot displays differentially expressed genes between the two groups combination with 463 out of 24,601 metabolic pathways showing significant differences. (F) Proposed interaction map of transcriptomic and metabolomic data regulated by IL4I1

Fig. 2

Effect of IL4I1 on CD19 CAR-T and PD-1 Inhibitor Combination Therapy. The in vitro co-culture model consisted of CAR-T cells, Pfeiffer cells, and M-THP1/M-THP1 (IL4I1 KO)/M-THP1 (IL4I1 overexpression) in a ratio of 1:3:1, with 36 µg/mL of PD-1 inhibitor added. After 24 h (or 48 h for cytotoxicity) of incubation, the following analyses were conducted: (A) Impact of IL4I1 overexpression on cytotoxicity; (B) Impact of IL4I1 knockout on cytotoxicity; (C) CD4/CD8 ratio and subtype analysis in the IL4I1 KO group and control group; (D) Quantification of the CD4/CD8 ratio and subtype analysis; (E) Proportion of CAR-T cells expressing PD-1, TIM-3, and LAG-3; (F) Quantification of the expression of PD-1, TIM-3, and LAG-3 on CAR-T cells

Fig. 3

IL4I1 Enhances Immune Suppression via the IDO1-AHR-Kyn Metabolic Pathway. The in vitro co-culture model consisted of CAR-T cells, Pfeiffer cells, and M-THP1/M-THP1 (IL4I1 KO)/M-THP1 (IL4I1 overexpression) in a ratio of 1:3:1, with the addition of 5 mM IDO inhibitor D-1MT and 36 µg/mL PD-1 inhibitor. After 24 h (or 48 h for cytotoxicity) of incubation, the following analyses were conducted: (A-C) Protein expression of IDO1 in cell lysates was analyzed by western blot; (D-E) Protein expression of kynurenine (Kyn) treated with the IDO inhibitor in the co-culture model; (F) Immunofluorescence detection of Kyn treated with the IDO inhibitor and PD-1 inhibitor in the co-culture model; (G-H) Protein expression of Kyn in cell lysates analyzed by western blot in the co-culture model; (I) Immunofluorescence detection of Kyn treated with the IDO inhibitor and PD-1 inhibitor in the co-culture model

Fig. 4

IL4I1 Inhibition in Macrophages Impedes CAR-T Treatment In Vivo. (A) The formation and progression of tumors in three groups of mice were monitored using bioluminescence imaging: the control group, the CAR-T + M-THP1 (IL4I1 KO) + PD-1 inhibitor group, and the CAR-T + M-THP1 (control) + PD-1 inhibitor group. Day 0 was established when engraftment was confirmed following the injection of Pfeiffer cells. (B) The percentage of CD19 CAR-T cells in the bone marrow over time was measured by flow cytometry. (C) Survival rates of the three mouse groups. Data in the bar graphs represent the mean ± SD values from three experimental repeats (P < 0.05; *, P < 0.01, n = 3)