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. 2024 Jan 22;16(2):1845-1859.
doi: 10.18632/aging.205463. Epub 2024 Jan 22.

Activation of Notch-1 signaling pathway in macrophages to secrete PD-L1 and regulate cytotoxicity of CAR-T cells in diffuse large B-cell lymphoma

Weijing Li  1 , Lili Wu  1 , Chen Huang  1 , Hongqing Ma  2 , Lianjing Wang  1 , Wei Liu  1 , Lihong Liu  1
Affiliations

Activation of Notch-1 signaling pathway in macrophages to secrete PD-L1 and regulate cytotoxicity of CAR-T cells in diffuse large B-cell lymphoma

Weijing Li et al. Aging (Albany NY). .

Abstract

Objective: To investigate the mechanism of action of the Notch-1/IRE1/XBP1s signaling pathway in diffuse large B-cell lymphoma (DLBCL).

Methods: The expressions of relevant proteins were detected by Western blotting. The effect of myeloid-specific knockout of Notch-1 on lymphoma progression was observed by mouse tumor transplantation and imaging. The apoptosis of chimeric antigen receptor T-cell therapy (CAR-T) cells were detected by flow cytometry, and the proliferation of CAR-T cells was detected by wound healing assay and cell counting kit-8 (CCK8) assay.

Results: Lymphoma cells mediated the Notch-1 signaling pathway in bone marrow-derived macrophages and promoted the activation of STAT3 and STAT6 in bone marrow-derived macrophages. Myeloid-specific knockout of Notch-1 could inhibit the progression of lymphoma. Lymphoma cells enhanced the expression of p-PERK, p-IRE1α, ATF6, IL-6, IL-4, p-AKT, CD9, CD63 and PD-L1 in bone marrow-derived macrophages by mediating the Notch-1 signaling pathway. Knockout of Notch-1 in macrophages alleviated, to some extent, the suppression of killing activity of CAR-T cells, while activation of Notch-1 in macrophages inhibited proliferation and promoted apoptosis of CAR-T cells. The PD-L1 antibody significantly restored the cytotoxicity and proliferation of CAR-T cells, and inhibited their apoptosis.

Conclusion: Activation of the Notch-1/IRE1/XBP1s signaling pathway in myeloid macrophages promotes the secretion of IL-6 and IL-4 as well as PD-L1, thereby inhibiting the activity and proliferation of CAR-T cells and promoting their apoptosis.

Keywords: CAR-T cells; Notch-1/IRE1/XBP1s signaling pathway; PD-L1; diffuse large B-cell lymphoma; macrophages.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare no conflicts of interest related to this study.

Figures

Figure 1
Figure 1
Western blotting of Notch-1. (A) Protein bands of Notch-1; (B) Relative protein expression of Notch-1. (WT group vs. KO group; WT + lymphoma cell group vs. KO + lymphoma cell group; WT + lymphoma cell + IXA4 group vs. KO + lymphoma cell + IXA4 group; **P < 0.01, nsP > 0.05; N = 3).
Figure 2
Figure 2
Western blotting of p-STAT3 and p-STAT6. (A) Protein bands of p-STAT3 and p-STAT6; (B) Relative protein expressions of p-STAT3 and p-STAT6. (WT group vs. KO group; WT + lymphoma cell group vs. KO + lymphoma cell group; WT + lymphoma cell + IXA4 group vs. KO + lymphoma cell + IXA4 group; **P < 0.01, nsP > 0.05; N = 3).
Figure 3
Figure 3
Western blotting of p-PERK, p-IRE1α and ATF6. (A) Protein bands of p-PERK, p-IRE1α and ATF6; (B) Relative protein expressions of p-PERK, p-IRE1α and ATF6. (WT group vs. KO group; WT + lymphoma cell group vs. KO + lymphoma cell group; WT + lymphoma cell + IXA4 group vs. KO + lymphoma cell + IXA4 group; **P < 0.01, nsP > 0.05; N = 3).
Figure 4
Figure 4
Western blotting of IL-4 and IL-6. (A) Protein bands of IL-4 and IL-6; (B) Relative protein expressions of IL-4 and IL-6. (**P < 0.01; N = 3).
Figure 5
Figure 5
Western blotting of AKT signaling pathway-related proteins, exosome-associated proteins and PD-L1. (A) Protein bands of p-AKT, CD9, CD63 and PD-L1; (B) Relative protein expressions of p-AKT, CD9, CD63 and PD-L1. (WT + lymphoma cell + CAR-T group vs. KO + lymphoma cell + CAR-T group; WT + lymphoma cell + CAR-T + PD-L1 neutralizing antibody group vs. KO + lymphoma cell + CAR-T + PD-L1 neutralizing antibody group; **P < 0.01, nsP > 0.05; N = 3).
Figure 6
Figure 6
Imaging of the effect of mouse tumor transplantation and myeloid-specific knockout Notch-1 on lymphoma progression and scale plot of CD4+/CD8+. (A) Results of mouse tumor transplantation and imaging; (B) Fluorescence area in each group; (C) Ratio diagram of CD4+/CD8+. (WT group vs. KO group; WT + lymphoma cell group vs. KO + lymphoma cell group; WT + lymphoma cell + IXA4 group vs. KO + lymphoma cell + IXA4 group; **P < 0.01, nsP > 0.05; N = 3).
Figure 7
Figure 7
CAR-T cell proliferation assay. (A) Colony formation assay; (B) Data statistics of number of colonies; (C) CCK8 assay. (WT + lymphoma cell + CAR-T group vs. KO + lymphoma cell + CAR-T group; WT + lymphoma cell + CAR-T + PD-L1 neutralizing antibody group vs. KO + lymphoma cell + CAR-T + PD-L1 neutralizing antibody group; **P < 0.01, nsP > 0.05; N = 3).
Figure 8
Figure 8
Flow cytometry of CAR-T cell apoptosis. (A) CAR-T cell apoptosis; (B) Data statistics of number of apoptotic CAR-T cells. (WT + lymphoma cell + CAR-T group vs. KO + lymphoma cell + CAR-T group; WT + lymphoma cell + CAR-T + PD-L1 neutralizing antibody group vs. KO + lymphoma cell + CAR-T + PD-L1 neutralizing antibody group; **P < 0.01, nsP > 0.05; N = 3).
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