IL-1 receptor–associated kinase-3 acts as an immune checkpoint in myeloid cells to limit cancer immunotherapy
Gürcan Tunalı, … , Irineos Papakyriacou, Yumeng Mao
Gürcan Tunalı, … , Irineos Papakyriacou, Yumeng Mao
Published February 9, 2023
Citation Information: J Clin Invest. 2023;133(7):e161084. https://doi.org/10.1172/JCI161084.
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Research Article Immunology Oncology

IL-1 receptor–associated kinase-3 acts as an immune checkpoint in myeloid cells to limit cancer immunotherapy

Abstract

Inflammatory mediators released by cancer cells promote the induction of immune suppression and tolerance in myeloid cells. IL-1 receptor–associated kinase-3 (IRAK3) is a pseudokinase that inhibits IL-1/TLR signaling, but its role in patients treated with immune checkpoint blockade (ICB) therapy remains unclear. Using RNA-Seq data from the IMvigor210 trial, we found that tumors with high IRAK3 expressions showed enriched antiinflammatory pathways and worse clinical response to ICB therapy. Upon IRAK3 protein deletion with CRISPR/Cas9, primary human monocytes displayed altered global protein expression and phosphorylation in quantitative proteomics and released more proinflammatory cytokines in response to stimulation. Bone marrow–derived macrophages from an IRAK3 CRISPR KO mouse model demonstrated a proinflammatory phenotype and enhanced sensitivity to TLR agonists compared with WT cells. IRAK3 deficiency delayed the growth of carcinogen-induced and oncogene-driven murine cancer cells and induced enhanced activation in myeloid cells and T cells. Upon ICB treatment, IRAK3-KO mice showed enrichment of TCF1+PD-1+ stem-like memory CD8+ T cells and resulted in superior growth inhibition of immunologically cold tumors in vivo. Altogether, our study demonstrated what we believe to be a novel cancer-driven immune tolerance program controlled by IRAK3 in humans and mice and proposed its suitability as an immunotherapy target.

Authors

Gürcan Tunalı, Marta Rúbies Bedós, Divya Nagarajan, Patrik Fridh, Irineos Papakyriacou, Yumeng Mao

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Figure 2

Genetic deletion of IRAK3 modulates primary human monocytes.

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Genetic deletion of IRAK3 modulates primary human monocytes. Human prima...
Human primary monocytes isolated from healthy blood donors or the human monocytic THP1 cell line were transfected with recombinant Cas9 and RNP complexes targeting the human IRAK3 gene. Deletion of the IRAK3 protein in human monocytic THP1 cells and primary monocytes was confirmed using (A) flow cytometry and (B) Western blotting. Cells transfected with Cas9 and the tracrRNA were used as controls. Representative histogram and blots from 3 experiments. (C) Primary human monocytes transfected with RNP complex with or without the IRAK3-targeting gRNA were cultured in 100 ng/mL rhGM-CSF and activated with LPS at 0.5 ng/mL. The resulted expression of the IRAK3 protein was shown in at least 3 donors (mean ± SD, unpaired t test, *P < 0.05, ***P < 0.001). (D) Control or IRAK3 KO THP1 cells were cocultured with primary human T cells from at least 5 donors in presence of micro-beads coated with αCD3/CD28 antibodies. The proliferation of CD4+ or CD8+ T cells was assessed using flow cytometry after 4 days (mean ± SD, unpaired t tests. *P < 0.05, **P < 0.01). (E) Primary human monocytes isolated from fresh buffy coats were transfected with RNP complex with or without the IRAK3-targeting gRNA. Cells were harvested after 4 days and cocultured with allogeneic CD3+ primary human T cells at indicated ratios. The proliferation of T cells was assessed after 5 days by flow cytometry, data were analyzed using unpaired t tests. *P < 0.05, **P < 0.01. Representative donor of 3 biological repeats. (F) Global protein expression in control or IRAK3-KO primary human monocytes from 4 donors after treatment with 1 μg/mL LPS for 45 minutes were determined using quantitative proteomics analysis and upregulated pathways in KO cells were shown. (G) Proteins were clustered by community analysis and grouped based on their functions. Expressions of individual proteins in IRAK3-KO versus control were shown by colors. Red, upregulated in KO; blue, downregulated in KO; purple, only detected in control; yellow, only detected in KO. The outline of the proteins and the community they belong to were annotated according to the biological functions described in the graph. Statistical tests were performed using paired student’s t tests and corrected by the False Discovery Rate.