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LILRB3 supports acute myeloid leukemia development and regulates T-cell antitumor immune responses through the TRAF2–cFLIP–NF-κB signaling axis

Nature Cancer volume 2, pages 1170–1184 (2021)Cite this article

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Abstract

Leukocyte immunoglobulin-like receptor B (LILRB), a family of immune checkpoint receptors, contributes to acute myeloid leukemia (AML) development, but the specific mechanisms triggered by activation or inhibition of these immune checkpoints in cancer is largely unknown. Here we demonstrate that the intracellular domain of LILRB3 is constitutively associated with the adaptor protein TRAF2. Activated LILRB3 in AML cells leads to recruitment of cFLIP and subsequent NF-κB upregulation, resulting in enhanced leukemic cell survival and inhibition of T-cell-mediated anti-tumor activity. Hyperactivation of NF-κB induces a negative regulatory feedback loop mediated by A20, which disrupts the interaction of LILRB3 and TRAF2; consequently the SHP-1/2-mediated inhibitory activity of LILRB3 becomes dominant. Finally, we show that blockade of LILRB3 signaling with antagonizing antibodies hampers AML progression. LILRB3 thus exerts context-dependent activating and inhibitory functions, and targeting LILRB3 may become a potential therapeutic strategy for AML treatment.

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Fig. 1: LILRB3 promotes monocytic AML progression.
Fig. 2: LILRB3 increases the sensitivity of monocytic AML cells to cytotoxic T cells.
Fig. 3: LILRB3 enhances NF-κB signaling and monocytic AML survival via TRAF2.
Fig. 4: LILRB3 interacted with TRAF2.
Fig. 5: LILRB3 enhancement of NF-κB signaling depends on cFLIP.
Fig. 6: LILRB3 negatively regulates NF-κB signaling with strong LPS stimulation.
Fig. 7: Blocking anti-LILRB3 antibody prevents monocytic AML development.
Fig. 8: Anti-LILRB3-blocking antibody prevents development of patient-derived AML.

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Data availability

The RNA-seq datasets generated in the present study have been deposited in the National Center for Biotechnology Information’s Sequence Read Archive (SRA) database with the SRA accession no. SRP292554. Source data for Figs.1–8 and Extended Data Figs. 1, 2 and 4–7 have been provided. All other data supporting the findings of the present study are available from the corresponding author on reasonable request.

References

  1. Robert, C. et al. Nivolumab in previously untreated melanoma without BRAF mutation. N. Engl. J. Med. 372, 320–330 (2015).

    Article  CAS  PubMed  Google Scholar 

  2. Robert, C. et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 372, 2521–2532 (2015).

    Article  CAS  PubMed  Google Scholar 

  3. Postow, M. A., Callahan, M. K. & Wolchok, J. D. Immune checkpoint blockade in cancer therapy. J. Cin. Oncol. 33, 1974 (2015).

    Article  CAS  Google Scholar 

  4. Curran, E. K., Godfrey, J. & Kline, J. Mechanisms of immune tolerance in leukemia and lymphoma. Trends Immunol. 38, 513–525 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Click, Z. R., Seddon, A. N., Bae, Y. R., Fisher, J. D. & Ogunniyi, A. New Food and Drug Administration-approved and emerging novel treatment options for acute myeloid leukemia. Pharmacotherapy 38, 1143–1154 (2018).

    Article  PubMed  Google Scholar 

  6. Trowsdale, J., Jones, D. C., Barrow, A. D. & Traherne, J. A. Surveillance of cell and tissue perturbation by receptors in the LRC. Immunol. Rev. 267, 117–136 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Daeron, M., Jaeger, S., Du Pasquier, L. & Vivier, E. Immunoreceptor tyrosine-based inhibition motifs: a quest in the past and future. Immunol. Rev. 224, 11–43 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Takai, T., Nakamura, A. & Endo, S. Role of PIR-B in autoimmune glomerulonephritis. J. Biomed. Biotechnol. 2011, 275302 (2011).

    Article  PubMed  Google Scholar 

  9. Katz, H. R. Inhibition of inflammatory responses by leukocyte Ig-like receptors. Adv. Immunol. 91, 251–272 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Kang, X. et al. Inhibitory leukocyte immunoglobulin-like receptors: immune checkpoint proteins and tumor sustaining factors. Cell Cycle 15, 25–40 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Hirayasu, K. & Arase, H. Functional and genetic diversity of leukocyte immunoglobulin-like receptor and implication for disease associations. J. Human Genet. 60, 703–708 (2015).

    Article  CAS  Google Scholar 

  12. Deng, M. et al. Leukocyte immunoglobulin-like receptor subfamily B (LILRB): therapeutic targets in cancer. Antibody Therapeut. 4, 16–33 (2021).

  13. van der Touw, W., Chen, H. M., Pan, P. Y. & Chen, S. H. LILRB receptor-mediated regulation of myeloid cell maturation and function. Cancer Immunol. Immunother. 66, 1079–1087 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Carosella, E. D., Rouas-Freiss, N., Roux, D. T., Moreau, P. & LeMaoult, J. HLA-G: an immune checkpoint molecule. Adv. Immunol. 127, 33–144 (2015).

    Article  CAS  PubMed  Google Scholar 

  15. John, S. et al. A novel anti-LILRB4 CAR-T cell for the treatment of monocytic AML. Mol. Ther. 26, 2487–2495 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zheng, J. et al. Inhibitory receptors bind ANGPTLs and support blood stem cells and leukaemia development. Nature 485, 656 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kang, X. et al. The ITIM-containing receptor LAIR1 is essential for acute myeloid leukaemia development. Nat. Cell Biol. 17, 665–677 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Deng, M. et al. LILRB4 signalling in leukaemia cells mediates T cell suppression and tumour infiltration. Nature 562, 605 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gui, X. et al. Disrupting LILRB4/APOE interaction by an efficacious humanized antibody reverses T-cell suppression and blocks AML development. Cancer Immunol. Res. 7, 1244–1257 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Anami, Y. et al. LILRB4-targeting antibody–drug conjugates for the treatment of acute myeloid leukemia. Mol. Cancer Ther. https://doi.org/10.1158/1535-7163.mct-20-0407 (2020).

  21. Li, Z. et al. LILRB4 ITIMs mediate the T cell suppression and infiltration of acute myeloid leukemia cells. Cell Mol. Immunol. https://doi.org/10.1038/s41423-019-0321-2 (2019).

  22. Churchill, H. R. O. et al. Leukocyte immunoglobulin-like receptor B1 and B4 (LILRB1 and LILRB4): highly sensitive and specific markers of acute myeloid leukemia with monocytic differentiation. Cytometry B Clin. Cytom. https://doi.org/10.1002/cyto.b.21952 (2020).

  23. Bergstrom, C. P. et al. The association of leukocyte immunoglobulin-like receptor subfamily B-4 expression in acute myeloid leukemia and central nervous system involvement. Leuk. Res. 100, 106480 (2021).

    Article  CAS  PubMed  Google Scholar 

  24. Barkal, A. A. et al. Engagement of MHC class I by the inhibitory receptor LILRB1 suppresses macrophages and is a target of cancer immunotherapy. Nat. Immunol. 19, 76–84 (2018).

    Article  CAS  PubMed  Google Scholar 

  25. Chen, H. M. et al. Blocking immunoinhibitory receptor LILRB2 reprograms tumor-associated myeloid cells and promotes antitumor immunity. J. Clin. Invest. 128, 5647–5662 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Tedla, N. et al. Activation of human eosinophils through leukocyte immunoglobulin-like receptor 7. Proc. Natl Acad. Sci. USA 100, 1174–1179 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Coxon, C. H., Geer, M. J. & Senis, Y. A. ITIM receptors: more than just inhibitors of platelet activation. Blood 129, 3407–3418 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sloane, D. E. et al. Leukocyte immunoglobulin-like receptors: novel innate receptors for human basophil activation and inhibition. Blood 104, 2832–2839 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Yeboah, M. et al. LILRB3 (ILT5) is a myeloid cell checkpoint that elicits profound immunomodulation. JCI Insight https://doi.org/10.1172/jci.insight.141593 (2020).

  30. Renauer, P. et al. Genome-wide association study identifies susceptibility loci in IL6, RPS9/LILRB3, and an intergenic locus on chromosome 21q22 in Takayasu’s arteritis. Arthr. Rheumatol. 67, 1361 (2015).

    Article  CAS  Google Scholar 

  31. Jones, D. C. et al. Allele-specific recognition by LILRB3 and LILRA6 of a cytokeratin 8-associated ligand on necrotic glandular epithelial cells. Oncotarget 7, 15618–15631 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Pfistershammer, K. et al. Allogeneic disparities in immunoglobulin-like transcript 5 induce potent antibody responses in hematopoietic stem cell transplant recipients. Blood 114, 2323–2332 (2009).

    Article  CAS  PubMed  Google Scholar 

  33. Tiscornia, G., Tergaonkar, V., Galimi, F. & Verma, I. M. CRE recombinase-inducible RNA interference mediated by lentiviral vectors. Proc. Natl Acad. Sci. USA 101, 7347–7351 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sanchez-Correa, B. et al. Cytokine profiles in acute myeloid leukemia patients at diagnosis: survival is inversely correlated with IL-6 and directly correlated with IL-10 levels. Cytokine 61, 885–891 (2013).

    Article  CAS  PubMed  Google Scholar 

  35. MacEwan, D. J. TNF receptor subtype signalling: differences and cellular consequences. Cell. Signal. 14, 477–492 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Syken, J., GrandPre, T., Kanold, P. O. & Shatz, C. J. PirB restricts ocular-dominance plasticity in visual cortex. Science 313, 1795–1800 (2006).

    Article  CAS  PubMed  Google Scholar 

  37. Hasegawa, K. et al. An immunocompetent mouse model for MLL/AF9 leukemia reveals the potential of spontaneous cytotoxic T-cell response to an antigen expressed in leukemia cells. PLoS ONE 10, e0144594 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Kawasaki, T. & Kawai, T. Toll-like receptor signaling pathways. Front. Immunol. 5, 461 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Shaw, P. J., Lamkanfi, M. & Kanneganti, T. D. NOD‐like receptor (NLR) signaling beyond the inflammasome. Eur. J. Immunol. 40, 624–627 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Taniguchi, K. & Karin, M. NF-κB, inflammation, immunity and cancer: coming of age. Nat. Rev. Immunol. 18, 309–324 (2018).

    Article  CAS  PubMed  Google Scholar 

  41. Cannons, J. L., Bertram, E. M. & Watts, T. H. Cutting edge: profound defect in T cell responses in TNF receptor-associated factor 2 dominant negative mice. J. Immunol. 169, 2828–2831 (2002).

    Article  CAS  PubMed  Google Scholar 

  42. Nishitoh, H. et al. ASK1 is essential for JNK/SAPK activation by TRAF2. Mol. Cell 2, 389–395 (1998).

    Article  CAS  PubMed  Google Scholar 

  43. Reinhard, C., Shamoon, B., Shyamala, V. & Williams, L. T. Tumor necrosis factor alpha-induced activation of c-jun N-terminal kinase is mediated by TRAF2. EMBO J. 16, 1080–1092 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Golks, A., Brenner, D., Krammer, P. H. & Lavrik, I. N. The c-FLIP–NH2 terminus (p22-FLIP) induces NF-κB activation. J. Exp. Med. 203, 1295–1305 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kataoka, T. & Tschopp, J. N-terminal fragment of c-FLIP (L) processed by caspase 8 specifically interacts with TRAF2 and induces activation of the NF-κB signaling pathway. Mol. Cell. Biol. 24, 2627–2636 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hughes, M. A. et al. Co-operative and hierarchical binding of c-FLIP and caspase-8: a unified model defines how c-FLIP isoforms differentially control cell fate. Mol. Cell 61, 834–849 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Chang, D. W. et al. c-FLIPL is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis. EMBO J. 21, 3704–3714 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Fritsch, M. et al. Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis. Nature 575, 683–687 (2019).

    Article  CAS  PubMed  Google Scholar 

  49. Bertheloot, D., Latz, E. & Franklin, B. S. Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cell. Mol. Immunol. 18, 1106–1121 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Wu, G. & Zhang, C. C. Membrane protein CAR promotes hematopoietic regeneration upon stress. Haematologica https://doi.org/10.3324/haematol.2019.243998 (2020).

  51. Pereira, S. & Lowell, C. The Lyn tyrosine kinase negatively regulates neutrophil integrin signaling. J. Immunol. 171, 1319–1327 (2003).

    Article  CAS  PubMed  Google Scholar 

  52. Medvedev, A. E. & Vogel, S. N. Overexpression of CD14, TLR4, and MD-2 in HEK 293T cells does not prevent induction of in vitro endotoxin tolerance. J. Endotoxin Res. 9, 60–64 (2003).

    Article  CAS  PubMed  Google Scholar 

  53. Micheau, O., Lens, S., Gaide, O., Alevizopoulos, K. & Tschopp, J. NF-κB signals induce the expression of c-FLIP. Mol. Cell. Biol. 21, 5299–5305 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Shembade, N. & Harhaj, E. W. Regulation of NF-κB signaling by the A20 deubiquitinase. Cell. Mol. immunol. 9, 123–130 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Li, L., Soetandyo, N., Wang, Q. & Ye, Y. The zinc finger protein A20 targets TRAF2 to the lysosomes for degradation. Biochim. Biophys. Acta Mol. Cell Res. 1793, 346–353 (2009).

    Article  CAS  Google Scholar 

  56. Song, H. Y., Rothe, M. & Goeddel, D. V. The tumor necrosis factor-inducible zinc finger protein A20 interacts with TRAF1/TRAF2 and inhibits NF-kappaB activation. Proc. Natl Acad. Sci. USA 93, 6721–6725 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Tao, M.-H. & Morrison, S. L. Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region. J. Immunol. 143, 2595–2601 (1989).

    Article  CAS  PubMed  Google Scholar 

  58. Hristodorov, D., Fischer, R. & Linden, L. With or without sugar? (A) glycosylation of therapeutic antibodies. Mol. Biotechnol. 54, 1056–1068 (2013).

    Article  CAS  PubMed  Google Scholar 

  59. Lo, M. et al. Effector-attenuating substitutions that maintain antibody stability and reduce toxicity in mice. J. Biol. Chem. 292, 3900–3908 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Dietrich, J., Cella, M. & Colonna, M. Ig-like transcript 2 (ILT2)/leukocyte Ig-like receptor 1 (LIR1) inhibits TCR signaling and actin cytoskeleton reorganization. J. Immunol. 166, 2514–2521 (2001).

    Article  CAS  PubMed  Google Scholar 

  61. Cella, M. et al. A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen processing. J. Exp. Med. 185, 1743–1751 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Sawanobori, M. et al. Expression of TNF receptors and related signaling molecules in the bone marrow from patients with myelodysplastic syndromes. Leukemia Res. 27, 583–591 (2003).

    Article  CAS  Google Scholar 

  63. Barrow, A. D. & Trowsdale, J. The extended human leukocyte receptor complex: diverse ways of modulating immune responses. Immunol. Rev. 224, 98–123 (2008).

    Article  CAS  PubMed  Google Scholar 

  64. Rodríguez, M. et al. NF-κB signal triggering and termination by tumor necrosis factor receptor 2. J. Biol. Chem. 286, 22814–22824 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Ye, H. & Wu, H. Thermodynamic characterization of the interaction between TRAF2 and tumor necrosis factor receptor peptides by isothermal titration calorimetry. Proc. Natl Acad. Sci. USA 97, 8961–8966 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Deng, M. et al. A motif in LILRB2 critical for Angptl2 binding and activation. Blood 124, 924–935 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank W. Xiong and H. Deng for their technical support, and G. Salazar for editing the manuscript. This work was supported by the National Cancer Institute (grant no. 1R01 CA248736), Leukemia and Lymphoma Society (grant no. TRP 6629-21), US Army Medical Research and Materiel Command (grant no. W81XWH2010793), the Cancer Prevention and Research Institute of Texas (grant nos. RP180435, RP150551 and RP190561), the Welch Foundation (grant no. AU-0042-20030616) and Immune-Onc Therapeutics, Inc. (Sponsored Research grant no. 111077).

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Authors and Affiliations

  1. Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Guojin Wu, Heyu Chen, Jingjing Xie, Mi Deng, Xiaoye Liu, Zhigang Lu & Cheng Cheng Zhang

  2. Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX, USA

    Yixiang Xu, Robbie D. Schultz, Xun Gui, Ningyan Zhang & Zhiqiang An

  3. Division of Pediatric Hematology–Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Samuel John

  4. Department of Immunochemistry, Research Institute for Microbial Diseases and Laboratory of Immunochemistry, World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan

    Hisashi Arase

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Contributions

G.W. developed the experimental protocol, designed, performed and analyzed experiments, and wrote the manuscript. C.C.Z. directed the project, interpreted the results and wrote the manuscript. Y.X. prepared the antibodies, evaluated the properties of antibodies in vitro, prepared RNA-seq samples, provided extensive discussion and wrote the manuscript. R.S screened and sequenced the antibody from scFv library. H.C., J.X. and X.L. conducted part of the animal experiments. M.D. and H.A. provided technical support. X.G. purified the antibodies. S.J. prepared clinical samples. Z.L. analyzed TCGA clinical data. N.Z. and Z.A. directed antibody production and evaluation of antibody properties, and contributed extensive discussions.

Corresponding author

Correspondence to Cheng Cheng Zhang.

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Competing interests

The Board of Regents of the University of Texas System has filed a patent application covering ‘Methods for identifying LILRB-blocking antibodies’. C.C.Z., G.W., H.C., M.D., Z.A. and N.Z. are listed as inventors. The patent application has been exclusively licensed to Immune-Onc Therapeutic, Inc. by the Board of Regents of the University of Texas System. Z.A., N.Z. and C.C.Z. hold equity in and have Sponsored Research Agreements with Immune-Onc Therapeutics, Inc. Z.A. is a Scientific Advisory Board member with Immune-Onc Therapeutics, Inc. The remaining authors declare no competing interests.

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Peer review information Nature Cancer thanks Iannis Aifantis, Ross Levine and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 LILRB3 enhances AML cell survival and promotes monocytic AML progression.

a, Analysis of LILRB3 and LILRB4 expression in patient AML samples (n = 35) as determined by flow cytometry. b, The cell death of THP-1 cells cultured with coated anti-LILRB3 or IgG in presence of DMSO, ABT199 (1 μM) or AZA (10 μM). (n = 3 independent cell cultures). c, Knockdown of LILRB3 in AML cell lines does not affect cell growth in culture (n = 3 independent cell cultures). d, THP-1 cells expressing Tet-on Cre and loxp U6 driven shRNAs were treated with Dox (1 μg/ml) for one day, and surface LILRB3 expression was analyzed by flow cytometry one week later. e, Percentages of dead cells in AML cultures treated with anti-LILRB3 antibody or IgG in the presence of different concentrations of TNFα (n = 3 independent cell cultures). f, Percentages of dead cells in THP-1 cells treated with anti-LILRB3 antibody or IgG in the presence of anti-TNFα (5 μg/ml) or control IgG (n = 3 independent cell cultures). g, Percentages of GFP+ AML cells in peripheral blood (PB), bone marrow (BM), spleen (SPL), and liver of mice transplanted with C1498 AML cells expressing B3-FL or B3del ICD (n = 4 mice). h, Survival curve of the mice treated as in panel e. i, THP-1 cell growth in plates coated anti-LILRB3 or IgG (n = 3 independent cell cultures). j, Serial colony-forming unit (CFU) replating with MLL-AF9 mouse AML cells (n = 3 independent cell cultures). k, Percentages of dead cells in U937 cells overexpressing LILRB3 or a control vector (n = 3 wells). The data are presented as mean ± s.e.m, and p values were calculated by two-tailed t-test except for h by log-rank test.

Source data

Extended Data Fig. 2 LILRB3 increases the survival of monocytic AML cells against cytotoxic T cells.

a, Percentages of CD4 and CD8 T cells in spleens of mice injected with mouse IgG or anti-mCD8 (10 mg/kg). b, CFU assays (MethoCult™ GF M3434) of regular BM cells mixed with mouse T cells specific to MLL-AF9 AML cells (T-AF9) or non-specific T cells (T-LPS) (n = 3 independent cell cultures). c, Expression of INFγ,TNFα, and PD-1 on CD4 and CD8 T cells from spleens of mice engrafted with MLL-AF9 AML cells expressing LILRB3 FL or LILRB3 with intracellular domain truncation.

Source data

Extended Data Fig. 3 LILRB3 enhances NF-κB signaling but not JNK signaling.

a, KEGG analysis of the top 20 processes affected by LILRB3 in mouse MLL-AF9 AML cells with whole-genome RNA-seq analysis. RNA was isolated from mouse MLL-AF9 AML cells expressing B3-FL or B3del ICD. “Down” and “Up” indicate genes expressed at lower or higher levels in AML cells that express B3del ICD versus those that express B3-FL. b, GSEA of the correlation between NF-κB signaling and LILRB3 in mouse MLL-AF9 AML cells (p values were calculated by Kolmogorov Smirnov (K-S) test in GSEA analysis). c, LILRB3 does not enhance the JNK signaling. GSEA of gene expression in THP-1 cells cultured in plates coated with anti-LILRB3 antibody or IgG.

Extended Data Fig. 4 TRAF2 and cFLIP interact, stimulate NF-κB signaling, and increase resistance of AML cells to the killing of cytotoxic T cells.

a, Relative NF-κB activities in 293 T cells co-transfected with NF-κB reporter plus empty vector, p22-FLIP, p43-CFLIP, or full-length cFLIP (n = 3 independent experiments). b, Relative NF-κB activities in 293 T cells co-transfected with NF-κB reporter plus empty vector or tet-on cFLIP in the presence of dox (n = 3 independent experiments). c, Co-immunoprecipitation assay of exogenous expressed FLAG-cFLIP and HA-TRAF2 in 293 T cells. d, e, Overexpression of TRAF2 and cFLIP increase the resistance of monocytic AML cells to cytotoxic T cells. CFSE-stained THP-1 cells that overexpress TRAF2 or empty vector (EV) (d) or cFLIP or empty vector (e) were co-cultured with activated T cells at the different ratios for 12 h and cell death was quantified. Left: Plots of percentage of dead cells versus E:T ration. Right: FACS analyses with E:T ratio of 2 (n = 3 independent experiments). f, West blotting of pMLKL (pS358) and MLKL in THP-1 cells treated with coated IgG or anti-LILRB3 for 12 h. g, Percentages of dead cells in THP-1 cells treated with anti-LILRB3 antibody or IgG in the presence of DMSO or NF-κB inhibitor QNZ (10 μM) (n = 3 independent cell cultures). The data are presented as mean ± s.e.m, and p values were calculated by two-tailed t-test.

Source data

Extended Data Fig. 5 LILRB3 balances NF-κB signaling with TRAF2 and SHP1/2.

a, Relative luciferase activity from THP-1-Lucia™ cells at different times after activation with anti-LILRB3 antibody or IgG (n = 3 individual samples). b, TRAF2 mRNA levels in AML cell lines and normal monocytes (n = 3 independent experiments) c, The percentage of GFP+ MLL-AF9 AML cells (with PirB knockout) expressing B3-FL or B3del ICD in peripheral blood (PB), bone marrow (BM), spleen (SPL), and liver in mice treated with PBS or LPS (n = 4 independent mice). d, Survival of mice engrafted with AML cells as treated in panel d (n = 4 independent mice). The data are presented as mean ± s.e.m, and p values were calculated by two-tailed t-test except for e by log-rank test. e, Mechanistic scheme of LILRB3 signaling. Without ligand-induced crosslinking of LILRB3, TRAF2 remains associated with LILRB3 but does not stimulate downstream signaling. When NF-κB signaling is at a low level, upon ligand-induced crosslinking of LILRB3, TRAF2 recruits cFLIP, and cFLIP is cleaved to p22-FLIP by caspase 8 (whose activity can be inhibited by zVAD-FMK). p22-FLIP binds to the IKK complex and stimulates NF-κB signaling. Meanwhile, after ligand binding to LILRB3, the ITIMs of LILRB3 are phosphorylated, which recruits SHP-1 and SHP-2. When there is a high level of NF-κB signaling stimulated by other cues (for example, LPS), higher expression of cFLIP and A20 (TNFAIP3) is induced. Increased cFLIP inhibits caspase 8 activity, and A20 disrupts the interaction between TRAF2 and LILRB3. Thus the inhibitory effect of LILRB3 on NF-κB signaling mediated by SHPs becomes dominant.

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Extended Data Fig. 6 Development of anti-LILRB3 blocking antibodies for suppressing AML development.

a, Upper: Flow chart of strategy for development of fully humanized antibodies against LILRB3. Lower: The identified antibodies were tested in the LILRB3 chimeric receptor reporter cell assay. b, ELISA results for LILRB3 binders. c, EC50 values of the anti-LILRB3 antibodies based on ELISA. d, Affinities of antibodies #32, #33, #67, and #45 to LILRB3 as determined by Octet. e, Cross-reactivity of the anti-LILRB3 antibodies with LILRAs evaluated with LILRA binding analyses. f, Cross-reactivity of the anti-LILRB3 antibodies with other LILRBs evaluated with LILRB binding analyses. g, Interaction with TRAF2 contributes to the effect of LILRB3 on AML development. CFU assay of MLL-AF9 AML cells expressing wild-type LILRB3 or mutant LILRB3 with mutations disrupting TRAF2 binding (AAA, QEE509-511AAA) or disrupting SHP-1/2 interactions (Y596/626 F) in the presence of control or anti-LILRB3 antibodies (n = 3 independent cell cultures). h, Evaluation of the effect of Fc-mediated effector functions of anti-LILRB3. Upper: Schematic of treatment. Lower: Percentages of GFP + MLL-AF9 mouse AML cells in PB, BM, SPL and LV of mice transplanted with AML cells expressing B3-FL or B3delICD and injected with IgG or anti-LILRB3 #6 (n = 4 independent mice). i, Survival of mice as treated in panel h. j, NF-κB signaling target gene expression (measured by qPCR) in THP-1 cells from BM of xenografted NSG mice treated with anti-LILRB3 #1NA or IgG (n = 3 independent experiments). The data are presented as mean ± s.e.m, and p values were calculated by two-tailed t-test except for i by log-rank test.

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Extended Data Fig. 7 Anti-LILRB3 #1N297A antibody did not affect normal hematopoiesis and leukocytosis.

a, Schematic of generation of myeloid-specific LILRB3 transgenic mice. b, c, LILRB3 is expressed on myeloid cells in peripheral blood (PB), spleen (SPL) and bone marrow (BM) of LysM-Cre driven LILRB3 transgenic mice, which were treated by anti-LILRB3 #1 antibody (n = 2 mice) or IgG (n = 3 mice). Shown are representative flow cytometry plots (b) and result summary (c).

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Wu, G., Xu, Y., Schultz, R.D. et al. LILRB3 supports acute myeloid leukemia development and regulates T-cell antitumor immune responses through the TRAF2–cFLIP–NF-κB signaling axis. Nat Cancer 2, 1170–1184 (2021). https://doi.org/10.1038/s43018-021-00262-0

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