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344 Human macrophages lacking NF-κB p50 display increased proinflammatory cytokine expression
  1. Theresa Barberi and
  2. Alan D Friedman
  1. Johns Hopkins University School of Medicine, Baltimore, MD, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background The NF-κB family of transcription factors plays a key role in inflammation. Proinflammatory signals induce IκB degradation to release cytoplasmic NF-κB p50:p65 heterodimers, which enter the nucleus and activate gene expression. NF-κB p50:p50 homodimers have 60-fold reduced affinity for IκB, and under basal conditions they repress proinflammatory genes. Multiple cancers grow slower in syngeneic p50(-/-) mice, or in p50(f/f);Lys-Cre mice lacking p50 in mature myeloid cells. Murine tumor macrophages lacking p50 display increased expression of a subset of proinflammatory (M1) genes, including IL-1β, IL-12β, and TNFα, and reduced expression of a subset of suppressive (M2) genes, which is associated with increased numbers of both total and activated tumor-infiltrating T cells.1–4 As a therapeutic approach, we find that adoptive transfer of immature myeloid cells lacking p50 (p50-IMC) slows the growth of syngeneic murine prostate cancer, pancreatic ductal carcinoma, and neuroblastoma tumors, when given after a dose of 5-fluorouracil.4 5 Immature rather than mature cells are utilized to favor tumor localization and retain dendritic cell potential. We have now sought to optimize development of human p50-IMC and determine whether macrophages that develop from these cells also manifest a more pro-inflammatory gene expression pattern.

Methods Human marrow CD34+ cells were nucleofected with Cas9:sgRNAs targeting the NFKB1 gene encoding p50, or with a non-targeting sgRNA. Cells were expanded in serum-free media that included SCF, TPO, FL, and IL-6, followed by transfer to serum-free media supplemented with M-CSF alone to produce monocytes, and finally to IMDM with human AB serum to generate macrophages. IFNγ or IL-4 were added for 24 hr to induce M1 or M2 polarization, followed by RNA isolation and qRT-PCR. Myeloid maturation in M-CSF was monitored by flow cytometry.

Results Highly efficient NFKB1 gene editing was confirmed by DNA analysis and Western blotting. Exome sequencing identified off-target edits affecting protein-coding regions of 27 genes, with 0.5–5.4% frequency; none are predicted to be transforming. Upon transfer of gene-edited cells to M-CSF, CD11b+CD14+ myeloid cells gradually accumulate and represent ~66% of the cell population after five days, with 2% granulocytes and rare erythroid cells and lymphocytes. Macrophages lacking human p50 manifested increased expression of a subset of M1 mRNAs, including IL-1β, IL-12β, and TNFα, and reduced expression of a subset of M2 mRNAs upon exposure to either IL-4 or IFNγ.

Conclusions Findings presented identify and validate a means to generate human p50-IMC for clinical application.

Acknowledgements This work was supported by grants to A.D.F. from the Maryland Stem Cell Research Fund (2020-MSCRFD-5380) and from the Department of Defense Prostate Cancer Research Program (W81XWH-21–1-0671).


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  2. Barberi T, Martin A, Suresh R, Barakat DJ, Harris-Bookman S, Drake CG, Lim M, Friedman AD. Absence of host NF-κB p50 induces murine glioblastoma tumor regression, increases survival, and decreases T cell induction of tumor-associated macrophage M2 polarization. Can. Immunol. Immunoth. 2018;67:1491–1503.

  3. Porta C, Ippolito A, Consonni FM, Carrero L, Celesti G, Correale C, Grizzi F, Pasqualini F, Tartari S, Rinaldi M, Bianchi P, Balzac F, Vetrano S, Turco E, Hirsch E, Laghi L, Sica A. Pro-tumor steering of cancer inflammation by p50 NF-κB enhances colorectal cancer progression. Cancer Immunol. Res. 2018;6:578–593.

  4. Suresh R, Barakat DJ, Barberi T, Zheng L, Jaffee EM, Pienta KJ, Friedman AD.NF-κB p50-deficient immature myeloid cell (p50-IMC) adoptive transfer slows the growth of murine prostate and pancreatic ductal carcinoma. J. Immunother. Cancer 2020;8:e000244.

  5. Cui C, Barberi T, Suresh R, Friedman AD. Adoptive transfer of immature myeloid cells lacking NF-κB p50 (p50-IMC) impedes the growth of MHC-matched high-risk neuroblastoma. Mol. Oncol. 2021;15:1783–1796.

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