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Early-stage myeloid-derived suppressor cell count: Basophil exclusion matters

2019; Elsevier BV; Volume: 144; Issue: 4 Linguagem: Inglês

10.1016/j.jaci.2019.06.027

1097-6825

Fabrice Uhel, Imane Azzaoui, Simon Le Gallou, Thierry Fest, Karin Tarte, Mikaël Roussel,

Inflammation biomarkers and pathways

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells characterized by their immune-suppressive functions.1Bronte V. Brandau S. Chen S.-H. Colombo M.P. Frey A.B. Greten T.F. et al.Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.Nat Commun. 2016; 7: 12150Crossref PubMed Scopus (1266) Google Scholar Circulating MDSCs are increased in inflammatory diseases, sepsis,2Uhel F. Azzaoui I. Grégoire M. Pangault C. Dulong J. Tadie J.M. et al.Early expansion of circulating granulocytic myeloid-derived suppressor cells predicts development of nosocomial infections in patients with sepsis.Am J Respir Crit Care Med. 2017; 196: 315-327Crossref PubMed Scopus (89) Google Scholar and numerous cancers, including lymphoid malignancies3Azzaoui I. Uhel F. Rossille D. Pangault C. Dulong J. Le Priol J. et al.T-cell defect in diffuse large B-cell lymphomas involves expansion of myeloid-derived suppressor cells.Blood. 2016; 128: 1081-1092Crossref PubMed Scopus (82) Google Scholar and solid tumors. Their number increases with disease severity and predicts response to treatment and overall survival in various cancers.3Azzaoui I. Uhel F. Rossille D. Pangault C. Dulong J. Le Priol J. et al.T-cell defect in diffuse large B-cell lymphomas involves expansion of myeloid-derived suppressor cells.Blood. 2016; 128: 1081-1092Crossref PubMed Scopus (82) Google Scholar, 4Lang S. Bruderek K. Kaspar C. Höing B. Kanaan O. Dominas N. et al.Clinical relevance and suppressive capacity of human myeloid-derived suppressor cell subsets.Clin Cancer Res. 2018; 24: 4834-4844Crossref PubMed Scopus (101) Google Scholar In septic patients, polymorphonuclear (PMN)-MDSCs are specifically associated with the occurrence of nosocomial infections.2Uhel F. Azzaoui I. Grégoire M. Pangault C. Dulong J. Tadie J.M. et al.Early expansion of circulating granulocytic myeloid-derived suppressor cells predicts development of nosocomial infections in patients with sepsis.Am J Respir Crit Care Med. 2017; 196: 315-327Crossref PubMed Scopus (89) Google Scholar Consequently, these cells are becoming important biomarkers in several clinical situations and need as such to be accurately quantified. However, despite efforts in recent years to unify the nomenclature of MDSCs, it remains difficult to standardize their gating strategy by flow cytometry because of their lack of fully specific phenotypic markers. In human PBMCs, monocytic-MDSCs are defined as CD14pos HLA-DRlow CD15neg and PMN-MDSCs as CD11bpos CD14neg CD15pos (or CD66bpos) low-density granulocytes.1Bronte V. Brandau S. Chen S.-H. Colombo M.P. Frey A.B. Greten T.F. et al.Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.Nat Commun. 2016; 7: 12150Crossref PubMed Scopus (1266) Google Scholar Recently, a third subset, so-called early-stage subset of MDSCs (e-MDSCs) comprising more immature progenitors, has been identified as Linneg (CD3/CD14/CD15/CD19/CD56) HLA-DRneg CD33pos CD11bpos cells lacking myeloid lineage markers of monocytic-MDSCs and PMN-MDSCs.1Bronte V. Brandau S. Chen S.-H. Colombo M.P. Frey A.B. Greten T.F. et al.Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.Nat Commun. 2016; 7: 12150Crossref PubMed Scopus (1266) Google Scholar, 5Fleming V. Hu X. Weber R. Nagibin V. Groth C. Altevogt P. et al.Targeting myeloid-derived suppressor cells to bypass tumor-induced immunosuppression.Front Immunol. 2018; 9: 398Crossref PubMed Scopus (256) Google Scholar The existence of e-MDSCs has been demonstrated in renal cell carcinoma, epithelial ovarian cancer, and head and neck cancer, but their suppressive function remains unproven.4Lang S. Bruderek K. Kaspar C. Höing B. Kanaan O. Dominas N. et al.Clinical relevance and suppressive capacity of human myeloid-derived suppressor cell subsets.Clin Cancer Res. 2018; 24: 4834-4844Crossref PubMed Scopus (101) Google Scholar, 6Okła K. Czerwonka A. Wawruszak A. Bobiński M. Bilska M. Tarkowski R. et al.Clinical relevance and immunosuppressive pattern of circulating and infiltrating subsets of myeloid-derived suppressor cells (MDSCs) in epithelial ovarian cancer.Front Immunol. 2019; 10: 691Crossref PubMed Scopus (38) Google Scholar, 7Najjar Y.G. Rayman P. Jia X. Pavicic Jr., P.G. Rini B.I. Tannenbaum C. et al.Myeloid-derived suppressor cell subset accumulation in renal cell carcinoma parenchyma is associated with intratumoral expression of IL1β, IL8, CXCL5, and Mip-1α.Clin Cancer Res. 2017; 23: 2346-2355Crossref PubMed Scopus (88) Google Scholar Basophils represent 0.5% to 1% of peripheral leukocytes in normal conditions. They can be isolated within the PBMC fraction after a density gradient centrifugation and are CD33pos CD11bpos HLA-DRneg CD3neg CD14neg CD19neg CD56neg, and for the most part CD15neg (ie, Linneg).8Toba K. Koike T. Shibata A. Hashimoto S. Takahashi M. Masuko M. et al.Novel technique for the direct flow cytofluorometric analysis of human basophils in unseparated blood and bone marrow, and the characterization of phenotype and peroxidase of human basophils.Cytometry A. 1999; 35: 249-259Google Scholar Thus, their phenotype overlaps with that of e-MDSCs, thereby leading to potential overestimation of respective cell-type counts. To address this issue, we first analyzed peripheral blood from healthy donors (HDs; n = 8) by flow cytometry using CD203c and CRTH2 antibodies ordered from Miltenyi Biotec (Bergich Gladbach, Germany), Lin-1 (CD3, CD14, CD16, CD19, CD20, CD56) from Becton Dickinson (BD Biosciences, San Jose, Calif), and HLA-DR, CD33, and CD11b from Beckman Coulter (Brea, Calif). We found that an average of 92.6% (range, 78.5%-98.2%) of Lin-1neg HLA-DRneg CD33pos CD11bpos cells expressed CD123 (IL-3 receptor α-chain) and CRTH2 (CD294, receptor for prostaglandin D2), consistently with a basophil phenotype (Fig 1, A).9Varricchi G. Raap U. Rivellese F. Marone G. Gibbs B.F. Human mast cells and basophils—how are they similar how are they different?.Immunol Rev. 2018; 282: 8-34Crossref PubMed Scopus (58) Google Scholar In addition, the expression of CD203c (ecto-nucleotide pyrophosphatase phosphodiesterase, specifically expressed by basophils and mast cells upon activation) was induced on CRTH2pos CD123pos cells from HDs (n = 3) after 15 minutes of stimulation by an anti-IgE antibody (Allergenicity kit, Beckman Coulter) (Fig 1, B). Moreover, these cells released histamine after activation by anti-IgE, as measured by ELISA (data not shown). To confirm these findings, we sorted Lin-1neg HLA-DRneg CD33pos CD11bpos cells from an HD sample (FacsAriaIII, BD Biosciences). Microscopic examination of cytospin slides stained with May-Grünwald-Giemsa and captured at 1000× magnification revealed a mixture of basophils (characterized by their large purple-black granules and double-lobed nucleus) and immature myeloid cells with agranular cytoplasm-containing vacuoles (presumably e-MDSCs) (Fig 1, C). Finally, and in accordance with a previous article,8Toba K. Koike T. Shibata A. Hashimoto S. Takahashi M. Masuko M. et al.Novel technique for the direct flow cytofluorometric analysis of human basophils in unseparated blood and bone marrow, and the characterization of phenotype and peroxidase of human basophils.Cytometry A. 1999; 35: 249-259Google Scholar we confirmed on 8 samples (n = 6 HD and n = 2 diffuse large B-cell lymphoma [DLBCL] samples) that on average only 9.4% (range, 4.8% to 14%) of basophils expressed CD15 (Fig 1, D). Consequently, the Linneg HLA-DRneg CD33pos phenotype encompasses various basophils, which are not excluded by the CD15 marker. Because circulating e-MDSCs are increased in various types of cancers,4Lang S. Bruderek K. Kaspar C. Höing B. Kanaan O. Dominas N. et al.Clinical relevance and suppressive capacity of human myeloid-derived suppressor cell subsets.Clin Cancer Res. 2018; 24: 4834-4844Crossref PubMed Scopus (101) Google Scholar, 6Okła K. Czerwonka A. Wawruszak A. Bobiński M. Bilska M. Tarkowski R. et al.Clinical relevance and immunosuppressive pattern of circulating and infiltrating subsets of myeloid-derived suppressor cells (MDSCs) in epithelial ovarian cancer.Front Immunol. 2019; 10: 691Crossref PubMed Scopus (38) Google Scholar, 7Najjar Y.G. Rayman P. Jia X. Pavicic Jr., P.G. Rini B.I. Tannenbaum C. et al.Myeloid-derived suppressor cell subset accumulation in renal cell carcinoma parenchyma is associated with intratumoral expression of IL1β, IL8, CXCL5, and Mip-1α.Clin Cancer Res. 2017; 23: 2346-2355Crossref PubMed Scopus (88) Google Scholar we further analyzed 20 DLBCL samples at diagnosis from a cohort already published by our group.3Azzaoui I. Uhel F. Rossille D. Pangault C. Dulong J. Le Priol J. et al.T-cell defect in diffuse large B-cell lymphomas involves expansion of myeloid-derived suppressor cells.Blood. 2016; 128: 1081-1092Crossref PubMed Scopus (82) Google Scholar The research protocol was conducted under French legal guidelines and fulfilled the requirements of the local institutional ethics committee; all patients provided written informed consent (BMS-LyTRANS study; clinicaltrials.gov: NCT01287923). Clinical characteristics of patients with DLBCL enrolled in this cohort are listed in Table I. Within the Lin-1neg HLA-DRneg CD33pos CD11bpos population, we defined basophils and e-MDSCs as CD123pos and CD123neg fractions, respectively (Fig 1, E). Basophils represented 68.3% (range, 4.1% to 97.6%) of Lin-1neg HLA-DRneg CD33posCD11bpos cells (Fig 1, E).Table IPatients' characteristics of the lymphoma cohortCharacteristicPatients (n = 20)Average age (y) (range)54 (32-72)Male (n)12Female (n)8IPI (n) 0-16 23 37 4-54Cell of origin (n) GCB5 Non-GCB9 Unknown6GCB, Germinal center B cells; IPI, International Prognostic Index. Open table in a new tab GCB, Germinal center B cells; IPI, International Prognostic Index. Altogether, our results show that the Linneg HLA-DRneg CD33pos CD11bpos phenotype includes a heterogeneous population of functionally active mature basophils and immature myeloid cells morphologically consistent with e-MDSCs. The presence of basophils, often in large proportions, might account for the absence of clear suppressive phenotype in previous functional assays targeting e-MDSCs.4Lang S. Bruderek K. Kaspar C. Höing B. Kanaan O. Dominas N. et al.Clinical relevance and suppressive capacity of human myeloid-derived suppressor cell subsets.Clin Cancer Res. 2018; 24: 4834-4844Crossref PubMed Scopus (101) Google Scholar, 6Okła K. Czerwonka A. Wawruszak A. Bobiński M. Bilska M. Tarkowski R. et al.Clinical relevance and immunosuppressive pattern of circulating and infiltrating subsets of myeloid-derived suppressor cells (MDSCs) in epithelial ovarian cancer.Front Immunol. 2019; 10: 691Crossref PubMed Scopus (38) Google Scholar, 7Najjar Y.G. Rayman P. Jia X. Pavicic Jr., P.G. Rini B.I. Tannenbaum C. et al.Myeloid-derived suppressor cell subset accumulation in renal cell carcinoma parenchyma is associated with intratumoral expression of IL1β, IL8, CXCL5, and Mip-1α.Clin Cancer Res. 2017; 23: 2346-2355Crossref PubMed Scopus (88) Google Scholar Therefore, in addition to the recent recommendations,1Bronte V. Brandau S. Chen S.-H. Colombo M.P. Frey A.B. Greten T.F. et al.Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards.Nat Commun. 2016; 7: 12150Crossref PubMed Scopus (1266) Google Scholar we suggest to include basophil-specific exclusion markers such as CRTH2/CD294 or CD123 in the reference panel, to refine the quantification of e-MDSCs and to better define the clinical relevance of these cells. We acknowledge Drs Patricia Amé-Thomas and Beatrice Ly-Sunnaram for their help in histamine quantification and image capture and the Centre of Ressources Biologiques of Rennes (BB-0033-00056) for managing samples.