CXCL8 secreted by immature granulocytes inhibits WT hematopoiesis in chronic myelomonocytic leukemia
Paul Deschamps, Margaux Wacheux, Axel Gosseye, Margot Morabito, Arnaud Pagès, Anne-Marie Lyne, Alexia Alfaro, Philippe Rameau, Aygun Imanci, Rabie Chelbi, Valentine Marchand, Aline Renneville, Mrinal M. Patnaik, Valerie Lapierre, Bouchra Badaoui, Orianne Wagner-Ballon, Céline Berthon, Thorsten Braun, Christophe Willekens, Raphael Itzykson, Pierre Fenaux, Sylvain Thépot, Gabriel Etienne, Emilie Elvira-Matelot, Francoise Porteu, Nathalie Droin, Leïla Perié, Lucie Laplane, Eric Solary, Dorothée Selimoglu-Buet
Paul Deschamps, Margaux Wacheux, Axel Gosseye, Margot Morabito, Arnaud Pagès, Anne-Marie Lyne, Alexia Alfaro, Philippe Rameau, Aygun Imanci, Rabie Chelbi, Valentine Marchand, Aline Renneville, Mrinal M. Patnaik, Valerie Lapierre, Bouchra Badaoui, Orianne Wagner-Ballon, Céline Berthon, Thorsten Braun, Christophe Willekens, Raphael Itzykson, Pierre Fenaux, Sylvain Thépot, Gabriel Etienne, Emilie Elvira-Matelot, Francoise Porteu, Nathalie Droin, Leïla Perié, Lucie Laplane, Eric Solary, Dorothée Selimoglu-Buet
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Research Article Hematology

CXCL8 secreted by immature granulocytes inhibits WT hematopoiesis in chronic myelomonocytic leukemia

Abstract

Chronic myelomonocytic leukemia (CMML) is a severe myeloid malignancy with limited therapeutic options. Single-cell analysis of clonal architecture demonstrates early clonal dominance with few residual WT hematopoietic stem cells. Circulating myeloid cells of the leukemic clone and the cytokines they produce generate a deleterious inflammatory climate. Our hypothesis is that therapeutic control of the inflammatory component in CMML could contribute to stepping down disease progression. The present study explored the contribution of immature granulocytes (iGRANs) to CMML progression. iGRANs were detected and quantified in the peripheral blood of patients by spectral and conventional flow cytometry. Their accumulation was a potent and independent poor prognostic factor. These cells belong to the leukemic clone and behaved as myeloid-derived suppressor cells. Bulk and single-cell RNA-Seq revealed a proinflammatory status of iGRAN that secreted multiple cytokines of which CXCL8 was at the highest level. This cytokine inhibited the proliferation of WT but not CMML hematopoietic stem and progenitor cells (HSPCs) in which CXCL8 receptors were downregulated. CXCL8 receptor inhibitors and CXCL8 blockade restored WT HSPC proliferation, suggesting that relieving CXCL8 selective pressure on WT HSPCs is a potential strategy to slow CMML progression and restore some healthy hematopoiesis.

Authors

Paul Deschamps, Margaux Wacheux, Axel Gosseye, Margot Morabito, Arnaud Pagès, Anne-Marie Lyne, Alexia Alfaro, Philippe Rameau, Aygun Imanci, Rabie Chelbi, Valentine Marchand, Aline Renneville, Mrinal M. Patnaik, Valerie Lapierre, Bouchra Badaoui, Orianne Wagner-Ballon, Céline Berthon, Thorsten Braun, Christophe Willekens, Raphael Itzykson, Pierre Fenaux, Sylvain Thépot, Gabriel Etienne, Emilie Elvira-Matelot, Francoise Porteu, Nathalie Droin, Leïla Perié, Lucie Laplane, Eric Solary, Dorothée Selimoglu-Buet

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Figure 6

CXCL8 specifically inhibits the growth of WT CD34+ cells.

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CXCL8 specifically inhibits the growth of WT CD34+ cells. (A) Cell outpu...
(A) Cell output of CMML CD34+ cells in liquid culture for 3 days in the presence of indicated doses of CXCL8 (ng/ml); ratio to untreated samples. Data are represented as means ± SD. n = 5 per group. One-way ANOVA, Dunnett’s multiple comparison. (B) Total colony output of CD34+ cells cultured in methylcellulose in the presence of indicated doses of CXCL8 (ng/ml) for 14 days. CB, cord blood (n = 6). Adult, healthy donor bone marrow CD34+ cells (n = 5) or CMML samples (n = 7); ratio to untreated samples. Data are represented as means ± SD. One-way ANOVA, Dunnett’s multiple comparison. (C) Cord blood (n = 6 upper panels) and healthy donor bone marrow (n = 5 lower panels) CD34+ cells were cultured in methylcellulose for 14 days to generate CFU-GM (left panel) and CFU-E (middle panel) colonies in the absence or presence of indicated concentrations of CXCL8. Output of treated relative to untreated cells. Right panel, fractions of CFU-GM and CFU-E were represented together. Data are represented as means ± SD. One-way ANOVA, Dunnett’s multiple comparison. (D) CXCR1 and CXCR2 mRNA expression assessed by RNA-Seq of CD34+ cells sorted from healthy donors (n = 7) and CMML patient (n = 12) bone marrow. Mann-Whitney U test. (E) Flow cytometry analysis of CXCR1 and CXCR2 at the surface of healthy donor (CTL, n = 5) and CMML patient (n = 18) CD34+ cells. Left panels, fraction of cells expressing the studied receptors; right panels, within positive cells, mean fluorescence intensity of each receptor. Mann-Whitney U test. (F) Cell output of healthy donor CD34+ in liquid culture in the absence or presence of 10 ng/mL CXCL8, 10 μM ladarixin (LADA), or 10 μM reparixin (REPA). Data are represented as means ± SD. n = 8. One-way ANOVA, Tukey’s multiple comparison. (G) Total colony output of healthy donor CD34+ cultured in methylcellulose in the absence or presence of CXCL8 (10 ng/mL), iGRAN supernatant, or reparixin (10 μM); ratio related to untreated samples. n = 6. Data are represented as means ± SD. One-way ANOVA, Tukey’s multiple comparison. (H) The same experiment was performed by using a CXCL8 neutralizing antibody (NAB, 5 μg/mL). n = 5. Data are represented as means ± SD. One-way ANOVA, Tukey’s multiple comparison.*P < 0.05; **P < 0.01; ***P < 0.001.