IL-10 Rescues CLL Survival through Repolarization of Inflammatory Nurse-like Cells

Abstract

Tumor-associated macrophages (TAMs) in chronic lymphocytic leukemia (CLL) are also called nurse-like cells (NLC), and confer survival signals through the release of soluble factors and cellular contacts. While in most patient samples the presence of NLC in co-cultures guarantees high viability of leukemic cells in vitro, in some cases this protective effect is absent. These macrophages are characterized by an "M1-like phenotype". We show here that their reprogramming towards an M2-like phenotype (tumor-supportive) with IL-10 leads to an increase in leukemic cell survival. Inflammatory cytokines, such as TNF, are also able to depolarize M2-type protective NLC (decreasing CLL cell viability), an effect which is countered by IL-10 or blocking antibodies. Interestingly, both IL-10 and TNF are implied in the pathophysiology of CLL and their elevated level is associated with bad prognosis. We propose that the molecular balance between these two cytokines in CLL niches plays an important role in the maintenance of the protective phenotype of NLCs, and therefore in the survival of CLL cells.

Keywords: CLL; IL-10; Nurse-like cells; TNF; polarization; survival.

Figure 1

Non protective NLC for CLL cells harbour an M1-like phenotype. (A,B). Flow cytometry analysis of the percentage of viability (Annexin V/7-AAD negative cells) of CLL cells from cultures of PBMC from CLL patients after 14 days. (One representative experiment (A) and data for PBMC from 35 CLL patients separated in two groups, one with high CLL cells viability and one with low CLL cells viability (B)). (C–E) Surface markers expressed by NLC analysed by flow cytometry at 14 days of cultures of PBMC from CLL patients with low (red) or high (green) in vitro CLL cells viability, comparing to the unstained (US: white) and the isotypic (IC: blue) controls (D) one representative experiment; (E) compilation of MFI ratios (marker/isotypic control) of five donors. (F) Fluorescence imaging of co-cultures of NLC (red staining) and CLL cells (green staining), at 1 min and 120 min, for patients with high (left) and low (right) in vitro viability of CLL cells. * indicates p < 0.05.

Figure 2

IL-10 rescues viability of CLL cells from patients with low protective NLC. (A) Surface markers expressed by NLC analysed by flow cytometry at 14 days of cultures of PBMC from CLL patient with low in vitro CLL cells viability, untreated (UT: red) or treated with IL-10 (grey), comparing to the unstained (US: white) and the isotypic (IC: blue) controls. Representative histograms from five independent experiments. (B,C) Percentage of the CLL cells viability at 14 days of culture of PBMC from CLL patient with low or high in vitro CLL cells viability untreated (UT) or treated with IL-10 (B) one representative experiment; (C) eight and five independent experiments for high viability and low viability cultures respectively). (D,E) Phagocytosis of CLL cells (blue dots) by NLC (red) visualized (D) upper: untreated; lower: IL-10 treated and quantified (E) thanks to the green pHrodo fluorescence. * indicates p < 0.05.

Figure 3

TNF depolarizes protective NLC, leading to a decrease of CLL cells viability. (A) Surface markers expressed by NLC analysed by flow cytometry at 14 days of CLL’s PBMC cultures incubated or not (UT: green) with TNF at day zero (orange) or at day six (yellow), compared to the unstained (US: white) and the isotypic (IC: blue) controls. Representative histograms from eight independent experiments. (B,C) Phagocytosis of CLL cells (blue dots) by NLC (red) is visualized. (B) upper: untreated; lower: TNF treated and quantified (C) thanks to the green pHrodo fluorescence. (D,E) Percentage of CLL cells viability at 14 days of culture of CLL PBMC-incubated or not (UT) with TNF at day zero or at day six. (D) one representative experiment. (E) A total of 19 independent experiments for TNF treatment at day zero and 10 independent experiments at day six. (F) Co-culture experiments: percentage of the CLL cells viability cultivated alone (white) or co-cultivated with autologous control NLC (untreated, green) or depolarized NLC (previously treated with TNF; orange). * indicates p < 0.05.

Figure 4

IL-10 repolarizes TNF-depolarized NLC which induce the increase of CLL cells viability. (A,B) Flow cytometry analysis of the viability (Annexin V/7-AAD negative cells) of CLL cells from cultures of CLL’s PBMC incubated or not (UT) with TNF at day zero following by treatment or not with IL-10 at day five. (A) one representative experiment; (B) five independent experiments. (C) Percentage of the CLL cells viability alone (white) or in a CLL’s PBMC culture without treatment (green) or in co-culture with autologous depolarized NLC previously treated with TNF (orange) or with autologous repolarized NLC (treated with TNF at day zero then with IL-10 at day five; purple) (14 independent experiments). (D) Surface markers expressed by NLC analysed by flow cytometry at 14 days of CLL’s PBMC cultures incubated or not (low viability, UT: red) with TNF at day zero (orange) or with TNF plus an IgG control (blue), or with TNF plus an anti-TNF antibody (green), compared to the isotypic control (IC: dark blue). (E,F) Percentage of the CLL cells viability at day 14 in CLL’s PBMC cultures untreated (E) or treated with TNF at day zero (F) without antibodies (red) or in the presence of IgG negative control (blue) or anti-IL-10 antibody (grey) or anti-TNF antibody (light green) or the combo anti-IL-10 antibody plus anti-TNF antibody (pale yellow) (nine independent experiments). * indicates p < 0.05.