Neutrophils as Suppressors of T Cell Proliferation: Does Age Matter?

Abstract

Whereas, neutrophils have long been considered to mainly function as efficient innate immunity killers of micro-organisms at infected sites, they are now recognized to also be involved in modulation of adaptive immune responses. Immature and mature neutrophils were reported to have the capacity to suppress T cell-mediated immune responses as so-called granulocyte-myeloid-derived suppressor cells (g-MDSCs), and thereby affect the clinical outcome of cancer patients and impact the chronicity of microbial infections or rejection reactions in organ transplantation settings. These MDSCs were at first considered to be immature myeloid cells that left the bone marrow due to disease-specific signals. Current studies show that also mature neutrophils can exert suppressive activity. In this study we investigated in a robust T cell suppression assay whether immature CD11b+ myeloid cells were capable of MDSC activity comparable to mature fully differentiated neutrophils. We compared circulating neutrophils with myeloid cell fractions from the bone marrow at different differentiation stages. Our results indicate that functional MDSC activity is only becoming detectable at the final stage of differentiation, depending on the procedure of cell isolation. The MDSC activity obtained during neutrophil maturation correlated with the induction of the well-known highly mobile and toxic effector functions of the circulating neutrophil. Although immature neutrophils have been suggested to be increased in the circulation of cancer patients, we show here that immature neutrophils are not efficient in suppressing T cells. This suggests that the presence of immature neutrophils in the bloodstream of cancer patients represent a mere association or may function as a source of mature neutrophils in the tumor environment but not a direct cause of enhanced MDSC activity in cancer.

Keywords: MDSC activity; T cell suppression; cell isolation; neutrophils; progenitors.

Figure 1

Sorted neutrophil progenitor cells from bone marrow do not exert MDSC activity. Neutrophil progenitors from bone marrow were isolated via FACS sorting based on CD11b and CD16 expression under cold conditions and with a small nozzle. (A) Purified CFSE-labeled T cells from healthy donors (n = 6) were cultured with anti-CD3 and anti-CD28 antibodies (white bars), and in presence of mature neutrophils from control donors (black bars, n = 6) or sorted neutrophil progenitors from bone marrow (gray bars, n = 3) and/or indicated stimuli. Cells were harvested after 5–6 days and analyzed by flow cytometry for CFSE dilution among CD4⁺ T cells. (B) The surface marker expression of FPR-1 and fMLF binding (top panel, n = 3–8), TNF receptor I and II (center panel, n = 4) and TLR4 (bottom panel, n = 4) was measured by flow cytometry analysis of mature neutrophils from blood (black bar) and neutrophil progenitors from bone marrow. (C) Mature neutrophils and neutrophil progenitors were stimulated with the indicated stimuli and production of H₂O₂ was determined by measuring Amplex Red conversion into fluorescent Resorufin (n = 3). Error bars indicate SEM; ^****p < 0.0001.

Figure 2

FACS sorting causes an impairment in neutrophil function. (A,B) Neutrophils were left unsorted (black bars, n = 6) or sorted based on CD11b and CD16 expression under cold conditions and a small nozzle (A, gray bars, n = 3) or based on size (FSC/SSC) under RT conditions and a big nozzle (B, gray bars, n = 5). Cells were stimulated with the indicated stimuli and production of H₂O₂ was determined by measuring Amplex Red conversion into fluorescent Resorufin. (C) Purified CFSE-labeled T cells from healthy donors were cultured with anti-CD3 and anti-CD28 antibodies (white bars), and in presence of unsorted (black bars) or sorted (gray bars) mature neutrophils from control donors and/or indicated stimuli (n = 3). Sort was based on size (FSC/SSC) under RT conditions and a big nozzle. Cells were harvested after 5–6 days and analyzed by flow cytometry for CFSE dilution among CD4⁺ T cells. Error bars indicate SEM; ***p < 0.001.

Figure 3

Neutrophil progenitors isolated by density centrifugation show a less effective MDSC activity. (A) Mature neutrophils (black bars) and neutrophil progenitors from the bone marrow pellet fraction after density centrifugation (gray bars) were stimulated with the indicated stimuli and production of H₂O₂ was determined by measuring Amplex Red conversion into fluorescent Resorufin (n = 3). (B) Purified CFSE-labeled T cells from healthy donors were cultured with anti-CD3 and anti-CD28 antibodies (white bars, n = 6), and in presence of mature neutrophils from blood (black bars, n = 6) or neutrophil progenitors from the bone marrow pellet (gray bars, n = 3) and/or indicated stimuli. Cells were harvested after 5–6 days and analyzed by flow cytometry for CFSE dilution among CD4⁺ T cells. Error bars indicate SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

Only the CD16⁺ neutrophil progenitors show MDSC activity, ROS production and degranulation. (A,B) CD16 positive cells were isolated via MACS isolation from mature neutrophils from blood and neutrophil progenitors from BM pellet. (A) Purified CFSE-labeled T cells from healthy donors (n = 4) were cultured with anti-CD3 and anti-CD28 antibodies (white bars), and in presence of mature neutrophils from control donors (black bars, n = 6), CD16⁺ mature neutrophils (gray bars, n = 6), total BM pellet fraction (dark green bars, n = 4), CD16⁺ (green bars, n = 4) or CD16⁻ (light green bars, n = 4) progenitors from BM pellet and/or indicated stimuli. Cells were harvested after 5–6 days and analyzed by flow cytometry for CFSE dilution among CD4⁺ T cells. (B) The indicated cell fractions were stimulated with the indicated stimuli and production of H₂O₂ was determined by measuring Amplex Red conversion into fluorescent Resorufin (n = 4–6). Error bars indicate SEM; *p < 0.05, **p < 0.01, ****p < 0.0001.