Immunoglobulin-like transcript 3 is expressed by myeloid-derived suppressor cells and correlates with survival in patients with non-small cell lung cancer

Abstract

Myeloid-derived suppressor cells (MDSCs) play an important role in immune suppression and accumulate under pathologic conditions such as cancer and chronic inflammation. They comprise a heterogeneous population of immature myeloid cells that exert their immunosuppressive function via a variety of mechanisms. Immunoglobulin-like transcript 3 (ILT3) is a receptor containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) that can be expressed on antigen-presenting cells and is an important regulator of dendritic cell tolerance. ILT3 exists in a membrane-bound and a soluble form and can interact with a yet unidentified ligand on T cells and thereby induce T-cell anergy, regulatory T cells, or T suppressor cells. In this study, we analyzed freshly isolated peripheral blood mononuclear cells (PBMCs) of 105 patients with non-small cell lung cancer and 20 healthy controls and demonstrated for the first time that ILT3 is expressed on MDSCs. We show that increased levels of circulating MDSCs correlate with reduced survival. On the basis of ILT3 cell surface expression, an ILT3^low and ILT3^high population of polymorphonuclear (PMN)-MDSCs could be distinguished. Interestingly, in line with the immunosuppressive function of ILT3 on dendritic cells, patients with an increased proportion of PMN-MDSCs and an increased fraction of the ILT3^high subset had a shorter median survival than patients with elevated PMN-MDSC and a smaller ILT3^high fraction. No correlation between the ILT3^high subset and other immune variables was found. ILT3 expressed on MDSCs might reflect a previously unknown mechanism by which this cell population induces immune suppression and could therefore be an attractive target for immune intervention.

Keywords: APC, antigen-presenting cell; CD85k; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; HC, healthy control; ILT3, immunoglobulin-like transcript 3; LILRB4; LIR-5; MDSC, myeloid-derived suppressor cell; MFI, mean fluorescence intensity; MO-MDSC, monocytic MDSC; NFκB, nuclear factor κB; NSCLC, non-small cell lung carcinoma; PBMC, peripheral blood mononuclear cell; PMN-MDSC, polymorphonuclear MDSC; Treg, regulatory T cell; Ts, T suppressor cell; immune suppression; immunoglobulin-like transcript 3; myeloid-derived suppressor cells; non-small cell lung cancer; overall survival; sILT3, soluble ILT3.

Figure 1.

Levels of circulating MO-MDSCs and PMN-MDSCs are increased in patients with non-small cell lung cancer. (A) Flow cytometry was performed on freshly isolated PBMCs from NSCLC patients and healthy controls. The gating strategy for determination of circulating MO-MDSCs and PMN-MDSCs is depicted. After gating the live cells, CD16^high cells were excluded. MO-MDSCs were characterized as CD14⁺CD11b⁺HLA-DR^lowCD15⁺ and PMN-MDSCs were characterized as CD14⁻CD11b⁺HLA-DR^lowCD15⁺. MDSC levels were determined as a proportion of live cells. (B). Frequency of MO-MDSCs and PMN-MDSCs in PBMCs was significantly higher in NSCLC patients than in healthy controls. **P < 0.01; ***P < 0.001; Mann-Whitney U test. MDSC, myeloid-derived suppressor cell; MO-MDSC, monocytic MDSC; NSCLC, non-small cell lung carcinoma; PBMC, peripheral blood mononuclear cell; PMN-MDSC, polymorphonuclear MDSC.

Figure 2.

ILT3 expression on myeloid-derived suppressor cells. (A) Flow cytometric data of a representative patient, displayed as density plot based on ILT3 and CD33 expression. Left panel: PMN-MDSCs, right panel: MO-MDSCs. (B) Histograms of 4 different patients with ILT3 expression of PMN-MDSCs (shaded) compared to the expression within the CD11b⁻CD14⁻ population (dashed line, mainly lymphocytes) and CD11b⁺CD14⁺ population (dotted line, mainly monocytes). Proportions of the ILT3^high fraction are displayed as percentage of PMN-MDSCs. ILT3, immunoglobulin-like transcript 3; MDSC, myeloid-derived suppressor cell; MO-MDSC, monocytic MDSC; PMN-MDSC, polymorphonuclear MDSC.

Figure 3.

ILT3^high proportion of PMN-MDSCs in patients with non-small cell lung cancer. (A) ILT3^high proportions of PMN-MDSCs were significantly higher in NSCLC patients than in healthy controls. ***P < 0.001, Student t test. (B) Correlations between the proportion of ILT3^high PMN-MDSC and various immune subsets in NSCLC patients were analyzed with the Spearman rho test. None of the tests revealed a significant correlation (P > 0.05 for all analyses). ILT3, immunoglobulin-like transcript 3; MDSC, myeloid-derived suppressor cell; MO-MDSC, monocytic MDSC; PMN-MDSC, polymorphonuclear MDSC.

Figure 4.

Serum sILT3 in patients with non-small cell lung cancer. (A) Soluble ILT3 was measured by ELISA in serum samples of healthy controls (n=8) and patients with Stage IV NSCLC (n=30). Levels of sILT3 were significantly higher in NSCLC patients compared to healthy controls. * P < 0.05, Student t test. (B) sILT3 levels of NSCLC patients did not correlate with the fraction of ILT3^high cells of PMN-MDSC (Spearman rho test). (C) Correlations between level of sILT3 in serum and various immune subsets in NSCLC patients were analyzed with the Spearman rho test. None of the tests revealed a significant correlation (P > 0.05 in all analyses). ELISA, enzyme-linked immunosorbent assay; ILT3, immunoglobulin-like transcript 3; MDSC, myeloid-derived suppressor cell; MO-MDSC, monocytic MDSC; NSCLC, non-small cell lung carcinoma; PMN-MDSC, polymorphonuclear MDSC; sILT3, soluble ILT3.

Figure 5.

Survival curves of NSCLC patient groups according to the proportion of MDSCs. NSCLC patients were divided into 2 groups based on the mean value + 2SD of healthy controls. (A) Survival of patients with elevated versus low PMN-MDSC levels (B) Survival of patients with elevated versus low MO-MDSC levels. MDSC, myeloid-derived suppressor cell; MO-MDSC, monocytic MDSC; NSCLC, non-small cell lung carcinoma; PMN-MDSC, polymorphonuclear MDSC.

Figure 6.

NSCLC patient survival based on ILT3 fractions of MDSC. (A) Survival of patients with an elevated percentage of ILT3^high PMN-MDSC versus patients with a low percentage of ILT3^high PMN-MDSCs. The survival curves were not significantly different. (B) No correlation was found between the level of total PMN-MDSCs and the percentage of ILT3^high PMN-MDSCs (Spearman rho; P = 0.38). For the curves in panels C and D, patients were divided based on the frequency of PMN-MDSC and the percentage of ILT3^high PMN-MDSCs. Cutoff values were the median value of all patients to create equally sized groups. (C) In patients with high levels of PMN-MDSC, the fraction of ILT3^high cells correlated significantly with overall survival. (D) In patients with low levels of PMN-MDSC, the percentage of ILT3^high PMN-MDSCs did not significantly contribute to overall survival. The curves were compared with a log-rank test, stratified for treatment arm. ILT3, immunoglobulin-like transcript 3; PBMC, peripheral blood mononuclear cells; MO-MDSC, monocytic myeloid-derived suppressor cell; PMN-MDSC, polymorphonuclear myeloid-derived suppressor cell.