Myeloid-Derived Suppressor Cells in Lung Transplantation

Abstract

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immune cells from the myeloid lineage. MDSCs expand in pathological situations, such as chronic infection, cancer, autoimmunity, and allograft rejection. As chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplantation (LTx), MDSCs may play a role in its pathophysiology. We assessed phenotype and frequency of MDSCs in peripheral blood from lung transplant recipients and its relationship to post-transplant complications and immunosuppression. Granulocytic (G)-MDSC were identified and quantified by flow cytometry of blood from 4 control subjects and 20 lung transplant patients (stable n = 6, infection n = 5; CLAD n = 9). G-MDSC functionality was assessed in vitro by their capability to block CD4 and CD8 T cell proliferation. More G-MDSC could be assessed using EDTA tubes compared to heparin tubes (p = 0.004). G-MDSC were increased in stable lung transplant recipients vs. non-transplant controls (52.1% vs. 9.4%; p = 0.0095). The infection or CLAD groups had lower G-MDSCs vs. stable recipients (28.2%p = 0.041 and 33.0%; p = 0.088, respectively), but were not different among CLAD phenotypes. G-MDSC tended to correlate with cyclosporine A and tacrolimus levels (r² = 0.18; r² = 0.17). CD4 and CD8 cells proliferation decreased by 50 and 80% if co-cultured with MDSCs (1:6 and 1:2 MDSC:T-cell ratio, respectively). In conclusion, circulating MDSCs are measurable, functional and have a G-MDSC phenotype in lung transplant patients. Their frequency is increased in stable patients, decreased during post-transplant complications, and related to level of immunosuppression. This study may pave the way for further investigations of MDSC in the context of lung transplantation.

Keywords: allograft; blood; chronic rejection; immunosuppression; infection; lung transplantation; myeloid-derived suppressor cells; phenotypes.

Figure 1

Gating Strategy to determine MDSC phenotype. (A) The low-density fraction of PBMC was stained with specific markers to differentiate between G-MDSCs (CD66b/CD33) and M-MDSCs (HLA-DR/CD14). (B) Different coatings of blood tubes (EDTA vs. Heparin) affect the MDSC cell numbers. (C) Exemplary FACS plots of the healthy controls and different LTx patient groups.

Figure 2

G-MDSC percentages measured in blood of lung transplant recipients and healthy controls. (A) the effect of LiHe vs. EDTA tubes on G-MDSC percentages; (B) G-MDSC in healthy controls and lung transplant recipients who were stable, had an infection or were diagnosed with CLAD; (C) CLAD sub-phenotypes in BOS and RAS. (D,E) G-MDSC correlated with CNI level of the patients.

Figure 3

G-MDSCs isolated from lung transplant patients functionally supress T cell proliferation. The suppressive effect of CD66b⁺-MACS-isolated MDSCs (isolated from lung transplant recipients; 1 with CLAD and 1 stable) on CFSE labeled T cell CD4⁺ (green) and CD8⁺ (purple) proliferation. (A) Different ratios of MDSC vs. T cells (1:6 and 1:2) were assessed and compared with T cell proliferation without MDSCs. P0 represents undivided cells, P1 cells divided 1 time; P2 cells divided twice and so on. T cell proliferation ratio is portion of divided cells over all cells. The bar graphs represent the proliferation index compared to control conditions (n = 2).