The Frequency and Effect of Granulocytic Myeloid-Derived Suppressor Cells on Mycobacterial Survival in Patients With Tuberculosis: A Preliminary Report

Abstract

Introduction: Protective host responses in those exposed to or infected with tuberculosis (TB) is thought to require a delicate balance between pro-inflammatory and regulatory immune responses. Myeloid-derived suppressor cells (MDSCs), regulatory cells that dampen T-cell function, have been described in cancer and other infectious diseases but there are limited data on their role in TB.

Methods: Peripheral blood was obtained from patients with active pulmonary TB and participants with presumed latent TB infection (LTBI) from Cape Town, South Africa. MDSC frequency was ascertained by flow cytometry. Purified MDSCs were used to assess (i) their suppressive effect on T-cell proliferation using a Ki67 flow cytometric assay and (ii) their effect on mycobacterial containment by co-culturing with H37Rv-infected monocyte-derived macrophages and autologous pre-primed effector T-cells with or without MDSCs. Mycobacterial containment was measured by plating colony forming units (CFU).

Results: MDSCs (CD15⁺HLA-DR^-CD33⁺) had significantly higher median frequencies (IQR) in patients with active TB (n=10) versus LTBI (n= 10) [8.2% (6.8-10.7) versus 42.2% (27-56) respectively; p=0.001]. Compared to MDSC-depleted peripheral blood mononuclear and effector T cell populations, dilutions of purified MDSCs isolated from active TB patients suppressed T-cell proliferation by up to 72% (n=6; p=0.03) and significantly subverted effector T-cell-mediated containment of H37Rv in monocyte-derived macrophages (n=7; 0.6% versus 8.5%; p=0.02).

Conclusion: Collectively, these data suggest that circulating MDSCs are induced during active TB disease and can functionally suppress T-cell proliferation and subvert mycobacterial containment. These data may inform the design of vaccines and immunotherapeutic interventions against TB but further studies are required to understand the mechanisms underpinning the effects of MDSCs.

Keywords: MDSC; biomarkers; immunology; myeloid derived suppressor cells; tuberculosis.

Figure 1

Frequency of myeloid derived suppressor cells (MDSC) in the peripheral blood compartment of participants with presumed-latent infection and active TB. (A) Flow cytometry gating strategy. Cells were first gated on CD14^-CD33⁺ cells, and within this population HLA DR^low/-CD15⁺ cells were identified. (B) frequency of CD14^-CD33⁺HLA-DR^low/-CD15⁺ MDSCs before and after stimulation with PPD and PHA. p-value of <0.05 were considered significant (Mann-Whitney unpaired t-test). ***p-value <0.0001.

Figure 2

Functional capabilities of myeloid derived suppressor cells (MDSC) was tested. (A) Illustrates the flow cytometry gating strategy. (B) Outlines suppression of PPD-driven T-cell proliferation by MDSCs at different ratios. Effector T-cells were co-cultured with MDSCs at a ratio of 4:1, 2:1 and 1:1, respectively. The percentage proliferation was measured using Ki67 flow cytometry staining. (C) Mycobacterial containment assays using peripheral blood cells from active TB patients to determine the effect of MDSCs from TB patients on mycobacterial survival. Briefly, monocyte-derived macrophages (MDMs) were infected with H37Rv and then co-cultured with PPD pre-primed effector T-cells with or without MDSCs for 24 hours. The impact of MDSCs on mycobacterial containment was assessed by plating surviving intracellular bacteria, and the magnitude of mycobacterial containment is expressed as % of the reference control (MDM only) for each experimental condition. *p-value of <0.05 were considered significant (Wilcoxon matched pairs test).