TNF-α and TGF-β counter-regulate PD-L1 expression on monocytes in systemic lupus erythematosus

Abstract

Monocytes in patients with systemic lupus erythematosus (SLE) are hyperstimulatory for T lymphocytes. We previously found that the normal program for expression of a negative costimulatory molecule programmed death ligand-1 (PD-L1) is defective in SLE patients with active disease. Here, we investigated the mechanism for PD-L1 dysregulation on lupus monocytes. We found that PD-L1 expression on cultured SLE monocytes correlated with TNF-α expression. Exogenous TNF-α restored PD-L1 expression on lupus monocytes. Conversely, TGF-β inversely correlated with PD-L1 in SLE and suppressed expression of PD-L1 on healthy monocytes. Therefore, PD-L1 expression in monocytes is regulated by opposing actions of TNF-α and TGF-β. As PD-L1 functions to fine tune lymphocyte activation, dysregulation of cytokines resulting in reduced expression could lead to loss of peripheral T cell tolerance.

Figure 1. PD-L1 levels correlated with TNF-α in remission and with TGF-β in patients with active disease.

(A) and (B) Levels of TNF-α and TGF-β detected in supernatants from PBMC cultured overnight without stimulation. Horizontal lines represent mean values. Cytokine levels between healthy controls and SLE patient groups were compared using the Wilcoxon-Mann-Whitney test. Significance was assigned where p<0.05; N.S., not significant. (C) PD-L1 protein levels on monocytes in the same culture was assayed by flow cytometry; TNF-α positively correlated with PD-L1 expression on monocytes in SLE patients in remission (dashed line). (D) TGF-β negatively correlated with PD-L1 protein in SLE patients with active disease (solid line). Correlations were analyzed by the Spearman's rank correlation test.

Figure 2. PD-L1 surface protein on SLE APCs is differentially regulated by TNF-α and TGF-β.

(A) Control PBMC were treated for 24 hours. Expression of PD-L1 on CD14^highCD11c⁺ monocytes was assayed by flow cytometry, and is expressed as fold induction over untreated cells. (B) Induction of PD-L1 on monocytes in SLE PBMC treated for 24 hours or cultured in media alone. Induction of PD-L1 protein by cytokine treatments was tested for significance by the Wilcoxon signed-rank test. Results were derived from multiple independent experiments.

Figure 3. Expression of PD-L1 on isolated CD14⁺ cells is not entirely dependent on lymphocytes.

(A) and (C) Representative histograms demonstrate PD-L1 induction on total PBMC or isolated CD14⁺ cells from a control subject and an active SLE patient. Protein expression was assayed by flow cytometry after culturing cells for 24 hours without stimulation. (B) and (D) PD-L1 expression on monocytes gated from total PBMC compared to isolated monocytes and myeloid DCs in eight healthy controls and eight SLE patients. (E) and (F) Fold induction of PD-L1 MFI on isolated CD14⁺ SLE monocytes treated with TNF-α or with anti-TGF-β mAb. (G) and (H) Fold induction of PD-L1 MFI on isolated CD14⁺ monocytes from healthy donors treated with anti-TNF-α mAb or with recombinant TGF-β. Induction was quantified by dividing the MFI of cytokine-treated samples by that of untreated cells, and tested for significance by the Wilcoxon signed-rank test.

Figure 4. Monocytes produce TNF-α to induce expression of PD-L1.

(A) TNF-α was assayed in supernatants from isolated CD14⁺ monocytes cultured overnight without stimulation. Horizontal lines represent mean values. (B) PD-L1 protein levels on monocytes in the same culture was assayed by flow cytometry; TNF-α positively correlated with PD-L1 expression on monocytes in SLE patients in remission. (C) TGF-β protein levels in monocyte supernatants. (D) Correlation between TGF-β and PD-L1 protein levels on monocytes. Cytokine levels between healthy controls and SLE patient groups were compared using the Wilcoxon-Mann-Whitney test. Correlations were determined by the Spearman's rank correlation test.

Figure 5. PD-L1 mRNA is induced by TNF-α and suppressed by TGF-β.

Total PBMC were cultured for 24 hours. PD-L1 mRNA expression was assayed by RT-qPCR for each subject in triplicate. (A) Relative PD-L1 mRNA expression in controls and lupus patients was represented by the scatter plot. Horizontal lines represent medians. Baseline mRNA expression was set at 5×10⁻³. Expression of PD-L1 mRNA in control subjects and SLE patients were compared using the Wilcoxon-Mann-Whitney test. P>0.05 between all groups. (B) PD-L1 mRNA in SLE PBMC treated with recombinant TNF-α. (C) and (D) PD-L1 mRNA levels in healthy donor PBMC treated with anti-TNF-α or TGF-β. Induction of PD-L1 mRNA by cytokines was tested for significance by the Wilcoxon signed-rank test.