Synovial T cell hyporesponsiveness to myeloid dendritic cells is reversed by preventing PD-1/PD-L1 interactions

Abstract

Introduction: The aim of this study was to investigate PD-1/PD-L1 involvement in the hyporesponsiveness of rheumatoid arthritis (RA) synovial fluid (SF) CD4 T cells upon stimulation by thymic stromal lymphopoietin (TSLP)-primed CD1c myeloid dendritic cells (mDCs).

Methods: Expression of PD-1 on naïve (Tn), central memory (Tcm) and effector memory (Tem) CD4 T cell subsets was assessed by flow cytometry. PD-L1 expression and its regulation upon TSLP stimulation of mDCs from peripheral blood (PB) and SF of RA patients were investigated by quantitative RT-PCR and flow cytometry. The involvement of PD-1/PD-L1 interactions in SF T cell hyporesponsiveness upon (TSLP-primed) mDC activation was determined by cell culture in the presence of PD-1 blocking antibodies, with or without interleukin 7 (IL-7) as a recognized suppressor of PD-1 expression.

Results: PD-1 expression was increased on CD4 T cells derived from SF compared with PB of RA patients. TSLP increased PD-L1 mRNA expression in both PB and SF mDCs. PD-L1 protein expression was increased on SF mDCs compared with PB mDCs and was associated with T cell hyporesponsiveness. Blockade of PD-1, as well as IL-7 stimulation, during cocultures of memory T cells and (TSLP-primed) mDCs from RA patients significantly recovered T cell proliferation.

Conclusion: SF T cell hyporesponsiveness upon (TSLP-primed) mDC stimulation in RA joints is partially dependent on PD-1/PD-L1 interactions, as PD-1 and PD-L1 are both highly expressed on SF T cells and mDCs, respectively, and inhibiting PD-1 availability restores T cell proliferation. The potential of IL-7 to robustly reverse this hyporesponsiveness suggests that such proinflammatory cytokines in RA joints strongly contribute to memory T cell activation.

Figure 1

CD4 T cells derived from synovial fluid of rheumatoid arthritis patients are hyporesponsive upon (TSLP-primed) myeloid dendritic cell stimulation in contrast to peripheral blood-derived CD4 T cells. Thymic stromal lymphopoietin (TSLP)-primed myeloid dendritic cells (mDCs) from peripheral blood (PB) as well as both mDCs and TSLP-primed mDCs from synovial fluid (SF) of rheumatoid arthritis (RA) patients strongly activate PB-derived CD4 T cells, whereas SF-derived CD4 T cells are hyporesponsive upon (TSLP-primed) mDC activation. (A) mDC:T cell ratio 1:5, paired analysis of CD4 T cells and mDCs of a representative donor are shown. (B) mDC:T cell ratio 1:10 derived from PB of five RA patients. (C) mDC:T cell ratio 1:10 derived from SF of five RA patients. *P < 0.05 (statistically significant difference). CPM, counts per minute.

Figure 2

Memory CD4 T cells, in particular synovial fluid–derived T cell subsets, express increased PD-1 levels. (A) CD4 T cells derived from synovial fluid (SF) of rheumatoid arthritis (RA) patients hardly contain any naïve T (Tn) cells and (B) express increased PD-1 levels. Representative histogram of PD-1 expression on PB and SF CD4 T cells of one donor is shown. Dashed line represents autofluorescence. (C) PD-1 expression is increased on central memory (Tcm) and effector memory (Tem) T cells in comparison with naïve T cells (Tn cells) from peripheral blood (PB; n = 9, both P < 0.01) and SF (n = 7, both P < 0.05). PD-1 expression was strongly upregulated on all CD4 T cell subsets derived from SF versus PB of RA patients. *P < 0.05, **P < 0.01 and ***P < 0.001 (statistically significant differences). MFI, mean fluorescence intensity; PD-1, Programmed death 1.

Figure 3

PD-L1 is upregulated by TSLP and highly expressed on synovial CD1c myeloid dendritic cells. (A) Thymic stromal lymphopoietin (TSLP) stimulation of myeloid dendritic cells (mDCs) significantly upregulates PD-L1 mRNA expression (peripheral blood (PB)–derived and synovial fluid (SF)–derived mDCs of rheumatoid arthritis (RA) patients; circles represent unstimulated mDCs, squares represent TSLP-stimulated mDCs, n = 5) and (B) PD-L1 protein expression. Representative histrogram of PD-L1 expression on TSLP-stimulated mDCs (filled dark grey area) and unstimulated mDCs (transparent) from PB of one donor is shown. Dashed line represents autofluorescence. (C, D) PD-L1 is expressed to a higher extent on CD1c mDCs derived from SF (n = 8) compared with PB (n = 9) of RA patients. (C) Representative histogram of PD-L1 expression on PB mDCs (filled light grey area; solid line) and SF mDCs (transparent area; solid line) of one donor is shown. Dashed line represents autofluorescence for PB mDCs (filled light grey area) and SF mDCs (transparent). (D) Mean percentages and MFI of PD-L1 expression of PB and SF mDCs (n = 9 and n = 8, respectively). *P < 0.05, **P < 0.01, and ***P < 0.001 (statistically significant differences). MFI, Mean fluorescence intensity; PD-L1, Programmed death ligand 1.

Figure 4

Hyporesponsiveness of memory CD4 T cells from rheumatoid arthritis patients upon TSLP-primed myeloid dendritic cell stimulation is at least partially reversed by PD-1 blockade. (A) Blockade of PD-1/PD-L1 interactions by anti-PD-1 antibody (1 μg/ml) in (TSLP-primed) myeloid dendritic cell (mDC) driven memory CD4 T cell activation from peripheral blood (PB) (n = 4, filled symbols) and synovial fluid (SF) (n = 4, open symbols) partially restores the T cell proliferative capacity. (B) Memory CD4 T cell hyporesponsiveness from SF (n = 4) is strongly reversed by interleukin-7 (IL-7), a cytokine that largely downregulates PD-1. Additional PD-1 blockade by anti-PD-1 antibody further reversed the downregulated proliferation. Statistical differences between these conditions were assessed using the paired-samples t-test. *P < 0.05 (statistically significant difference). PD-1, Programmed death 1; PD-L1, Programmed death ligand 1; TSLP, Thymic stromal lymphopoietin.