Tumor necrosis factor alpha neutralization attenuates immune checkpoint inhibitor-induced activation of intermediate monocytes in synovial fluid mononuclear cells from patients with inflammatory arthritis

Abstract

Objective: During treatment with immune checkpoint inhibitors (ICI) such as the anti-PD-1 antibody pembrolizumab, half of patients with pre-existing inflammatory arthritis experience disease flares. The underlying immunological mechanisms have not been characterized. Here, we investigate the effect of pembrolizumab on cells involved in inflammation and destruction in the synovial joint and how immunosuppressive treatments affect the pembrolizumab-induced immune reactions.

Methods: We included synovial fluid mononuclear cells (SFMCs, n = 28) and peripheral blood mononuclear cells (PBMCs, n = 6) from patients with rheumatoid arthritis and peripheral spondyloarthritis and PBMCs from healthy controls (n = 6). Fibroblast-like synovial cells (FLSs) were grown from SFMCs. The in vitro effect of pembrolizumab was tested in SFMCs cultured for 48 h, FLS-PBMC co-cultures and in SFMCs cultured for 21 days (inflammatory osteoclastogenesis). Cells and supernatants were analyzed by ELISA, flow cytometry, and pro-inflammatory multiplex assay. Finally, the effect of the disease-modifying anti-rheumatic drugs (DMARDs) adalimumab (TNFα inhibitor), tocilizumab (IL-6R inhibitor), tofacitinib (JAK1/JAK3 inhibitor), and baricitinib (JAK1/JAK2 inhibitor) on pembrolizumab-induced immune reactions was tested.

Results: Pembrolizumab significantly increased monocyte chemoattractant protein-1 (MCP-1) production by arthritis SFMCs (P = 0.0031) but not by PBMCs from patients or healthy controls (P = 0.77 and P = 0.43). Pembrolizumab did not alter MMP-3 production in FLS-PBMC co-cultures (P = 0.76) or TRAP secretion in the inflammatory osteoclastogenesis model (P = 0.28). In SFMCs, pembrolizumab further increased the production of TNFα (P = 0.0110), IFNγ (P = 0.0125), IL-12p70 (P = 0.0014), IL-10 (P = 0.0100), IL-13 (P = 0.0044), IL-2 (P = 0.0066), and IL-4 (P = 0.0008) but did not change the production of IL-6 (P = 0.1938) and IL-1 (P = 0.1022). The SFMCs treated with pembrolizumab showed an increased frequency of intermediate monocytes (P = 0.044), and the MCP-1 production increased only within the intermediate monocyte subset (P = 0.028). Lastly, adalimumab, baricitinib, and tofacitinib treatment were able to attenuate the pembrolizumab-induced MCP-1 production (P = 0.0004, P = 0.033, and P = 0.025, respectively), while this was not seen with tocilizumab treatment (P = 0.75).

Conclusion: Pembrolizumab specifically activated intermediate monocytes and induced the production of several cytokines including TNFα but not IL-6. These findings indicate that flares in patients with pre-existing inflammatory arthritis involve monocyte activation and could be managed with TNFα neutralization.

Keywords: Arthritis; Cytokine; Immunotherapy; Monocyte.

Fig. 1

MCP-1 production following treatment with pembrolizumab. A–C MCP-1 production in SFMCs (n = 16, 3 SpA patients, 5 PsA patients, and 8 RA patients) and PBMCs (n = 6, 3 SpA patients and 3 RA patients) from patients and healthy controls (n = 6) cultured for 48 h untreated (UT) or treated with pembrolizumab (Pembro) or LPS. D–F Data were normalized to untreated cultures and expressed as ratios. Data is presented as median with interquartile range. Log-transformed ratios were analyzed with the paired Student’s t-test. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001

Fig. 2

MMP-3 and TRAP production following treatment with pembrolizumab. A MMP-3 production in the FLS-PBMC co-culture cultured for 48 h untreated (UT) or treated with pembrolizumab (Pembro) or LPS (n = 6, 3 SpA patients and 3 RA patients). B TRAP activity in SFMCs cultured for 21 days untreated (UT) or treated with pembrolizumab (Pembro) or LPS (n = 14, 7 SpA patients and 7 RA patients). C, D Data were normalized to untreated cultures and expressed as ratios. Data is presented as median with interquartile range. Log-transformed ratios were analyzed with the paired Student’s t-test. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001, ****P-value < 0.0001

Fig. 3

Distribution of the monocyte subsets among SFMCs following treatment with pembrolizumab (n = 4, 2 patients with SpA and 2 patients with PsA). A Representative CD14 vs. CD16 dot plot of monocyte subsets (CD14+CD16−: classical monocytes; CD14+CD16+: intermediate monocytes) in untreated cultures (UT), pembrolizumab treated cultures (Pembro), and LPS-treated cultures (LPS). B Upper panel: Frequency of classical monocytes in each culture. Lower panel: Data were normalized to untreated cultures and expressed as ratios. C Upper panel: Frequencies of intermediate monocytes in each culture. Lower panel: Data were normalized to untreated cultures and expressed as ratios. Data is presented as median with interquartile range. Log-transformed ratios were analyzed with the paired Student’s t-test. *P-value < 0.05, **P-value < 0.01

Fig. 4

MCP-1 production in the monocyte subsets following treatment with pembrolizumab in SFMCs from arthritis patients (n = 4, 2 SpA patients and 2 PsA patients). A Representative dotplots of MCP-1 production in classical monocytes. B Representative dot plots of MCP-1 production in intermediate monocytes. C Upper panel: Frequency of MCP-1+ cells in classical monocytes in each culture. Lower panel: Data were normalized and expressed as ratios. D Upper panel: Frequency of MCP-1+ cells in intermediate monocytes in each culture. Lower panel: Data were normalized and expressed as ratios. All data are expressed as median with interquartile range. Log-transformed ratios were analyzed with the paired Student’s t-test. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. UT, untreated; Pembro, pembrolizumab

Fig. 5

Production of different pro-inflammatory cytokines by SFMC cultures following treatment with pembrolizumab (n = 11). Production of TNF, IL-6, IFN-γ, IL-12p70, IL-10, IL-1β, IL-2, IL-13, and IL-4 in SFMCs from arthritis patients cultured for 48 h untreated (UT) or treated with pembrolizumab (Pembro) (8 RA patients, 2 PsA patients, and 1 SpA patient). Data were normalized to untreated cultures and expressed as ratios. Cytokine measurements were excluded if levels were above or below the detection range. All data are expressed as median with interquartile range. Log-transformed ratios were analyzed with the paired Student’s t-test. *P-value< 0.05, **P-value < 0.01, ***P-value< 0.001

Fig. 6

MCP-1 production in SFMC cultures treated with pembrolizumab combined with different DMARDs. A MCP-1 production in SFMCs cultured for 48 h untreated (UT) or treated with pembrolizumab (Pembro), pembrolizumab + adalimumab (Ada), pembrolizumab + tocilizumab (Toci) (n = 10, 2 RA patients, 4 PsA patients, and 4 SpA patients). A MCP-1 production in SFMCs cultured for 48 h untreated (UT) or treated with DMSO, pembrolizumab (Pembro), pembrolizumab + tofacitinib (Tofa), or pembrolizumab + baricitinib (Bari) (n = 7, 3 PsA patients and 4 SpA patients). C, D Data were normalized to untreated cultures and expressed as ratios. Data is expressed as median with interquartile range.. Log-transformed ratios were analyzed with the paired Student’s t-test. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. DMSO, dimethyl sulfoxide; Pembro, pembrolizumab; Ada, adalimumab; Toci, tocilizumab; Tofa, tofacitinib; Bari, baricitinib

Fig. 7

The main findings. Pembrolizumab inhibits the engagement of PD-1 with PD-L1. Removing this immunological brake in SFMC cultures resulted in differentiation of monocytes into intermediate monocytes. The intermediate monocytes showed an increased production of CCL-2 (MCP-1) and TNFα upon pembrolizumab stimulation. No increase was seen in IL-6 production. Inhibiting TNFα production with adalimumab decreased pembrolizumab-induced MCP-1 production. Blocking IL-6 secretion with tocilizumab did not decrease the pembrolizumab-induced immune reactions. Illustration made with BioRendor