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Impact of agonistic LAG‐3 antibody on proinflammatory cytokine secretion by PBMCs. (A) dcSSc (n = 8) PBMCs were prestimulated with CD3/CD28 for 24 hours in monocultures or (B) cocultured with allogeneic dcSSc fibroblasts. The cultures were treated with either an LAG‐3 Q22 agonistic antibody (LAG‐3 Ag) or an isotype control (LAG‐3 Isotype) for 48 hours. In monocultures, the agonistic LAG‐3 Ag significantly reduced the levels of IFNγ, IL‐1β, IL‐4, and TNFα. In cocultures, IFNγ, IL‐12p70, IL‐13, IL‐2, IL‐4, and TNFα were decreased significantly. In both setups, IL‐10 levels were significantly increased by the addition of agonistic LAG‐3 isotype–treated samples. Data are presented as mean with SD Q23 (* P < 0.05, ** P < 0.01). Ag, agonistic antibody; dcSSc, diffuse cutaneous systemic sclerosis; IFNγ, interferon γ; IL, interleukin; LAG‐3, lymphocyte Q24 activation gene 3; ns, not significant; NT, no treatment; PBMC, peripheral blood mononuclear cell; TNFα, tumor necrosis factor α (1425 pg/mL vs 1847 pg/mL; P = 0.001); compared to the control IgG, there was still a notable increase in IL‐10 production (8.33 pg/mL vs 6.10 pg/mL; P = 0.01). No significant changes were observed in the production of IL‐1β or IL‐6 (Figure ). Agonistic LAG‐3 antibodies suppress type I IFN production and reduce fibroblast matrix production. PBMCs from patients with dcSSc (n = 8) were stimulated with CD3/CD28 for 24 hours and cultured with or without allogeneic dcSSc fibroblasts for 48 hours. The cultures were then treated with either the LAG‐3 Ag or the isotype control (LAG‐3 Isotype). (C, D) The LAG‐3 Ag significantly reduced the production of bioactive IFNα/β in monocultures and cocultures, as measured in HEK‐Blue cells, compared to the LAG‐3 isotype control. (E) In cocultures, levels of <t>type</t> <t>1</t> procollagen and (F) fibronectin were significantly reduced by LAG‐3 Ag. Data are presented as mean with SD (* P < 0.05, ** P < 0.01). Ag, agonistic antibody; dcSSc, diffuse cutaneous systemic sclerosis; IFN, interferon; LAG‐3, lymphocyte activation gene 3; ns, not significant; NT, no treatment; PBMC, peripheral blood mononuclear cell.
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Impact of agonistic LAG‐3 antibody on proinflammatory cytokine secretion by PBMCs. (A) dcSSc (n = 8) PBMCs were prestimulated with CD3/CD28 for 24 hours in monocultures or (B) cocultured with allogeneic dcSSc fibroblasts. The cultures were treated with either an LAG‐3 Q22 agonistic antibody (LAG‐3 Ag) or an isotype control (LAG‐3 Isotype) for 48 hours. In monocultures, the agonistic LAG‐3 Ag significantly reduced the levels of IFNγ, IL‐1β, IL‐4, and TNFα. In cocultures, IFNγ, IL‐12p70, IL‐13, IL‐2, IL‐4, and TNFα were decreased significantly. In both setups, IL‐10 levels were significantly increased by the addition of agonistic LAG‐3 isotype–treated samples. Data are presented as mean with SD Q23 (* P < 0.05, ** P < 0.01). Ag, agonistic antibody; dcSSc, diffuse cutaneous systemic sclerosis; IFNγ, interferon γ; IL, interleukin; LAG‐3, lymphocyte Q24 activation gene 3; ns, not significant; NT, no treatment; PBMC, peripheral blood mononuclear cell; TNFα, tumor necrosis factor α (1425 pg/mL vs 1847 pg/mL; P = 0.001); compared to the control IgG, there was still a notable increase in IL‐10 production (8.33 pg/mL vs 6.10 pg/mL; P = 0.01). No significant changes were observed in the production of IL‐1β or IL‐6 (Figure ). Agonistic LAG‐3 antibodies suppress type I IFN production and reduce fibroblast matrix production. PBMCs from patients with dcSSc (n = 8) were stimulated with CD3/CD28 for 24 hours and cultured with or without allogeneic dcSSc fibroblasts for 48 hours. The cultures were then treated with either the LAG‐3 Ag or the isotype control (LAG‐3 Isotype). (C, D) The LAG‐3 Ag significantly reduced the production of bioactive IFNα/β in monocultures and cocultures, as measured in HEK‐Blue cells, compared to the LAG‐3 isotype control. (E) In cocultures, levels of type 1 procollagen and (F) fibronectin were significantly reduced by LAG‐3 Ag. Data are presented as mean with SD (* P < 0.05, ** P < 0.01). Ag, agonistic antibody; dcSSc, diffuse cutaneous systemic sclerosis; IFN, interferon; LAG‐3, lymphocyte activation gene 3; ns, not significant; NT, no treatment; PBMC, peripheral blood mononuclear cell.

Journal: ACR Open Rheumatology

Article Title: Lymphocyte Activation Gene 3 Regulation of Profibrotic Cytokines and Type I Collagen Production in Patients With Systemic Sclerosis

doi: 10.1002/acr2.70120

Figure Lengend Snippet: Impact of agonistic LAG‐3 antibody on proinflammatory cytokine secretion by PBMCs. (A) dcSSc (n = 8) PBMCs were prestimulated with CD3/CD28 for 24 hours in monocultures or (B) cocultured with allogeneic dcSSc fibroblasts. The cultures were treated with either an LAG‐3 Q22 agonistic antibody (LAG‐3 Ag) or an isotype control (LAG‐3 Isotype) for 48 hours. In monocultures, the agonistic LAG‐3 Ag significantly reduced the levels of IFNγ, IL‐1β, IL‐4, and TNFα. In cocultures, IFNγ, IL‐12p70, IL‐13, IL‐2, IL‐4, and TNFα were decreased significantly. In both setups, IL‐10 levels were significantly increased by the addition of agonistic LAG‐3 isotype–treated samples. Data are presented as mean with SD Q23 (* P < 0.05, ** P < 0.01). Ag, agonistic antibody; dcSSc, diffuse cutaneous systemic sclerosis; IFNγ, interferon γ; IL, interleukin; LAG‐3, lymphocyte Q24 activation gene 3; ns, not significant; NT, no treatment; PBMC, peripheral blood mononuclear cell; TNFα, tumor necrosis factor α (1425 pg/mL vs 1847 pg/mL; P = 0.001); compared to the control IgG, there was still a notable increase in IL‐10 production (8.33 pg/mL vs 6.10 pg/mL; P = 0.01). No significant changes were observed in the production of IL‐1β or IL‐6 (Figure ). Agonistic LAG‐3 antibodies suppress type I IFN production and reduce fibroblast matrix production. PBMCs from patients with dcSSc (n = 8) were stimulated with CD3/CD28 for 24 hours and cultured with or without allogeneic dcSSc fibroblasts for 48 hours. The cultures were then treated with either the LAG‐3 Ag or the isotype control (LAG‐3 Isotype). (C, D) The LAG‐3 Ag significantly reduced the production of bioactive IFNα/β in monocultures and cocultures, as measured in HEK‐Blue cells, compared to the LAG‐3 isotype control. (E) In cocultures, levels of type 1 procollagen and (F) fibronectin were significantly reduced by LAG‐3 Ag. Data are presented as mean with SD (* P < 0.05, ** P < 0.01). Ag, agonistic antibody; dcSSc, diffuse cutaneous systemic sclerosis; IFN, interferon; LAG‐3, lymphocyte activation gene 3; ns, not significant; NT, no treatment; PBMC, peripheral blood mononuclear cell.

Article Snippet: Quantification of sLAG‐3 (LAG‐3 Human Enzyme‐Linked Immunosorbent Assay [ELISA] kit # BMS2211; Invitrogen), type 1 procollagenα (Human Pro‐Collagen I alpha 1 DuoSet ELISA Cat.DY6220‐05; R&D Systems), and fibronectin (Human Fibronectin DuoSet ELISA Cat. DY1918‐05; R&D Systems) in the plasma and supernatants was performed according to the manufacturer's protocol.

Techniques: Control, Activation Assay, Cell Culture