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P014 Tofacitinib and a selective Janus kinase 1 inhibitor are equally potent in suppressing human macrophage function and T-cell proliferation

L. De Vries*1, 2, M. Wildenberg2, V. Ludbrook3, G. D’Haens1, 
W. De Jonge2

1Academic Medical Centre (AMC), Gastroenterology & Hepatology, Amsterdam, Netherlands, 2Academic Medical Centre (AMC), Tytgat Institute for Liver and Intestinal Research, Amsterdam, Netherlands, 3GlaxoSmithKline, ImmunoInflammation Therapy Area, Stevenage, United Kingdom


Nonselective Janus kinase (JAK) inhibitors such as tofacitinib have shown efficacy in treatment of ulcerative colitis. Tofacitinib inhibits JAK1, JAK2, JAK3, and TYK2. Side effects observed in patients (eg, neutropenia, anaemia) are attributed to JAK2 signalling. These side effects, have led to the development of selective JAK inhibitors. It is unclear whether selective JAK1 or JAK3 inhibition is sufficient in suppressing inflammatory responses. We aimed to investigate the potency of a selective JAK1 inhibitor (JAK1i, GSK2586186), a JAK3 inhibitor (JAK3i, GSK2864192A), and tofacitinib (CP-690,550-10, Pfizer) to supress innate and adaptive immune responses in vitro.


Peripheral blood mononuclear cells (PBMCs), monocytes, and lymphocytes were isolated from buffy coats using Ficoll and Percoll separation. CD14+ monocytes (n = 6) were isolated with CD14+ micro beads and stimulated with LPS (100 ng/ml) and IFNγ (10 ng/ml) for 6 hours in presence or absence of JAK1i, JAK3i, or tofacitinib (10-1000 nM). Cytokine and chemokine production by monocytes was measured in the supernatant by ELISA. Lymphocytes (n = 3) were stimulated with anti-CD3/CD28 beads in the presence or absence of JAK1i, JAK3i, or tofacitinib in a concentration ranging from 10 to 10.000 nM. Cell viability was assessed using an MTS colorimetric assay. In addition, JAK1i, JAK3i, and tofacitinib (10-10.000 nM) were added in a mixed lymphocyte reaction (MLR), in which PBMCs of 2 donors are mixed in a 1:1 ratio and cultured for 6 days. In both experiments, T-cell proliferation was measured with a Tritium proliferation assay.


In human CD14+ monocyte-derived macrophages, JAK1i and tofacitinib, but not JAK3i, decreased CXCL10 secretion at 1000 nM (resp. p = 0.021, p = 0.021, p = 0.416), whereas TNFα, and IL6 secretion was unaffected by all inhibitors. When assessing the effect of tofacitinib, JAK1i, and JAK3i on T-cell proliferation using anti-CD3/CD28 beads, JAK1i, and tofacitinib equally inhibited T-cell proliferation at 1000 nM (resp. p = 0.010 and p = 0.006). JAK3i inhibited T-cell proliferation at 4000 nM (p = 0.004). At these doses, T-cell viability was not affected. In a MLR, JAK1i, and tofacitinib inhibited T-cell proliferation at 100 nM (both p = 0.000). JAK3i to inhibited T-cell proliferation at 2000 nM (p = 0.022).


In vitro, JAK1i and tofacitinib, but not JAK3i, inhibit CXCL10 secretion produced by IFNγ/LPS triggered human monocyte-derived macrophages and proliferation of human T-cells in a dose-dependent manner. JAK3i inhibits T-cell proliferation at a higher dose, without affecting T-cell viability.