Identification of enhanced IFN-γ signaling in polyarticular juvenile idiopathic arthritis with mass cytometry
Allison A. Throm, Halima Moncrieffe, Amir B. Orandi, Jeanette T. Pingel, Theresa L. Geurs, Hannah L. Miller, Allyssa L. Daugherty, Olga N. Malkova, Daniel J. Lovell, Susan D. Thompson, Alexei A. Grom, Megan A. Cooper, Stephen T. Oh, Anthony R. French
Allison A. Throm, Halima Moncrieffe, Amir B. Orandi, Jeanette T. Pingel, Theresa L. Geurs, Hannah L. Miller, Allyssa L. Daugherty, Olga N. Malkova, Daniel J. Lovell, Susan D. Thompson, Alexei A. Grom, Megan A. Cooper, Stephen T. Oh, Anthony R. French
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Research Article Immunology Inflammation

Identification of enhanced IFN-γ signaling in polyarticular juvenile idiopathic arthritis with mass cytometry

Abstract

Polyarticular juvenile idiopathic arthritis (JIA) is among the most challenging of the JIA subtypes to treat. Even with current biologic therapies, the disease remains difficult to control in a substantial subset of patients, highlighting the need for new therapies. The aim of this study was to use the high dimensionality afforded by mass cytometry with phospho-specific antibodies to delineate signaling abnormalities in immune cells from treatment-naive polyarticular JIA patients. Peripheral blood mononuclear cells were isolated from 17 treatment-naive polyarticular JIA patients, 10 of the patients after achieving clinical remission, and 19 healthy controls. Samples were stimulated for 15 minutes with IL-6 or IFN-γ and analyzed by mass cytometry. Following IFN-γ stimulation, increased STAT1 and/or STAT3 phosphorylation was observed in subsets of CD4 T cells and classical monocytes from treatment-naive patients. The enhanced IFN-γ signaling was associated with increased expression of JAK1 and SOCS1 in CD4 T cells. Furthermore, substantial heterogeneity in surface marker expression was observed among the subsets of CD4 T cells and classical monocytes with increased IFN-γ responsiveness. The identification of enhanced IFN-γ signaling in CD4 T cells and classical monocytes from treatment-naive polyarticular JIA patients provides mechanistic support for investigations into therapies that attenuate IFN-γ signaling in this disease.

Authors

Allison A. Throm, Halima Moncrieffe, Amir B. Orandi, Jeanette T. Pingel, Theresa L. Geurs, Hannah L. Miller, Allyssa L. Daugherty, Olga N. Malkova, Daniel J. Lovell, Susan D. Thompson, Alexei A. Grom, Megan A. Cooper, Stephen T. Oh, Anthony R. French

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Figure 2

Classical monocyte and CD4 T cell subsets respond more strongly to IFN-γ stimulation in treatment-naive patients than controls with enhanced STAT1 and STAT3 phosphorylation.

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Classical monocyte and CD4 T cell subsets respond more strongly to IFN-γ...
(A) Hierarchical clustering for IFN-γ–stimulated treatment-naive patient versus control PBMCs. Nonstratifying clusters are light blue for FDR < 0.05, p-STAT1 stratifying (different between patients and controls for a signaling molecule) clusters are purple, p-STAT3 stratifying clusters are orange, and clusters stratifying for both p-STAT1 and p-STAT3 are red. Asterisks indicate clusters visualized in C. (B) Heatmap depicting surface marker intensities for stratifying and nonstratifying classical monocyte and CD4 T cell clusters. (C) Arcsinh-transformed median phosphoprotein intensities for largest stratifying CD4 T cell and classical monocyte clusters (classical monocyte p-STAT1: t = 1.656, df = 32, P = 0.108; classical monocyte p-STAT3: t = 2.72, df = 32, P = 0.011; CD4 T cell p-STAT1: t = 3.04, df = 21, P = 0.006; CD4 T cell p-STAT3: t = 3.40, df = 32, P = 0.002). White circles denote treatment-naive patients, and black squares denote controls. Data represent mean ± SEM. n = 15 IFN-γ–stimulated treatment-naive patients, patients 4 and 5 in Table 1 did not have enough cells for an IFN-γ–stimulated sample and were excluded here; n = 19 controls. Statistical significance was determined with 2-tailed Student’s t test with Benjamini-Hochberg multiple hypothesis correction to account for 4 comparisons.