Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription.

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Presentation transcript:

Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of- function mutation Katja G. Weinacht, MD, PhD, Louis-Marie Charbonnier, PhD, Fayhan Alroqi, MD, Ashley Plant, MD, Qi Qiao, PhD, Hao Wu, PhD, Clement Ma, PhD, Troy R. Torgerson, MD, PhD, Sergio D. Rosenzweig, MD, PhD, Thomas A. Fleisher, MD, Luigi D. Notarangelo, MD, Imelda C. Hanson, MD, Lisa R. Forbes, MD, Talal A. Chatila, MD, MSc Journal of Allergy and Clinical Immunology Volume 139, Issue 5, Pages 1629-1640.e2 (May 2017) DOI: 10.1016/j.jaci.2016.11.022 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Journal of Allergy and Clinical Immunology 2017 139, 1629-1640 Journal of Allergy and Clinical Immunology 2017 139, 1629-1640.e2DOI: (10.1016/j.jaci.2016.11.022) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Hemoglobin and platelet count in response to pharmacotherapy. The graph depicts serum hemoglobin concentration in grams per deciliter (gray curve) and platelet count in 103 cells per microliter (black curve) in response to changes in steroid dose, as well as pharmacotherapy with ATG, the IL-1 receptor antagonist anakinra, cyclosporine, and ruxolitinib. Timing of packed red blood cell transfusions is indicated in dark gray. The prednisone dose is expressed in milligrams per kilogram body weight. Journal of Allergy and Clinical Immunology 2017 139, 1629-1640.e2DOI: (10.1016/j.jaci.2016.11.022) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 STAT1E545K mutation leads to hyperphosphorylation GOF. A, Sanger sequencing revealed a monoallelic c.1633G>A substitution in STAT1. B, Structural alignment between STAT1 structure and modeled E545K mutant structure (white). WT domains are colored (SH2 in green, dimerization domain in cyan, and IFN-γ peptide in orange) to highlight their position relative to residue 545 (WT structure in yellow and mutant in magenta). C, Total STAT1 expression in CD4+ T cells determined by means of flow cytometry in patient 1 and a control subject. D, Phospho-STAT1 (P-STAT1) expression in CD4+ T cells stimulated with IFN-β (20 ng/mL) and IFN-γ (20 ng/mL) in the patient and a control subject (top) and dose-response curve with increasing interferon concentrations (bottom). E, Dephosphorylation kinetics of phospho-STAT1 in response to deprivation of IFN-β and IFN-γ in CD4+ T cells represented as absolute mean fluorescence intensity (MFI; top) and normalized to maximum expression before deprivation (bottom). ***P < .001, 2-way ANOVA. Journal of Allergy and Clinical Immunology 2017 139, 1629-1640.e2DOI: (10.1016/j.jaci.2016.11.022) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 STAT1E545K GOF mutation results in exacerbated TH1/TC1 and TFH cell responses and impaired TH17/cytotoxic T type 17 (Tc17) immunity. A, Representative flow cytometric analyses of IL-17 and IFN-γ secretion by peripheral CD4+ and CD8+ T cells from the STAT1E545K patient and control subjects. B, Dot plots represent frequencies of IFN-γ– and IL-17–producing CD4+ and CD8+ T cells in each group (n = 8-9 individual control subjects and the STAT1E545K patient at 3 different time points). C, CXCR5 and PD1 expression in CD4+ T cells from patient 1 (STAT1E545K) and patient 2 (STAT1T385M) compared with healthy control subjects. D, CXCR3 and CCR6 expression in CD4+CXCR5+PD1+ T cells from patients 1 and 2 and healthy control subjects (gates shown above). *P < .05 and ***P < .001. Journal of Allergy and Clinical Immunology 2017 139, 1629-1640.e2DOI: (10.1016/j.jaci.2016.11.022) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Different JAK inhibitors variably inhibit STAT1 and STAT3 phosphorylation in vitro. A, Phospho-STAT1 (P-STAT1) expression on IFN-β stimulation in CD4+ T cells from a control subject and the STAT1E545K patient treated in vitro with 10 and 100 nmol/L concentrations of ruxolitinib (red curve) and tofacitinib (blue curve) or vehicle (dimethyl sulfoxide, black curve). Plain gray corresponds to unstimulated cells. B, Phospho-STAT1 mean fluorescence intensity (MFI) expressed as percentage of maximum vehicle-treated control CD4+ T cells shown in Fig 4, A. C, Phospho-STAT3 expression on IL-21 stimulation in CD4+ T cells from a control subject and the STAT1E545K patient treated in vitro with 10 and 100 nmol/L concentrations of ruxolitinib (red curve) and tofacitinib (blue curve) or vehicle. Plain gray corresponds to unstimulated cells. D, Phospho-STAT3 mean fluorescence intensity (MFI) expressed as a percentage of maximum vehicle-treated control CD4+ T cells shown in Fig 4, C. **P < .001 and ***P < .0001, 2-way ANOVA with posttest analysis. Journal of Allergy and Clinical Immunology 2017 139, 1629-1640.e2DOI: (10.1016/j.jaci.2016.11.022) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 In vivo ruxolitinib treatment differentially controls STAT1 phosphorylation. A, Expression of phospho-STAT1 (P-STAT1) in CD4+ T cells of the STAT1E545K patient and control subject before and after in vivo therapy with ruxolitinib after IFN-β stimulation. B, Histogram represents phospho-STAT1 mean fluorescence intensity (MFI) normalized to expression in control CD4+ T cells after IFN-β or IFN-γ stimulation before and after in vivo therapy with ruxolitinib. C, Expression of phospho-STAT3 in CD4+ T cells of the STAT1E545K patient and control subject before and after in vivo therapy with ruxolitinib after IL-21 stimulation. D, Histogram represents phospho-STAT3 MFI normalized to expression in control CD4+ T cells after IL-21 stimulation before and after in vivo therapy with ruxolitinib. *P < .05 and ***P < .001. Journal of Allergy and Clinical Immunology 2017 139, 1629-1640.e2DOI: (10.1016/j.jaci.2016.11.022) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Ruxolitinib treatment normalizes amplified TH1 and strengthens impaired TH17 responses. A, Expression of IFN-γ and IL-17 in CD4+ T cells of patient 1 before and after in vivo therapy with ruxolitinib at target dose after 3 to 4 months and greater than 12 months, respectively. B, Histograms represent frequencies of IFN-γ– and IL-17–producing CD4+ T cells shown in panel Fig 6, A. **P < .01 and ***P < .001, unpaired 2-tailed Student t test. C, Expression of IFN-γ and IL-17 in naive CD4+ T cells from a control subject and the STAT1E545K patient cultured under TH0, TH1, and TH17 conditions before and after initiation of ruxolitinib treatment. D, Histograms represent frequency of IFN-γ– and IL-17–producing CD4+ T cells under TH0, TH1, and TH17 conditions, respectively. Pretreatment samples were obtained at least 6 months after ATG and rituximab treatment. Posttreatment samples were obtained after at least 12 months of ruxolitinib treatment at target dose. One representative experiment of 2 is shown. **P < .01 and ***P < .001, unpaired 2-tailed Student t test (Fig 6, B) or 2-way ANOVA (Fig 6, D) with posttest analysis. Journal of Allergy and Clinical Immunology 2017 139, 1629-1640.e2DOI: (10.1016/j.jaci.2016.11.022) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Ruxolitinib treatment corrects the exacerbated TFH response in the STAT1E545K patient. A, CXCR5 and PD1 expression in CD4+ T cells in the STAT1E545K patient before and after treatment with ruxolitinib at target dose for 3 months compared with control subjects. B, Histograms represent frequencies of TFH cells in the STAT1E545K patient before and after treatment compared with control subjects. C, CXCR5 and ICOS expression in in vitro–differentiated TFH-like cells from the patient and control subjects stimulated with TGF-β1, IL-12, and IL-23 in the presence or absence of ruxolitinib. D, Histograms represent frequencies of in vitro–differentiated TFH cells with and without ruxolitinib in the patient and control subjects. E, ICOS mean fluorescence intensity (MFI) in in vitro–differentiated TFH cells with and without ruxolitinib in the patient and control subjects. **P < .01 and ***P < .001, 1-way (Fig 7, B, D, and E) and 2-way (Fig 7, D and E) ANOVA with posttest analysis. Journal of Allergy and Clinical Immunology 2017 139, 1629-1640.e2DOI: (10.1016/j.jaci.2016.11.022) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Hemoglobin and platelet count in response to pharmacotherapy. Graph depicts absolute reticulocyte count in 103 cells per microliter (gray curve) and absolute neutrophil count in cells per microliter (black curve) in response to changes in steroid dose, as well as therapy with ATG, the IL-1 receptor antagonist anakinra, cyclosporine, and ruxolitinib. Timing of packed red blood cell transfusions is indicated in dark gray. The prednisone dose is expressed in milligrams per kilogram body weight. Journal of Allergy and Clinical Immunology 2017 139, 1629-1640.e2DOI: (10.1016/j.jaci.2016.11.022) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 STAT1T385M mutation in patient 2 leads to hyperphosphorylation GOF. A, Phospho-STAT1 (P-STAT1) expression in CD4+ T cells stimulated with IFN-β and IFN-γ in the STAT1T385M patient (patient 2) and control subjects. B, Dose-response curve of STAT1 phosphorylation induced with IFN-β and IFN-γ in CD4+ T cells of patient 2 and control subjects. C, Dephosphorylation kinetics of phospho-STAT1 in response to deprivation of IFN-β and IFN-γ in CD4+ T cells represented as absolute mean fluorescence intensity (MFI; top) and normalized to maximum expression before deprivation (bottom). D, Phospho-STAT1 expression on IFN-β stimulation in CD4+ T cells of patient 2 and control subject treated in vitro with ruxolitinib (red curve) and tofacitinib (blue curve) or vehicle (dimethyl sulfoxide, black curve). Plain gray corresponds to unstimulated cells. E, Phospho-STAT1 and phospho-STAT3 MFI expressed as percentage of maximum vehicle-treated control CD4+ T cells shown in Fig E2, D, in response to increasing concentrations of ruxolitinib (red curve) and tofacitinib (blue curve). Journal of Allergy and Clinical Immunology 2017 139, 1629-1640.e2DOI: (10.1016/j.jaci.2016.11.022) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions