Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function

Abstract

Autoinflammatory disease can result from monogenic errors of immunity. We describe a patient with early-onset multi-organ immune dysregulation resulting from a mosaic, gain-of-function mutation (S703I) in JAK1, encoding a kinase essential for signaling downstream of >25 cytokines. By custom single-cell RNA sequencing, we examine mosaicism with single-cell resolution. We find that JAK1 transcription was predominantly restricted to a single allele across different cells, introducing the concept of a mutational "transcriptotype" that differs from the genotype. Functionally, the mutation increases JAK1 activity and transactivates partnering JAKs, independent of its catalytic domain. S703I JAK1 is not only hypermorphic for cytokine signaling but also neomorphic, as it enables signaling cascades not canonically mediated by JAK1. Given these results, the patient was treated with tofacitinib, a JAK inhibitor, leading to the rapid resolution of clinical disease. These findings offer a platform for personalized medicine with the concurrent discovery of fundamental biological principles.

Keywords: JAK inhibitors; JAK-STAT signaling; JAK1; cytokine signaling; inborn errors of immunity; monoallelic expression; mosaicis; precision medicine.

Graphical abstract

Figure 1

De Novo Mutation in JAK1 Identified in a Patient with Immunodysregulatory Syndrome (A) Schematic representing clinical history of the patient, with gray bars representing the kinetics of each disease feature. (B) Photograph of the dermatologic lesions on the arm. (C) Histology of the cecal mucosa showing expansion of the lamina propria secondary to increased inflammatory cell infiltrate, with eosinophils in the lamina propria and crypt epithelium (arrows). (D) Electron microscopy of a renal biopsy obtained during disease recurrence that demonstrates irregular glomerular basement membranes and subepithelial and intramembranous immune type dense deposits. (E) Patient’s family pedigree. (F) Whole-exome sequencing reads mapping to JAK1 locus c.2108, with variant nucleotides displayed in green. (G) Representative chromatograms from 3 independent experiments of Sanger sequencing of peripheral blood DNA to confirm c.2108 G > T JAK1. (H) Proportion of cells carrying the heterozygous JAK1 mutation, as estimated by digital droplet PCR with WT- and mutation-specific probes. DNA was obtained from bilateral cheek swabs, Ficoll-fractionated whole blood, and epithelial tissue isolated from a colonic biopsy (n = 1). (I) Model for the development of the de novo mutation and its distribution into all 3 germ layers. See also Figure S1.

Figure 2

S703I JAK1 Confers Constitutively Active and Hyperresponsive STAT Signaling (A) Localization of the S703I (red) mutation to the pseudokinase domain (blue) of JAK1, as represented in the linear sequence and the predicted structure, as modeled on TYK2. (B) Immunoblotting for STAT phosphorylation in U4C Cells (JAK1^−/−) reconstituted with lentiviruses for vector control (luciferase), WT JAK1, or S703I JAK1 and stimulated for 15 min with indicated doses of IFN-α (IU/mL). (C) qPCR from transduced U4C cells for IFN-stimulated gene expression (MX1 and RSAD2) at baseline and following 100 IU/mL of IFN-α for 8 h (n = 3). Columns represent means and error bars represent standard deviations. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, 2-tailed Student’s t test with Welch’s correction. (D and E) Stimulation of transduced U4C cells with IFN-γ (0.1 ng/mL) (D) or IL-6 (25 ng/mL) (E) for 15 min. (F and G) Derivation of B-EBV cells and isolation of JAK1 WT/WT and JAK1 S703I/WT cells from patient blood, followed by stimulation with IFN-α (100 IU/mL) (F) or IFN-γ (0.1 ng/mL) (G) for 15 min. All of the data are representative of at least 3 independent experiments. See also Figure S2.

Figure 3

S703I JAK1 Mediates Hyperactive STAT Phosphorylation by Transactivation of Partnering JAKs (A) Immunoprecipitation of JAK1 from JAK1-transduced U4C cells and immunoblotting for total phosphorylation (4G10) after stimulation with IFN-α (100 IU/mL) for 15 min. (B) Immunoprecipitation of JAK2 from patient-derived B-EBV cells and western blotting for phosphorylation at the activation loop after stimulation with IFN-γ (0.1 ng/mL) for 15 min. (C) Immunoprecipitation of TYK2 from JAK1-transduced U4C cells and western blotting for phosphorylation at the activation loop after stimulation with IFN-α (100 IU/mL) for 15 min. (D) Immunoprecipitation of JAK3 from patient-derived B-EBV cells and western blotting for total phosphorylation stimulation with IL-21 (50 ng/mL) for 15 min. (E) Surface staining of the IFNAR2 subunit by flow cytometry in U4C cells transduced with the indicated constructs. Lines indicate means and error bars represent SD. ^∗∗∗p < 0.001, ^****p < 0.0001, analysis of variance (ANOVA) with Tukey's post-hoc analysis. (F and G) Transduction of U4C cells with catalytically inactivated JAK1 (K908A), WT JAK1, S703I JAK1, or double-mutant JAK1 (K908A S703I), followed by stimulation with IFN-α (1,000 IU/mL) (F) or IFN-γ (1.0 ng/mL) (G). (H) Transduction of U4C cells with catalytically inactivated JAK1 (K908A), WT JAK1, A634D JAK1, or double-mutant JAK1 (K908A A634D), followed by stimulation with IFN-α (1000 IU/mL) or IFN-γ (1.0 ng/mL). (I) Inhibition of STAT phosphorylation by a 4-h treatment with saturating doses (1 and 10 μM) of JAK inhibitors followed by 15-min stimulation with IFN-α (1,000 IU/mL). All of the data are representative of at least 3 independent experiments. See also Figure S2.

Figure 4

CyTOF Analysis Reveals Cytokine-, STAT-, and Cell-Type-Specific Gain of Function (A) Representative (tSNE) plots generated from immunophenotyping CyTOF data of whole blood from 2 independent experiments. (B) Manually gated CyTOF populations from the whole blood of 5 separate healthy controls and the patient on 2 separate occasions (2X) were quantified as the percentage of single cells and expressed as relative frequency (patient/controls). The gray bars indicate means with standard deviations of healthy donors, and colored bars indicate means with standard deviations of patient. Multiple t tests performed correcting for multiple comparisons using the Holm-Sidak method. ^∗∗∗p < 0.001. (C) Relative MSI of phospho-STAT staining from intracellular phospho-CyTOF of whole blood from 4 healthy donors (n = 4) and the patient on 3 separate occasions (3X). The columns represent means and the error bars represent standard deviations. (D) Ex vivo stimulation with IFN-α (100 IU/mL), IL-2 (50 ng/mL), and IL-4 (50 ng/mL) for 15 min. The color intensity indicates the log2 fold-change in MSI over the unstimulated healthy control for each cell type (n = 1). Cent Mem, central memory; C Mono, classical monocyte; DN, double negative; DP, double positive; Eff Mem, effector memory; FC, fold change; HC, healthy control; ILC, innate lymphoid cells; I Mono, intermediate monocytes; MAIT, mucosal associate invariant T cells; mDC, myeloid dendritic cell; MSI, mean signal intensity; NC Mono, non-classical monocyte; P, patient; pDC, plasmacytoid dendritic cell; Th, T helper; . tSNE, t-distributed stochastic neighbor embedding. See also Figures S3 and S4.

Figure 5

Custom scRNA-Seq Maps JAK1 Allele Distribution, Transcriptomic Impact, and Expression Patterns (A) tSNE plots and cell-type assignments from scRNA-seq of patient PBMCs, with an inDrops platform adapted to target the mutant JAK1 transcript. n = 4,763 cells. (B) tSNE plots representing the subset of cells with sufficient JAK1 counts to be assigned putative JAK1 genotypes (based on transcript sequences). Cells in which any mutant transcript was detected above empirically determined thresholds (> 5 JAK1 transcripts) were assigned “S703I JAK1” (purple), while cells with only WT transcript detected were assigned “WT JAK1” (orange). (C) Doughnut charts quantifying allele distribution in cells meeting genotyping criteria (cell count in the center), as in (B). (D) Expression of the ISG IFI44L, a statistically significant differentially expressed gene in the comparison of WT JAK1 and S703I JAK1 genotyped cells. (E) Gene set scores for IFN-α signaling in CD14⁺ monocytes. (F) Number of unique transcripts detected per cell for the WT or S703I JAK1 allele (left) or a control variant GNLY rs12845 (right). The bubble size indicates the number of cells. The color coding indicates cells containing S703I JAK1 (purple), WT JAK1 without S703I JAK1 (orange), WT JAK1 with S703I JAK1 detected below threshold (yellow), or insufficient transcripts counts (gray). (G) Transcript genotyping of JAK1 rs2230587 from healthy control PBMCs (n = 96) by single-cell qPCR with allele-specific probes. The histogram represents the relative frequency of cells expressing binned allele ratios as quantified by oligonucleotide standards. (I) Single-cell qPCR transcript genotyping of control gene NACA (rs4902). See also Figure S5.

Figure 6

Treatment with Tofacitinib Rescues STAT Hyperphosphorylation and Resolves Clinical Disease (A) In vitro assessment of JAK-inhibitor efficacy after 4-h drug treatment for resolution of basal STAT phosphorylation in transduced U4Cs. (B) Similar analysis in patient-derived B-EBVs, with doses indicated. (C) Ex vivo inhibition with equimolar doses (500 nM) of ruxolitinib and tofacitinib for 4 h followed by IFN-α stimulation (1,000 IU/mL) for 15 min (n = 1). (D) C-Reactive Protein monitoring from patient sera over the course of treatment. Dotted line represents the upper limit of normal (ULN). (E) Erythrocyte sedimentation rate in patient blood over the course of treatment, with a dotted line indicating the ULN. (F) Gross appearance of skin before and 5 months after tofacitinib treatment (Tx). (Image of the arm also in Figure 1.) (G) Colonic biopsies before and 6 months post-therapy. (H) ISG expression in RNA from bulk PBMCs isolated throughout treatment (n = 1). The dotted line represents the average expression across 3 healthy controls. (I) Phospho-CyTOF analysis comparing relative changes in pSTAT MSI from before and 10 months post-therapy (n = 1). See also Figure S6.