Panel A shows longitudinal scatter plots. Blood cell counts in P1 remain largely within reference ranges. Scatter plots display thrombocytes, monocytes, eosinophils, neutrophils, and hemoglobin measurements from 2011 to 2019 against shaded reference intervals. Panel B shows longitudinal scatter plots. Immunoglobulin levels and CD21 low B-cell frequencies tracked. Scatter plots display IgM, IgE, IgA, IgG, and percentage of CD21 low B cells across multiple years with shaded reference ranges. Panel C shows computed tomography images. Thorax and abdomen imaging demonstrate intact internal anatomical structures without lymphoproliferation. Three computed tomography cross-sections show two thorax views and one abdomen view. Panel D shows flow cytometry gating plots. Sequential gating identifies T cells for downstream analysis. Density plots illustrate total cells, singlets, and CD3 positive T-cell gating strategy. Panel E shows an agarose gel image. PCR products detected in sorted TCR alpha beta and gamma delta cells. Gel lanes display amplified products from TCR alpha beta cells, TCR gamma delta cells, and water control. Panel F shows an agarose gel image. PCR products assessed in myeloid cell populations. Gel lanes display amplified products from monocytes, granulocytes, eosinophils, and water control. Panel G shows Sanger sequencing chromatograms. STAT5B sequence region examined across different cell types. Chromatograms display nucleotide traces from monocytes, granulocytes, and eosinophils with marked positions demonstrating absence of mutation. Panel H shows scatter plots. Interleukin-2 stimulated pSTAT5 responses compared between groups. Scatter plots compare pSTAT5 mean fluorescence intensity in TCR alpha beta and TCR gamma delta cells from healthy controls and P1. Panel I shows scatter plots. Baseline pSTAT5 levels assessed without stimulation. Scatter plots compare unstimulated pSTAT5 mean fluorescence intensity in TCR alpha beta and TCR gamma delta cells from healthy controls and P1.
Longitudinal blood and immune cell subpopulation counts in P1 prior to JAK inhibition therapy, CT scan, and screening for STAT5B GOF mutation in monocytes, granulocytes, and eosinophils. (A) Levels of thrombocytes, monocytes, eosinophils, neutrophils, and hemoglobin from hemograms performed between 2011 and 2019. Gray areas delineate the normal reference values for each parameter. (B) Immunoglobulin levels in different hemograms performed between 2011 and 2019. Gray areas delineate the normal reference values for each parameter. (C) CT scan of the thorax and abdomen performed in 2013. (D) Gating strategy to assess TCR, CD4, CD8, and pSTAT5 of T cells, which were isolated from PBMCs. Lymphocytes were sequentially gated according to FSC-A/SSC-A, followed by singlets (FSC-A/FSC-H and SSC-A/SSC-H). T cells were then gated as CD3+ cells. (E and F) Agarose gel electrophoresis of PCR product amplifying the p.Y665F STAT5B missense variant from cDNA of monocytes, granulocytes, and eosinophils. (E) Lane 1—ladder with size marker; lanes 2, 3, 4, 5—TCR αβ cells with different cDNA input; lanes 7, 8, 9, 10—TCR γδ cells with different amounts of cDNA input from the same P1-derived sample; lane 12—water. (F) Lane 1—ladder with size marker; lanes 2, 3—monocytes; lanes 4, 5—granulocytes; lanes 6, 7—eosinophils; lane 8—water; duplicates correspond to different cDNA amount inputs from the same P1-derived sample. (G) Sanger sequencing of p.Y665F STAT5B region amplified from cDNA of PBMC-derived freshly sorted monocytes (left), granulocytes (middle), and eosinophils (right). The data correspond to one experiment. (H and I) Summary of pSTAT5 MFI in TCR αβ or TCR γδ cells from five different HCs and P1 from five different time points. Cells were rested overnight and incubated in the presence (H) or absence (I) of IL-2. Unpaired t test. *P < 0.05. Every dot corresponds to an independent experiment. CT, computed tomography. Source data are available for this figure: SourceData FS1.