Clinical, genetic, and functional analysis of a mosaic STAT5B variant. (A) Multifocal, enhancing lesions with increased T2 fluid-attenuated inversion recovery (T2/FLAIR) hyperintensity in the brain at diagnosis in the pons and cervical spine. (B) ddPCR at diagnosis of STAT5B reference and STAT5B c.1883C>G VAFs in patient and parental DNA isolated from indicated tissues or FACS-sorted immune cells (T cells [CD3⁺], CD4 T cells [CD3⁺TCRαβ⁺CD4⁺], CD8 T cells [CD3⁺TCRαβ⁺CD8⁺], B cells [CD3⁻CD19⁺], monocytes [CD3⁻CD14⁺], natural killer [NK] cells [CD3⁻CD56⁺]). ddPCR (Bio-Rad) was performed with DNA from the indicated tissues with probes specific for the STAT5B c.1883C>G variant (FAM dye, custom design, Bio-Rad) and analyzed with the QX200 droplet reader and QX Manager software. Data represent two technical replicates and two separate buccal samples. (C) Patient and control peripheral blood mononuclear cells were stimulated with IL-2 for 20 min (Chiron, Proleukin) and pSTAT5 (pY694, Cat# 612599; BD Biosciences) assessed by flow cytometry (median fluorescence intensity, MFI) shown as unstimulated (shaded) versus stimulated (solid line) gated on live, CD45⁺CD19⁻CD14⁻CD3⁺CD8⁻CD4⁺ T cells. Assay was performed while the patient was off steroids. (D) STAT5B-RFP MFI measured in a reporter T cell line transfected with a GFP-tagged EV, or plasmids with WT STAT5B or the indicated STAT5B variants. RFP MFI was assessed in GFP⁺ cells that were unstimulated or stimulated with IL-7 for 24 h. Mean ± SEM of unstimulated and stimulated WT plasmids are indicated by the horizontal dashed lines and shading. The STAT5B reporter T cell line was established by transducing T28 cells with a STAT5B-RFP reporter lentivirus. The STAT5B reporter T cell line was then transfected with GFP-tagged STAT5B plasmids as indicated. Transfected cells were stimulated with IL-7 for 24 h, and RFP was measured by flow cytometry. Data shown are representative of three independent experiments. (E) Representative flow cytometry showing reduced CD3⁺CD4⁺ TCRαβ⁺ CXCR5⁺CD45RA⁻ cTfh cells (top row) and frequency of CD3⁺CD4⁺ TCRαβ⁺ CD25⁺CD127⁻ Treg cells (bottom row) in the patient compared with age-matched healthy controls at diagnosis and after 6 mo of tofacitinib. cTfh cells and Treg cells were pregated as CD45⁺ CD19⁻ CD14⁻ CD56⁻ TCRγδ⁻ CD3⁺ CD8⁻. (F) ddPCR for STAT5B c.1883C>G variant after 6 mo of therapy with tofacitinib in patient DNA isolated from whole blood or sorted cell populations. (G) ddPCR of patient peripheral blood DNA at diagnosis (left) and 20 mo after HCT (right) showing percentage of droplets with STAT5B c.1883C>G variant (Var) or WT STAT5B reference sequence (Ref). Data are representative of two technical replicates. NT, no template. Both, both templates. ddPCR, droplet digital polymerase chain reaction; EV, empty vector.