Panel A shows a clinical photograph from P1. Oral lesions visible on palate indicate mucosal inflammatory involvement. Panel B shows flow cytometry plots and a histogram. Expanded double-negative gamma delta T-cell population detected in patient. Panel C shows a flow cytometry density plot. Distinct alpha beta and gamma delta T-cell populations identified. Panel D shows scatter plots. Gamma delta cells relatively increased while alpha beta cells reduced in P1. Panel E shows a variant comparison diagram. STAT5B variant detected exclusively in gamma delta T cells of P1. Panel F shows Sanger sequencing chromatograms. Sequencing confirms presence of wild-type and mutant alleles. Panel G shows a summary table. STAT5B variant classified as mosaic gain-of-function mutation. Panel H shows a protein domain schematic. Y665F mutation localized within the SH2 domain. Panel I shows flow cytometry histograms. Enhanced pSTAT5 observed following interleukin-2 stimulation. Panel J shows scatter plots. Elevated pSTAT5-high gamma delta cells quantified in patient.
Identification of a STAT5 GOF mutation selectively in TCR γδ cells of P1. (A) Representative picture of the oral mucosa from P1 at diagnosis. The picture shows erosive stomatitis (red arrows). (B) Representative flow cytometry plots of CD4 and CD8 expression on total T cells from a HC and P1, followed by TCR γδ expression on CD4−CD8 DN cells. (C) Representative flow cytometry plots of TCR γδ and TCR αβ cells within live CD3+ PBMCs from P1. (D) Summary of T cells from HCs vs. P1 expressing TCR αβ (left) or TCR γδ (right). The analyzed samples from P1 were collected throughout 14 years; each dot corresponds to an independent experiment. Scatter plots of percentages and the median of seven independent measurements. Mann–Whitney test. ****P < 0.0001. (E) List of PID gene variants detected by WES from the genomic DNA of freshly sorted TCR αβ and TCR γδ cells. Found PID variants were filtered for CADD scores >15 and allele frequencies <0.05. The p.Y665F STAT5B missense variant was the only one found exclusively in TCR γδ cells. (F) Sanger sequencing of p.Y665F STAT5B region amplified from cDNA of freshly sorted TCR αβ (top panel) and TCR γδ (bottom panel) cells. A STAT5B-specific PCR product amplified from cDNA containing the region of the p.Y665F mutation was cloned into a plasmid and sequenced. In total, 23 colonies containing plasmids with the PCR product were sequenced and five colonies carried the STAT5B mutation (21%). Sanger sequencing results are shown from a colony containing wild-type STAT5B (top right panel) and a colony containing mutant STAT5B (bottom right panel). The data correspond to one experiment. (G) Overview for the p.Y665F (c.A1994T) STAT5B variant. (H) Scheme of STAT5B protein with the location of the p.Y665F STAT5B variant in the SH2 domain of STAT5B. (I) Representative histogram of pSTAT5 expression in TCR αβ (top) or TCR γδ (bottom) cells from HCs vs. P1 rested overnight and stimulated or not with IL-2 for 30 min. The matched isotype is shown in black. (J) Summary of percentages of pSTAT5 in TCR αβ or TCR γδ cells from HCs and P1 rested overnight and stimulated with IL-2 for 30 min. Mann–Whitney test. **P < 0.01. Each dot corresponds to an independent experiment. DN, double negative; PID, primary immunodeficiency.