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. 2024 Apr 5;44(4):85.
doi: 10.1007/s10875-024-01684-y.

Patients with STAT1 Gain-of-function Mutations Display Increased Apoptosis which is Reversed by the JAK Inhibitor Ruxolitinib

Laura Dotta  1 , Francesca Todaro  2 , Manuela Baronio  3 , Mauro Giacomelli  2 , Marinella Pinelli  2 , Martina Giambarda  4 , Beatrice Brognoli  4 , Silvia Greco  4 , Francesca Rota  4 , Manuela Cortesi  4 , Annarosa Soresina  4 , Daniele Moratto  2 , Cesare Tomasi  5 , Rosalba Monica Ferraro  2 , Silvia Giliani  2 , Raffaele Badolato  5
Affiliations

Patients with STAT1 Gain-of-function Mutations Display Increased Apoptosis which is Reversed by the JAK Inhibitor Ruxolitinib

Laura Dotta et al. J Clin Immunol. .

Abstract

Introduction: The signal transducer and activator of transcription (STAT1) gain-of-function (GOF) syndrome accounts for most cases of chronic mucocutaneous candidiasis but is characterized by a broader clinical phenotype that may include bacterial, viral, or invasive fungal infections, autoimmunity, autoinflammatory manifestations, vascular complications, or malignancies. The severity of lymphopenia may vary and influence the infectious morbidity.

Methods: In our cohort of seven STAT1-GOF patients, we investigated the mechanisms that may determine T lymphopenia, we characterized the interferon gene signature (IGS) and analyzed the effect of ruxolitinib in reverting the immune dysregulation.

Results: STAT1-GOF patients exhibited increased T lymphocyte apoptosis that was significantly augmented in both resting conditions and following stimulation with mitogens and IFNα, as evaluated by flow cytometry by Annexin V/ Propidium iodide assay. The JAK inhibitor ruxolitinib significantly reduced the IFNα-induced hyperphosphorylation of STAT1 and reverted the stimulation-induced T-cell apoptosis, in vitro. In two adult STAT1-GOF patients, the JAKinib treatment ameliorated chronic mucocutaneous candidiasis and lymphopenia. Most STAT1-GOF patients, particularly those who had autoimmunity, presented increased IGS that significantly decreased in the two patients during ruxolitinib treatment.

Conclusion: In STAT1-GOF patients, T lymphocyte apoptosis is increased, and T lymphopenia may determine higher risk of severe infections. The JAKinib target therapy should be evaluated to treat severe chronic candidiasis and lymphopenia, and to downregulate the IFNs in patients with autoinflammatory or autoimmune manifestations.

Keywords: IGS; JAK Inhibitor; Ruxolitinib; STAT1-GOF; T Lymphopenia; T-cell Apoptosis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Cellular apoptosis was measured with flow cytometry in T lymphocytes that were left unstimulated or stimulated for 48 h: the results are shown as percentage of early and late apoptotic cells that were positive to Annexin V-FITC. STAT1-GOF patients exhibited significantly increased apoptosis on basal condition (unstimulated) and following all combinations of stimuli, compared to healthy subjects that were tested in parallel. Statistical significance was assessed by the Mann Withney U test with two tails, *p < 0.05
Fig. 2
Fig. 2
IRF1 (a) and CASP3 (b) expression was assessed by Real-Time PCR assay in healthy controls (gray color) and STAT1-GOF patients (black color). STAT1-GOF patients showed increased expression of CASP3 following stimulation with anti-CD3/anti-CD8/IFNα (*p < 0,05). US, unstimulated
Fig. 3
Fig. 3
Phosphorylation of STAT1 (pSTAT1) after stimulation with IFNα was assessed by flow cytometry in STAT1-GOF patients (black color) and healthy controls (gray color). In STAT1-GOF patients the expected increase of pSTAT1 was statistically significant compared to healthy controls and was significantly reversed by the JAK inhibitor ruxolitinib with a dose-dependent effect. US, unstimulated
Fig. 4
Fig. 4
The effect of ruxolitinib on T lymphocyte apoptosis was evaluated in vitro with flow cytometry as percentage of early and late apoptotic cells that were positive to Annexin V after 48-hour stimulation: ruxolitinib significantly reduced stimulation-induced T cell apoptosis both in STAT1-GOF patients and healthy subjects (as assessed by the Mann-Whitney test)
Fig. 5
Fig. 5
Lymphocyte count in STAT1L283M P2 and STAT1L351F P3 treated with ruxolitinib. #: nadir of total lymphocyte count was observed in P3 during JAKinhib treatment when he had SARS-COV2 infection and pulmonary aspergillosis
Fig. 6
Fig. 6
Interferon gene signature (ISG) was measured in leukocytes of STAT1-GOF patients, in comparison to a pool of healthy controls. The relative gene expression levels were calculated using the comparative Ct (∆∆Ct) method and normalized to the expression of the housekeeping gene 18S. STAT1-GOF patients presented increased ISG compared to healthy subjects (a), that reduced or normalized during ruxolitinib treatment in both STAT1L351F P3 (b) and STAT1L283M P2 (c) (in STAT1L351F P3 t0 is before starting ruxolitinib, t + 1,2,3,4,5 correspond to + 1, +2, + 3, +8, + 12, and + 24 months, respectively; in STAT1L283M P2 t0 is before the initiation of the JAKinib, t + 1 is after 6 months of treatment). HD, healthy donor
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