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Stepwise Reversal of Immune Dysregulation Due to STAT1 Gain-of-Function Mutation Following Ruxolitinib Bridge Therapy and Transplantation

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Abstract

Purpose

Patients with heterozygous gain-of-function (GOF) mutations in STAT1 frequently exhibit chronic mucocutaneous candidiasis (CMC), immunodeficiency and autoimmune manifestations. Several treatment options including targeted therapies and hematopoietic stem cell transplantation (HSCT) are available for STAT1 GOF patients but modalities and outcomes are not well established. Herein, we aimed to unravel the effect of ruxolitinib as a bridge therapy in a patient with sporadic STAT1 T385M mutation to manage infections and other disease manifestations.

Methods

Peripheral blood mononuclear cells were isolated from the patient prior to, during ruxolitinib treatment and 6 months after HSCT. IFN-β-induced STAT1 phosphorylation/dephosphorylation levels and PMA/ionomycin-stimulated intracellular IL-17A/IFN-γ production in CD4+ T cells were evaluated. Differentially expressed genes between healthy controls and the patient prior to, during ruxolitinib treatment and post-transplantation were investigated using Nanostring nCounter Profiling Panel.

Results

Ruxolitinib provided favorable responses by controlling candidiasis and autoimmune hemolytic anemia in the patient. Dysregulation in STAT1 phosphorylation kinetics improved with ruxolitinib treatment and was completely normalized after transplantation. TH17 deficiency persisted after ruxolitinib treatment, but normalized following HSCT. Consistent with the impairment in JAK/STAT signaling, multiple immune related pathways were found to be dysregulated in the patient. At baseline, genes related to type I IFN-related pathways, antigen processing, T-cell and B-cell functions were upregulated, while NK-cell function and cytotoxicity related genes were downregulated. Dysregulated gene expression was partially improved with ruxolitinib treatment and normalized after transplantation.

Conclusion

Our findings suggest that improved disease management and immune dysregulatory profile can be achieved with ruxolitinib treatment before transplantation and this would be beneficial to reduce the risk of adverse outcome of HSCT.

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Acknowledgments

We thank Deniz Cansen Kahraman for the assistance in using the nCounter Nanostring analysis system.

Funding

This work was supported by the Scientific and Technological Research Council of Turkey (217S847 and 318S202) to S.B., and by National Institutes of Health grant R01 AI085090 to T.A.C.

Author information

Author notes

  1. Basak Kayaoglu, Nurhan Kasap and Naz Surucu Yilmaz contributed equally to this work. Mayda Gursel and Safa Baris contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Middle East Technical University, Ankara, Turkey

    Basak Kayaoglu, Naz Surucu Yilmaz, Busranur Geckin & Mayda Gursel

  2. Division of Allergy and Immunology, Marmara University, Istanbul, Turkey

    Nurhan Kasap, Sevgi Bilgic Eltan, Ahmet Ozen, Elif Karakoc-Aydiner & Safa Baris

  3. Istanbul Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, Istanbul, Turkey

    Nurhan Kasap, Sevgi Bilgic Eltan, Ahmet Ozen, Elif Karakoc-Aydiner & Safa Baris

  4. The Isil Berat Barlan Center for Translational Medicine, Istanbul, Turkey

    Nurhan Kasap, Sevgi Bilgic Eltan, Ahmet Ozen, Elif Karakoc-Aydiner & Safa Baris

  5. Division of Immunology, Boston Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Louis Marie Charbonnier & Talal A. Chatila

  6. Acıbadem Atakent Hospital, Pediatric Bone Marrow Transplantation Unit, Acibadem University, Istanbul, Turkey

    Arzu Akcay & Gulyuz Ozturk

  7. KanSiL, Department of Health Informatics, Graduate School of Informatics, Middle East Technical University, Ankara, Turkey

    Mayda Gursel

  8. Division of Pediatric Allergy/Immunology, Marmara University, Fevzi Çakmak Mah, No: 41, Pendik, Istanbul, Turkey

    Safa Baris

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  1. Basak Kayaoglu
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Contributions

B.K., T.A.C., M.G., and S.B. conceptualized the study and wrote the manuscript. B.K., N.Y., L.M.C., and B.G. performed the experiments. N.K., A.K., S.B.E., G.O., A.O., E.K.A., and S.B. followed up the patients, provided samples, and intellectually contributed to the manuscript.

Corresponding author

Correspondence to Safa Baris.

Ethics declarations

The study was approved by Ethics Committee of Marmara University, School of Medicine. Written informed consents were obtained from the patients and parents.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Informed consent for participation was obtained from all individuals.

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Informed publication consent was obtained from all participants.

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Supplementary information

Supplementary Table 1

The list of genes used for pathway z-score analysis. (XLSX 16 kb)

Figure S1

Representative flow cytometry plots illustrating IL-17 deficiency in the patient. IL-17A and IFN-γ production in CD4+ T cells of patient (P-Baseline) and healthy controls (HC) following stimulation with PMA (50ng/ml) and ionomycin (1 μg/ml) are shown. (PDF 105 kb)

Figure S2

Normalization of TFH percentage in the patient after ruxolitinib treatment. (A) Percentages of TFH (CXCR5+PD-1+) cells in CD4+ T cell gate in patient before (P-Baseline) and after ruxolitinib treatment (P-Ruxo) compared to healthy controls (HC) were assessed by flow cytometry. (PDF 176 kb)

Figure S3

Improvement in augmented STAT1 phosphorylation in the patient helper T cells with ruxolitinib treatment. STAT1 phosphorylation with or without IFN-γ (20ng/ml) stimulation was analyzed in patient cells before (P-Baseline) and 6 months after ruxolitinib treatment (P-Ruxo) by flow cytometry. (PDF 35 kb)

Figure S4

Restoration of STAT1 phosphorylation and dephosphorylation pattern in CD4+ T cells of the patient after HSCT. STAT1 phosphorylation was assessed in PBMCs stimulated with IFN-β for 15, 60 and 120 minutes as opposed to untreated healthy controls (HC). (PDF 514 kb)

Figure S5

Immune restoration after ruxolitinib and HSCT. (A) Comparison of IFN scores between patient, prior to (P-baseline), at 7 months of ruxolitinib therapy (P-Ruxo) and 6 months after HSCT (P-HSCT), and healthy controls. IFN score was calculated using median fold change from the values in healthy controls for the expression of 30 ISGs given in Figure 3C. (B) Comparison of antigen processing pathway z-score between patient and healthy controls. (C) Comparison of T cell function pathway z-score between patient and healthy controls. (D) Comparison of B cell function pathway z-score between patient and healthy controls. Asterisks (*) on bars show comparisons between patient and healthy controls. n.s., Not significant. **p <.01, ***p <.001 **** and p<.0001, Student unpaired 2-tailed t test for comparison between patient and healthy controls and Student paired 2-tailed t test for comparison within patient groups. (PDF 66 kb)

Figure S6

Reverse of NK cell function and cytotoxicity after ruxolitinib treatment and HSCT. (A) Comparison of NK cell function pathway z-score between patient, prior to (P-baseline), at 7 months of ruxolitinib therapy (P-Ruxo) and 6 months after HSCT (P-HSCT), and healthy controls. (B) Comparison of cytotoxicity pathway z-score between patient and healthy controls. Asterisks (*) on bars show comparisons between patient and healthy controls. n.s., Not significant. *p <.05, **p <.01, ***p <.001 **** and p<.0001, Student unpaired 2-tailed t test for comparison between patient and healthy controls and Student paired 2-tailed t test for comparison within patient groups. (PDF 34 kb)

Figure S7

Impact of ruxolitinib treatment and HSCT on SOCS1 and PD-L1 gene expression. (A) Number of probes detecting SOCS1 mRNAs in total RNA sample of patient, prior to (P-baseline), at 7 months of ruxolitinib therapy (P-Ruxo) and 6 months after HSCT (P-HSCT), and healthy controls are compared. (B) Number of probes detecting PD-L1 mRNAs in total RNA sample of patient, prior to (P-baseline), at 7 months of ruxolitinib therapy (P-Ruxo) and 6 months after HSCT (P-HSCT), and healthy controls are compared. Asterisks (*) on bars show comparisons between patient and healthy controls. n.s., Not significant. **p <.01, ***p <.001 **** and p<.0001, Student unpaired 2-tailed t test for comparison between patient and healthy controls and Student paired 2-tailed t test for comparison within patient groups. (PDF 32 kb)

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Kayaoglu, B., Kasap, N., Yilmaz, N.S. et al. Stepwise Reversal of Immune Dysregulation Due to STAT1 Gain-of-Function Mutation Following Ruxolitinib Bridge Therapy and Transplantation. J Clin Immunol 41, 769–779 (2021). https://doi.org/10.1007/s10875-020-00943-y

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