Recombinant IFN-γ1b Treatment in a Patient with Inherited IFN-γ Deficiency

Abstract

Purpose: Inborn errors of IFN-γ immunity underlie Mendelian susceptibility to mycobacterial disease (MSMD). Twenty-two genes with products involved in the production of, or response to, IFN-γ and variants of which underlie MSMD have been identified. However, pathogenic variants of IFNG encoding a defective IFN-γ have been described in only two siblings, who both underwent hematopoietic stem cell transplantation (HCST).

Methods: We characterized a new patient with MSMD by genetic, immunological, and clinical means. Therapeutic decisions were taken on the basis of these findings.

Results: The patient was born to consanguineous Turkish parents and developed bacillus Calmette-Guérin (BCG) disease following vaccination at birth. Whole-exome sequencing revealed a homozygous private IFNG variant (c.224 T > C, p.F75S). Upon overexpression in recipient cells or constitutive expression in the patient's cells, the mutant IFN-γ was produced within the cells but was not correctly folded or secreted. The patient was treated for 6 months with two or three antimycobacterial drugs only and then for 30 months with subcutaneous recombinant IFN-γ1b plus two antimycobacterial drugs. Treatment with IFN-γ1b finally normalized all biological parameters. The patient presented no recurrence of mycobacterial disease or other related infectious diseases. The treatment was well tolerated, without the production of detectable autoantibodies against IFN-γ.

Conclusion: We describe a patient with a new form of autosomal recessive IFN-γ deficiency, with intracellular, but not extracellular IFN-γ. IFN-γ1b treatment appears to have been beneficial in this patient, with no recurrence of mycobacterial infection over a period of more than 30 months. This targeted treatment provides an alternative to HCST in patients with complete IFN-γ deficiency or at least an option to better control mycobacterial infection prior to HCST.

Keywords: BCG; Inborn error of immunity; Interferon-gamma; Mycobacterium.

Fig. 1

A new patient with a private rare biallelic variant of IFNG. A Pedigree of the kindred. Each generation is indicated by a Roman numeral, and each individual is indicated by an Arabic numeral. The patient is indicated by a black square. M, mutated; WT, wild-type. B Skin of the patient at the age of 6 months, with a maculopapular rash. C Computed tomography scan of the thorax of the patient at the age of 6 months showing pleural effusion in the left hemithorax. D Timeline of the follow-up of the patient with clinical events (top) and treatment (bottom). INH, isoniazid; RFP, rifampicin; EMB, ethambutol. E Antiviral antibody responses to species for which at least one sample tested seropositive by PhIP-Seq, based on stringent in-house cutoff values, color-coded as indicated. “cI.V.IgG” and “pediatric CTLs” correspond to the mean response for samples from pooled patients on IVIG (n = 8) and pediatric controls (n = 111) with a mean age of 9 years (SD = 2 years), respectively. A hierarchical clustering of samples based on antibodies directed against viruses is indicated at the top. F Electropherograms for the sequencing of representative IFNG nucleotide sequences from the patient, his relatives (heterozygous), and a healthy control (homozygous wild-type). G Combined annotation depletion-dependent (CADD) score vs. minor allele frequency (MAF) for variants in translated regions of IFNG found in the homozygous state either in public databases (blue) or in patients with AR complete IFN-γ deficiency. The 95% mutation significance cutoff (MSC) is indicated by a dotted line. H Crystal homodimer of IFN-γ (PDB = 6E3K [41]) showing the location of the p.F75 residue (red)

Fig. 2

Autosomal recessive complete IFN-γ deficiency in the patient. A Western blot on total cell extracts from HEK293T cells left untransfected (NT, non-transfected) or transfected with the indicated plasmid, with an anti-DDK, anti-IFN-γ, or anti-GAPDH antibody used for detection. B Western blot on total cell extracts with and without prior treatment with PNGaseF from HEK293T cells untransfected or transfected with the plasmid indicated, with an anti-DDK, anti-IFN-γ, or anti-GAPDH antibody used for detection. C HLA-DR induction in SV40-fibroblasts with and without (NS, non-stimulated) treatment for 48 h with recombinant IFN-γ (IFN-γ1b) or with supernatant from untransfected HEK293T cells of HEK293T cells transfected with the plasmids indicated. D Western blot for IFN-γ and GAPDH on PHA-activated and anti-CD2/3/28 bead-activated blasts from two controls (CTLs) and the patient. E Intracellular flow cytometry for IFN-γ on PHA-activated T cells from the indicated patients or individuals after pretreatment with brefeldin in the indicated conditions (P/I, PMA/ionomycin). F HLA-DR induction in SV40-fibroblasts with and without (NS) treatment with supernatant from PHA-activated T-cell blasts from the indicated individuals or patients. G Secretion of IFN-γ, assessed in whole-blood assays, for the patient, the patient’s relatives, and healthy controls (local and travel), after activation with BCG (alone or in combination with IL-12, IL-23, or IFN-γ) or PMA/ionomycin (P/I)

Fig. 3

Efficacy and lack of immunogenicity of treatment with recombinant IFN-γ. A Quantification of IFN-γ levels in plasma from the patient before and 10 h after IFN-γ1b administration. B Sequencing of RNA extracted from the whole blood of three healthy controls (CTLs), healthy individuals with or without ex vivo treatment with IFN-γ1b for 8 h, and the patient before and 10 h after the subcutaneous administration of IFN-γ. C Quantification of IFN-γ levels in plasma from the patient before and 10 h after IFN-γ1b administration. Screening for autoantibodies against IFN-γ by D ELISA and E neutralization luciferase assays on plasma from the indicated controls or patients