IFNγR1 deficiency presenting with visceral leishmaniasis and Mycobacterium Avium infections mimicking HLH

To the Editor,

Interferon-gamma receptor-1 (IFN-γR1) deficiency is a rare inborn error of immunity. It can be inherited as an autosomal recessive (AR), partial or complete, or autosomal dominant (AD) trait and exhibits high allelic variability. Biallelic deleterious recessive variants of IFNGR1 that result in absent expression or defective downstream signaling of IFN-γR1 cause failure to activate macrophages in response to interferon-gamma (IFN-γ), rendering innate immunity ineffective in clearing intracellular pathogens. Complete AR IFN-γR1 deficiency is characterized by early childhood-onset and a severe phenotype with predisposition to life-threatening infections. Disseminated environmental mycobacteria and bacille Calmette-Guérin (BCG) are the most commonly reported pathogens; however, these patients are also vulnerable to a variety of intracellular pathogens including bacteria, viruses, fungi, and parasites.^1,2 IFN-γR1 deficiency can be a challenging diagnosis, requiring molecular and flow cytometry analysis and functional assessment of IFN-γR signaling. Due to the severity of prognosis, early recognition remains crucial. Here, we describe a patient with a distinctive clinical presentation of AR IFN-γR1 deficiency resulting from compound heterozygous IFNGR1 variants.

A 3-year, 6-month-old boy, who is the only child, was born at term to non-consanguineous Portuguese parents. He remained well in early childhood and received age-appropriate vaccinations, but later developed three episodes of bacterial pneumonia, one episode of viral gastroenteritis, and an episode of culture-negative septic arthritis between 18 months and 3 years of age. As he turned 3, he developed intermittent fevers associated with leukocytosis, neutrophilia, hypereosinophilia (Figure S1), and elevated inflammatory markers. Despite empirical antimicrobial therapy, his fevers became more frequent, and he developed lymphadenopathy and splenomegaly with micro-abscesses. IFN-γ release assay (QuantiFERON) showed an indeterminate response. A detailed workup was unrevealing for infections, and hematological and rheumatological conditions. Bone marrow biopsy showed granulocytic hyperplasia with eosinophilia without features of dysplasia. Lymph node biopsies showed reactive changes without evidence of malignancy (Figure S2). DNA sequencing targeted to a panel of primary immunodeficiency genes failed to uncover pathogenic variants. Analysis of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity by means of the dihydrorhodamine (DHR) flow cytometry assay showed normal neutrophilic oxidative burst. Empirical corticosteroids were started for a hyperinflammatory state of unclear etiology, which resulted in stabilization of fevers and inflammatory markers. A trial of tocilizumab was attempted but had to be discontinued due to severe thrombocytopenia. All antibiotics were discontinued due to lack of evidence of an infection. To characterize his disorder, he was referred to the National Institutes of Health (NIH) where he was enrolled in a research protocol (18-I-0041) approved by the NIH Institutional Review Board.

At admission, he was noticed to be febrile, lethargic, and failing to thrive (weight at 5^th percentile; height at 3^rd percentile for age). Initial laboratory evaluation showed neutrophilic leukocytosis, anemia of chronic disease, elevated inflammatory markers, indeterminate response in IFN-γ release assay, low-level CMV (<3.08 log10 lU/ml), EBV (2.45 log10 lU/ml), and HHV-6 (<2.4 log10) viremia. Immunophenotyping showed mild increase in CD3+ TCR-α/β⁺ CD4⁻ CD8⁻ double-negative T cells (275/mcl, normal: 24–223) and IgG hypergammaglobulinemia (2411 mg/dl, normal: 453–916), raising the suspicion for autoimmune lymphoproliferative syndrome (ALPS). Abdominal computed tomography showed hepatomegaly (12 cm), splenomegaly (14 cm), and lymphadenopathy. Bone marrow biopsy showed granulocytic hyperplasia, without evidence of hemophagocytosis or malignancy. Bone marrow PCR testing returned positive for Leishmania at cycle threshold (Ct) value of 35. While morphology and staining failed to identify Leishmania, this was considered to be a true infection due to living in Portugal which is an endemic area and demonstration of Leishmania in the dogs living with the child’s grandfather, whom he visited often. Because of this, he was started on daily intravenous amphotericin B. Trimethoprim-sulfamethoxazole (TMP-SMX) was also initiated for Pneumocystis jirovecii (PJP) prophylaxis, while he continued to be on maintenance corticosteroids.

Two weeks after presentation, he developed abrupt worsening of fever, elevation of C-reactive protein (CRP) (290 mg/L, normal: <5), ferritin (727 mcg/L, normal: 30–400), and triglyceride levels (285 mg/dl, normal: <150). Soluble CD25 levels were very high (15713 U/ml, normal: 144–1329), with elevated interleukin-18 (1340 pg/ml, normal: 89–540) and S100A8/S100A9 heterodimer (8248 ng/ml, normal: 16–3004), and normal soluble CD163 (1827 ng/ml, normal: 274–2580) and FAS-ligand levels (172 pg/ml, normal: 0–308). Due to the continued hyperinflammatory state, he was started on anakinra (an IL-1 receptor antagonist), which transiently stabilized fevers and inflammatory markers; however, he then developed respiratory distress. Repeat imaging showed new bilateral patchy pulmonary opacities and worsening splenomegaly (15 cm). PCR from bronchoalveolar lavage returned positive for PJP. Blood next-generation sequencing detected Mycobacterium chimera DNA sequences. Despite multimodal therapy including high-dose corticosteroids, anakinra, TMP-SMX, liposomal amphotericin B, and antimycobacterial therapy with rifabutin, ethambutol, and azithromycin, he continued to deteriorate with persistently elevated CRP (272 mg/L), rapidly increasing ferritin (2502 mcg/L) and fourfold increase in sCD25 levels (61457 U/ml) (Figure 1). He was noted to have very high serum IFN-γ (80360 pg/ml, normal: 0–41), elevated IL-12 (340 pg/ml, normal: 21–184) and TNF-α (28 pg/ml, normal: 0–4), and undetectable CXCL-9 levels (<31 pg/ml), which were concerning for an IFN-γ pathway defect.

FIGURE 1.

Longitudinal analysis of inflammatory biomarkers, and duration and timepoints of antimicrobial and immunomodulatory therapy during hospital course. All medication doses are expressed cumulatively as total daily dose. *Dose was increased during acute illness and weaned down to a physiologic dose at the time of discharge. Antimicrobials continued at time of discharge: Amphotericin B was decreased from 3 mg/kg/day for 3 weeks to 3 mg/kg every other day for 4 doses, then at time of discharge and concurrent with physiologic corticosteroid dosing to 4 mg/kg/week for 4 weeks (partially shown in graph), followed by suppressive IV therapy with 5 mg/kg/3 weekly (not shown). Sulfamethoxazole-trimethoprim was increased to treatment dose during acute illness for 21 days followed by reduction to prophylactic dosing, which was continued at time of discharge. Rifabutin, ethambutol, and azithromycin were continued at treatment dose for disseminated MAC infection

Whole-exome sequencing identified two heterozygous IFNGR1 variants including a novel frameshift deletion: NM_000416 c.622_623del, p.S208Tfs*21 (absent in public databases: gnomAD, ExAC, dbSNP, or 1000 Genomes Project databases), and a rare in-frame deletion: NM_000416 c.653_655del, p.E218del previously reported by Jouanguy et al.³ These variants were then confirmed by Sanger sequencing. Targeted Sanger sequencing detected the heterozygous variants, c.653_655del in mother and c.622_623del, p.S208Tfs*21 in father (Figure 2A). Previously reported p.E218del variant was noticed to cause a functional defect without impaired expression or structure of the molecule.³ The novel mutation p.S208Tfs*21 was predicted by MutationTaster to result in nonsense-mediated decay. Although this possibility was not tested experimentally, the frameshift mutation drastically alters the amino acid sequence of the regions of the molecule corresponding to the transmembrane and intracytoplasmic domain (Figure 2B),⁴ thereby contributing to impaired levels of IFN-γR1 on the cell surface demonstrated by flow cytometry (Figure 2C). Flow cytometric analysis showed markedly reduced expression of IFN-γR1 and normal IFN-γR2 on the surface of the patient’s peripheral blood mononuclear cells (Figure 2C). In vitro stimulation with IFN-γ demonstrated a complete absence of STAT-1 phosphorylation compared with a healthy control (Figure 2D).

FIGURE 2.

Demonstration of genetic, flow cytometric and radiographic changes in a patient with AR IFN-γR1 deficiency. (A) Sanger sequencing confirmation of IFNGR1 variants in patient and parents, arrows indicating sites of mutation. (B) Schematic illustration of the wild-type IFN-γR1 protein and the two variants: Variant 1: p.E218del and Variant 2: p.S208Tfs*21. (C) Flow cytometric analysis in patient compared with healthy control, showing significantly reduced IFN-γR1 expression and normal IFN-γR2 expression. (D) Flow cytometric analysis in patient compared with healthy control, showing absent STAT-1 phosphorylation in response to IFN-γ stimulation when gated on monocytes. (E) Computed tomography images comparing hepatosplenomegaly at the peak of illness and at time of recovery

The child was diagnosed with HLH mimic associated with visceral leishmaniasis and mycobacterial disease, as per the recommendations of the North American Consortium for histiocytosis.⁵ Due to rapid clinical worsening in the setting of HLH mimic, he was switched to the Janus kinase (JAK) inhibitor, ruxolitinib (5 mg/day corresponding to 0.4 mg/kg/day), and anakinra was discontinued. Rapid response to ruxolitinib was observed with resolution of fevers and hepatomegaly, and significant improvement in inflammatory markers and splenomegaly (Figure 2E, Figure 1). Ruxolitinib was discontinued after 3 weeks, and corticosteroids were weaned to physiologic dosing. He was continued on TMP-SMX prophylaxis until corticosteroid therapy was discontinued. Antimycobacterial therapy was continued for disseminated Mycobacterium chimera, which was later isolated in lower respiratory, blood, and bone marrow cultures, obtained at the time of florid clinical manifestations of the disease. Repeat blood PCR for Leishmania was negative. Liposomal amphotericin B therapy was gradually decreased to suppressive dosing (Figure 1), which was continued while awaiting definitive therapy with HLA-matched unrelated donor hematopoietic stem cell transplantation, planned in the near future.

In summary, we report a new case of AR IFN-γR1 deficiency due to compound heterozygous IFNGR1 variants including a novel frameshift deletion and a rare in-frame deletion, resulting in impaired IFN-γR1 expression and absent IFN-γR1 function with visceral leishmaniasis and disseminated Mycobacterium chimera infections complicated by HLH-like state. A similar HLH-like presentation has been previously reported in partial autosomal dominant IFN-γR1 deficiency.⁶ However, while in that case residual signaling through IFN-γR is maintained, this was not the case in our patient, suggesting that other IFN-γ-independent mechanisms of immune activation may have triggered the hyperinflammatory manifestations. As described by Humblet-Baron et al,⁷ our patient responded to targeted antimicrobial therapy and to a JAK inhibitor, likely by controlling CD8⁺ T-cell expansion and diminishing exaggerated response to a variety of cytokines. This case emphasizes the importance of utilizing flow assays and molecular assessment for timely diagnosis of this rare, potentially fatal primary immunodeficiency.

DATA AVAILABILITY STATEMENT

Flow cytometry data are available upon request.