Germline HAVCR2/TIM-3 Checkpoint Inhibitor Receptor Deficiency in Recurrent Autoinflammatory Myocarditis

Abstract

Myocarditis can be caused by viral infection, drug reaction or general inflammatory condition. To provide understanding on inflammatory myocarditis, we describe clinical, genetic, and immunological properties of a young male patient who suffered from recurrent myocarditis episodes since the age of four years. Electrocardiography, troponin I/T, echocardiography, myocardial magnetic resonance imaging and histological findings were consistent with recurrent myocarditis episodes. Homozygous c.245 A > G p.Tyr82Cys pathogenic variant in Hepatitis A Virus Cellular Receptor 2 (HAVCR2) gene encoding T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) receptor was found. Peripheral blood mononuclear cells were collected when the patient was asymptomatic; CD4⁺ and CD8⁺ T lymphoblasts, CD56⁺ natural killer cells and CD14⁺ monocytes were negative for surface TIM-3 expression. In vitro, TLR4 mediated interleukin-1β (IL-1β) response was high after LPS/ATP stimulation. Clinical symptoms responded to IL-1 receptor antagonist anakinra. TIM-3 p.Tyr82Cys CD4⁺ and CD8⁺ T cell proliferation in vitro was unrestrained. Findings on IL-2, interferon gamma, regulatory T cells, signal transducer and activator of transcription (STAT) 1, 3 and 4 phosphorylation, and PD-1 and LAG-3 checkpoint inhibitor receptor analyses were comparable to controls. We conclude that TIM-3 deficiency due to homozygous HAVCR2 c.245 A > G p.Tyr82Cys pathogenic variant in the patient described here is associated with autoinflammatory symptoms limited to early onset recurrent febrile myocarditis. Excessive IL-1β production and defective regulation of T cell proliferation may contribute to this clinical condition responsive to anakinra treatment.

Keywords: Autoinflammation; Inborn error in immunity; Interleukin-1; Myocarditis; T-lymphocytes.

Fig. 1

(A) Representative electrocardiographs (ECG, 50 mm/s) on days 1 to 5 of hospitalization during an episode of acute myocarditis at age 17 years (B) Late gadolinium enhancement pattern (arrows) consistent with myocarditis in cardiac MRI (C) Representative images of TIM-3 immunostaining in myocardium biopsies. TIM-3 staining was positive (red arrows) in the control and negative in the patient’s samples

Fig. 2

(A) TIM-3 surface and intracellular expression in CD4⁺ T cells. Graph shows quantified mean fluorescence index (MFI) values for cell surface and intracellular TIM-3 expression. (B) TIM-3 surface and intracellular expression in CD8⁺ T cells. Graph shows quantified MFI values for cell surface and intracellular TIM-3 expression. (C) TIM-3 surface and intracellular expression in NK cells. Graph shows quantified MFI values for cell surface and intracellular TIM-3 expression. (D) TIM-3 surface expression in CD14⁺ monocytes with a graph showing quantified MFI values for surface TIM-3 expression

Fig. 3

(A) PD-1 expression in 3-day PHA stimulated CD4⁺ and CD8⁺ T lymphoblasts. (B) LAG-3 expression in 3-day PHA stimulated CD4⁺ and CD8⁺ T lymphoblasts

Fig. 4

(A) IL-1β secretion was measured in LPS/ATP stimulated PBMCs of p.Tyr82Cys index and healthy controls. (B) Interferon-γ production was measured in 5-hour PMA stimulated CD4⁺ and CD8⁺ T cells. (C) STAT4 phosphorylation determined by flow cytometry; PHA and IL-2 pre-stimulated CD4⁺ and CD8⁺ T lymphoblasts were activated with IL-12 for STAT4 phosphorylation

Fig. 5

(A) Proliferation in CD4⁺ T lymphoblasts was determined by flow cytometry after 4-day stimulation with CD3/CD28 antibodies. (B) Proliferated populations of CD4⁺ and CD8⁺ T lymphoblasts after 4-day stimulation with CD3/CD28 antibodies (C) Proliferated populations of CD4⁺ and CD8⁺ T lymphoblasts after 4-day PHA stimulation. (D) Interleukin-2 (IL-2) production in CD4⁺ and CD8⁺ T cells after five-hour PMA stimulation