Clinical course
A 4-month-old Japanese female was admitted to our hospital with a developed fever and hepatosplenomegaly. Laboratory testing revealed pancytopenia (white blood cell [WBC] of 2.7 × 109/L, hemoglobin of 108 g/L, platelet of 14 × 109/L), liver dysfunction (aspartate transferase of 410 U/L and alanine aminotransferase of 111 U/L), elevated serum lactate dehydrogenase (2,909 U/L) and ferritin (12,122 U/ml), and severe coagulopathy (Table S1).
Bone marrow smear preparation revealed hemophagocytosis, but with no malignant cells. Chromosomal analysis of bone marrow cells revealed a normal karyotype. The case was diagnosed with HLH and was treated with dexamethasone, cyclosporine A, and prednisolone, which improve her condition. However, γ-globulin was gradually increased and thrombocytopenia proceeded along with the dose de-escalation.
She was suspected of ALPS at 2 years because of increased αβDNTs in her peripheral blood. However, no ALPS-related genes (FAS, FASL, CASP 10, and FADD) alteration were detected. A whole-exome sequencing was performed at the age of 2 years and 3 months, which detected a somatic mosaic variant of NRAS G13D, leading to RALD diagnosis. NRAS G13D mutation was observed as mosaicism in further analysis. Mutated allele was observed in the nails (0–22%) and blood cells (67%), including T cells, natural killer cells, and αβDNTs. Almost simultaneously, complement factors began to decrease, serum double-strand DNA antibody was increased, facial erythema and proteinuria appeared, and anti-dsDNA antibody turned positive. A renal biopsy revealed lupus type V, and she was diagnosed with systemic lupus erythematosus (SLE) and lupus nephritis. She presented monocytosis at 3 years and was tentatively diagnosed with JMML. These symptoms were improved with tacrolimus and remained in remission, although monocytes were repeatedly increased. However, monocytes markedly increased again at around 4 years of age. Bone marrow examination revealed mild dysplasia and high granulocyte-macrophage colony-stimulating factor (GM-CSF) sensitivity, which was compatible with the JMML diagnosis (Figure S1).
Around this time, she began to have repeated hospitalizations for shock state (Figure S1). Sometimes, the infection was the trigger, but at other times the cause was unclear, and autoinflammation associated with the primary disease was thought to trigger the condition.
Respiratory distress symptoms appeared at the age of 6 years, and a chest computed tomography scan revealed mottled shadows around the bronchioles (Fig. 1A). A thoracoscopic lung biopsy was performed at the age of 7 years, which revealed monocyte infiltration in the lung tissue.
She was diagnosed with acute exacerbation of JMML, accompanying pulmonary infiltration, and we decided to perform allogeneic HCT. She underwent HCT from an HLA fully matched unrelated donor. The conditioning regimen consisted of total body irradiation (3 Gy on day-3), busulfan (0.8 mg/kg × 4/day on days − 11 and − 10), L-PAM (90 mg/m2 × 2 from days − 5 and − 4), and Flu (30 mg/m2 from days − 9 to -6). Tacrolimus and short-term methotrexate (15 mg on day 3, 10 mg on day 7, and 10 mg on day 15) were employed for the prophylaxis of graft-versus-host disease (GVHD). Bone marrow transplantation (BMT) with 3 × 105 CD34 cells /kg was transfused. BMT with 4.8 × 106 total nucleus cells/kg was transfused. The case suffered several transplantation-related complications, including Grade 1 acute GVHD (mucositis, skin rash, fever, and diarrhea), which were detected on day 21 and were controllable with prednisolone. Hematological reconstitution was normally observed. A WBC count of > 1 × 109/L, a neutrophil count of > 500/µl, and a platelet count of > 50 × 109/L were observed on days 15, 26, and 39 after BMT, respectively. The αβDNTs had decreased at normal levels (Fig. 1B).
αβDNTs analysis
The αβDNTs of this case with RALD expressed CD45RO and chemokine receptors CCR4 and CCR6. However, the αβDNTs cells of the case with ALPS expressed few of these (Figs. 2 and 3). In contrast, CXCR3 was not expressed on αβDNTs of RALD but expressed on these of ALPS (Fig. 3).
CD45RO is the surface marker of memory T cells, and CCR4, CCR6, and CXCR3 can help discriminate between helper T cell subtypes: Th1: CCR4−CCR6−CXCR3+, Th2: CCR4+CCR6−CXCR3−, and Th17: CCR4+CCR6+CXCR3− (Supplementary Figure S2).
The αβDNTs of RALD were recognized as CD45RO+CCR4+CCR6+CXCR3− in general [14], thereby indicating similarity as Th17 other than CD4 expression. Th17 produces IL-17A and αβDNTs of ALPS produce IL-10 [15], thus we analyzed whether αβDNTs produce IL-17A and IL-10 or not. The αβDNTs of RALD produced IL-17A and ALPS did not produce it. Conversely, the αβDNTs of RALD did not produce IL-10. Hence, this RALD case produced IL-17A and little IL-10, while the two ALPS cases produced IL-10 without IL-17A (Fig. 4).
Moreover, we analyzed RORγt expression in the αβDNTs. RORγt is the master regulator transcription factor for Th17, and Th17 has intracellular RORγt expression [16, 17]. The αβDNTs of RALD had intracellular RORγt expression but not with ALPS (Fig. 5).