Subtypes of familial hemophagocytic lymphohistiocytosis in Japan based on genetic and functional analyses of cytotoxic T lymphocytes

Abstract

Background: Familial hemophagocytic lymphohistiocytosis (FHL) is a rare disease of infancy or early childhood. To clarify the incidence and subtypes of FHL in Japan, we performed genetic and functional analyses of cytotoxic T lymphocytes (CTLs) in Japanese patients with FHL.

Design and methods: Among the Japanese children with hemophagocytic lymphohistiocytosis (HLH) registered at our laboratory, those with more than one of the following findings were eligible for study entry under a diagnosis of FHL: positive for known genetic mutations, a family history of HLH, and impaired CTL-mediated cytotoxicity. Mutations of the newly identified causative gene for FHL5, STXBP2, and the cytotoxicity and degranulation activity of CTLs in FHL patients, were analyzed.

Results: Among 31 FHL patients who satisfied the above criteria, PRF1 mutation was detected in 17 (FHL2) and UNC13D mutation was in 10 (FHL3). In 2 other patients, 3 novel mutations of STXBP2 gene were confirmed (FHL5). Finally, the remaining 2 were classified as having FHL with unknown genetic mutations. In all FHL patients, CTL-mediated cytotoxicity was low or deficient, and degranulation activity was also low or absent except FHL2 patients. In 2 patients with unknown genetic mutations, the cytotoxicity and degranulation activity of CTLs appeared to be deficient in one patient and moderately impaired in the other.

Conclusions: FHL can be diagnosed and classified on the basis of CTL-mediated cytotoxicity, degranulation activity, and genetic analysis. Based on the data obtained from functional analysis of CTLs, other unknown gene(s) responsible for FHL remain to be identified.

Figure 1. Identification of STXBP2 mutations.

(A) Sequencing analysis of 4 patients with non-FHL2/3/4 and detection of 3 novel mutations in 2 of them: a compound heterozygous mutation of 292_294delGCG resulting in Ala98del at exon 5 (upper panel) and 88-1g>a in intron 2 (lower panel) in one patient (UPN28), and a homozygous mutation of 1243-1246AGTG resulting in Ser415ArgfsX6 at exon 15 in the other (UPN29). (B) Expression of STXBP2 cDNA in UPN28 with 88-1g>a mutation. Schematic representation of positions of the primers for RT-PCR and 88-1g>a mutation is shown in the upper panel, and for RT-PCR products from 88-1G>A mutation of STXBP2 in the lower panel. The expected 350-bp product of STXBP2 exons 1–5 was detected in a healthy control individual, whereas extra larger- and smaller-sized products were detected in UPN28 (arrow). (C) Sequence analysis revealed that the 88-1g>a mutation retained the entire intron 2 (243 bp) in the cDNA. This insertion is predicted to cause addition of 81 amino acids to the N-terminal region of the large Sec1 domain of the Munc18-2 protein (upper panel). Sequence analysis of the smaller fragment revealed that the mutation caused skipping of exon 3 (82 bp), resulting in a frame shift and translational arrest after an additional 20 amino acids (lower panel).

Figure 2. Western blot analysis of Munc18-2 protein expression.

Expression of Munc18-2 protein in each CD8⁺ T-cell line that had been stimulated with allogeneic B-LCL cells was analyzed by Western blotting using anti-Munc18-2 antibody. Munc18-2 protein was abundantly detected at 67 kDa in CTL lines established from healthy control individuals and 2 non-FHL2/3/4/5 patients (UPN30, and UPN31).

Figure 3. IFN-γ production by alloantigen-specific CD8⁺ T cell lines.

CD8⁺ T-cell lines were generated from the PBMCs of the patients with FHL and healthy individuals as controls by stimulation with allogeneic B-LCL (KI-LCL) cells. Responder cells were co-cultured with or without KI-LCL or TA-LCL, which shared no HLA antigens with KI-LCL, in the presence or absence of anti-HLA class I monoclonal antibody for 24 hours. IFN-γ production was measured by ELISA. All FHL patients showed normal production of IFN-γ. The HLA type of KI-LCL is HLA-A01/30, B13/17, Cw6/-, DRB1*0701/*0701, and that of TA-LCL is HLA-A24/26, B62/-, Cw4/w9, DRB1*0405/*0901. NS indicates PRF1 nonsense mutation.

Figure 4. Cytotoxicity of alloantigen-specific CD8⁺ T-cell lines.

CD8⁺ T-cell lines were generated from the PBMCs of the patients with FHL and 24 healthy individuals as controls by stimulation with allogeneic B-LCL (KI-LCL) cells. Their cytotoxicity was determined against allogeneic KI-LCL (clear circles) and against allogeneic TA-LCL (solid circles). All FHL patients showed various degrees of impairment of CTL-mediated cytotoxicity against allogeneic B-LCLs. NS indicates PRF1 nonsense mutation.

Figure 5. CD107a expression of alloantigen-specific CD8⁺ T-cell lines.

Flow cytometric analysis of CD107a expression was performed using CD8⁺ T-cell lines generated from a healthy individual and FHL patients, as detailed in the text. Left panel of each column shows CD107a expression in CD8⁺ T cells without any stimulation. Right panel of each column shows CD107a expression in CD8⁺ T cells stimulated with KI-LCL cells.