Defective cytotoxic lymphocyte degranulation in syntaxin-11 deficient familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients

Familial hemophagocytic lymphohistiocytosis (FHL) is typically an early onset, fatal disease characterized by a sepsislike illness with cytopenia, hepatosplenomegaly, and deficient lymphocyte cytotoxicity. Disease-causing mutations have been identified in genes encoding perforin (PRF1/FHL2), Munc13-4 (UNC13D/FHL3), and syntaxin-11 (STX11/FHL4). In contrast to mutations leading to loss of perforin and Munc13-4 function, it is unclear how syntaxin-11 loss-of-function mutations contribute to disease. We show here that freshly isolated, resting natural killer (NK) cells and CD8(+) T cells express syntaxin-11. In infants, NK cells are the predominant perforin-containing cell type. NK cells from FHL4 patients fail to degranulate when encountering susceptible target cells. Unexpectedly, IL-2 stimulation partially restores degranulation and cytotoxicity by NK cells, which could explain the less severe disease progression observed in FHL4 patients, compared with FHL2 and FHL3 patients. Since the effector T-cell compartment is still immature in infants, our data suggest that the observed defect in NK-cell degranulation may contribute to the pathophysiology of FHL, that evaluation of NK-cell degranulation in suspected FHL patients may facilitate diagnosis, and that these new insights may offer novel therapeutic possibilities.     

Macrophage activation syndrome and reactive hemophagocytic lymphohistiocytosis: the same entities?

PURPOSE OF THE REVIEW: One of the most perplexing features of systemic-onset juvenile rheumatoid arthritis is the association with macrophage activation syndrome, a life-threatening complication caused by excessive activation and proliferation of T cells and macrophages. The main purpose of the review is to summarize current understanding of the relation between macrophage activation syndrome and other clinically similar hemophagocytic disorders. RECENT FINDINGS: Clinically, macrophage activation syndrome has strong similarities with familial and virus-associated reactive hemophagocytic lymphohistiocytosis. The better understood familial hemophagocytic lymphohistiocytosis is a constellation of rare, autosomal recessive immune disorders. The most consistent immunologic abnormalities in patients with familial hemophagocytic lymphohistiocytosis are decreased natural killer and cytotoxic cell functions. In approximately one third of familial hemophagocytic lymphohistiocytosis patients, these immunologic abnormalities are secondary to mutations in the gene encoding perforin, a protein that mediates cytotoxic activity of natural killer and cytotoxic CD8+ T cells. Several recent studies have suggested that profoundly depressed natural killer cell activity and abnormal levels of perforin expression may be a feature of macrophage activation syndrome in systemic-onset juvenile rheumatoid arthritis as well. Although it has been proposed that in both hemophagocytic lymphohistiocytosis and macrophage activation syndrome, natural killer and cytotoxic cell dysfunction may lead to inadequate control of cellular immune responses, the exact nature of such dysregulation and the relation between macrophage activation syndrome and hemophagocytic lymphohistiocytosis still remain to be determined.     

Atypical familial hemophagocytic lymphohistiocytosis due to mutations in UNC13D and STXBP2 overlaps with primary immunodeficiency diseases

Background

Familial hemophagocytic lymphohistiocytosis is a genetic disorder of lymphocyte cytotoxicity that usually presents in the first two years of life and has a poor prognosis unless treated by hematopoietic stem cell transplantation. Atypical courses with later onset and prolonged survival have been described, but no detailed analysis of immunological parameters associated with typical versus atypical forms of familial hemophagocytic lymphohistiocytosis has been performed.

Design and Methods

We analyzed disease manifestations, NK-cell and T-cell cytotoxicity and degranulation, markers of T-cell activation and B-cell differentiation as well as Natural Killer T cells in 8 patients with atypical familial hemophagocytic lymphohistiocytosis due to mutations in UNC13D and STXBP2.

Results

All but one patient with atypical familial hemophagocytic lymphohistiocytosis carried at least one splice-site mutation in UNC13D or STXBP2. In most patients episodes of hemophagocytic lymphohistiocytosis were preceded or followed by clinical features typically associated with immunodeficiency, such as chronic active Epstein Barr virus infection, increased susceptibility to bacterial infections, granulomatous lung or liver disease, encephalitis or lymphoma. Five of 8 patients had hypogammaglobulinemia and reduced memory B cells. Most patients had a predominance of activated CD8⁺ T cells and low numbers of Natural Killer T cells. When compared to patients with typical familial hemophagocytic lymphohistiocytosis, NK-cell cytotoxicity and NK-cell and CTL degranulation were impaired to a similar extent. However, in patients with an atypical course NK-cell degranulation could be partially reconstituted by interleukin-2 and cytotoxic T-cell cytotoxicity in vitro was normal.

Conclusions

Clinical and immunological features of atypical familial hemophagocytic lymphohistiocytosis show an important overlap to primary immunodeficiency diseases (particularly common variable immunodeficiency and X-linked lymphoproliferative syndrome) and must, therefore, be considered in a variety of clinical presentations. We show that degranulation assays are helpful screening tests for the identification of such patients.     

An inframe perforin gene deletion in familial hemophagocytic lymphohistiocytosis is associated with perforin expression

Familial hemophagocytic lymphohistiocytosis is an autosomal recessive disease of early childhood manifested by hypercytokinemia and organ infiltration of macrophages and activated lymphocytes, and it is characterized by a fulminant clinical course. The molecular mechanism underlying this disease appears to be a deregulation of apoptosis of activated T cells and macrophages. Approximately 20-40% of patients with familial hemophagocytic lymphohistiocytosis reported worldwide had a perforin gene mutation. We report herein a novel perforin variant in the homozygous state in an Omani boy who was diagnosed 44 days after birth. Sequence analysis of the perforin gene coding region revealed a 12-base pair deletion (codon 284-287) resulting in the deletion of four amino acids in the membrane attack complex domain of the protein. This deletion maintains the reading frame of the perforin mRNA. Both parents were heterozygotes for this molecular defect. Flow-cytometric analysis revealed intracellular perforin expression at the lower end of the normal range in the cytotoxic T cells (CD3+/CD8+) and (CD3+/CD56+) and in around 50% of the natural killer cells (CD3-/CD56+). This is an additional example of a perforin variant which is associated with a significant level of cellular perforin expression and thus confirms that drastic reduction in its expression is not a constant feature in familial hemophagocytic lymphohistiocytosis type 2.     

Perforin defects of primary haemophagocytic lymphohistiocytosis in Japan

The perforin gene was analysed in 15 Japanese patients with primary haemophagocytic lymphohistiocytosis (HLH). Perforin gene defects were found in two out of eight patients with familial HLH (FHL), and one out of seven without affected siblings. Four novel mutations were identified. Compound heterozygous mutations (one FHL and one sporadic HLH) and only one allele mutation (one FHL) were defined. Flow cytometry revealed no perforin expression in CD8+ or CD56+ cells from a surviving patient with a mutation. The frequency of mutation was at least 20% of FHL in Japan. Flow cytometry for intracellular perforin may be useful for the screening of FHL2.     

Severe and progressive encephalitis as a presenting manifestation of a novel missense perforin mutation and impaired cytolytic activity

Mutations in the perforin gene cause familial hemophagocytic lymphohistiocytosis (FHL). The first symptoms of FHL are usually intractable fever, hepatosplenomegaly, and pancytopenia. Most FHL patients subsequently develop central nervous system (CNS) manifestations due to infiltration of tissues by activated lymphocytes and macrophages. We report 2 FHL patients with an atypical phenotype characterized by isolated severe neurologic symptoms mimicking chronic encephalitis and leading to an early death. Functional and molecular analyses revealed the same novel missense mutation in the perforin gene in both patients; this mutation affected the calcium-binding domain of the protein. This missense mutation did not affect perforin maturation or expression in cytotoxic cells but impaired in vitro cytotoxic activity. Diagnosis was delayed in both patients because of the initial neurologic expression and the normal expression of perforin in circulating lymphocytes. This emphasizes the importance of early diagnosis of this atypical form of FHL, as CNS involvement causes severe, irreversible encephalopathy. This observation also raises the question of the role of some mutations in the neurologic expression of FHL.     

Familial hemophagocytic lymphohistiocytosis associated with disseminated T-cell lymphoma: A report of two siblings

Summary Two siblings with evidence of disseminated T-cell lymphoma at the time of diagnosis of familial hemophagocytic lymphohistiocytosis (FHL) are reported, an association which has not been described previously. The first child with typical clinical and laboratory features of FHL died shortly after admission, before diagnosis could be established. Retrospective analysis of autoptic tissue revealed marked hemophagocytosis as well as morphological and immunohistochemical features suggestive of disseminated T-cell lymphoma. In the second child, FHL was diagnosed in time. Subsequent histologic investigation of bone marrow biopsies displayed a focal infiltration by T-cell lymphoma. DNA hybridization studies provided evidence of a monoclonal T-cell receptor beta chain gene rearrangement. Following conventional chemotherapeutic induction for FHL, the patient received an allogeneic bone marrow transplant (BMT) from a related healthy donor. Currently, 17 months after BMT, the boy is in unmaintained remission from FHL and T-cell lymphoma. The current pathogenetic concepts for FHL and a possible relationship between T-cell lymphoma and FHL are discussed.     

Late-onset cases of familial hemophagocytic lymphohistiocytosis with missense perforin gene mutations

Since the discovery of perforin gene mutations in familial hemophagocytic lymphohistiocytosis (FHL) type 2, heterogeneous features in FHL2 patients have been identified in a report of Feldmann et al. as the beginning. This study was conducted to determine the impact of characteristic gene mutations on late-onset (age > or = 7 years) hemophagocytic lymphohistiocytosis episodes. We analyzed perforin gene mutations in three late-onset cases from our registry in Japan and an additional 10 cases from the literature. Of the 13 cases with onset ages of a median of 10 (range 7-49) years, nine had homozygous and four had compound heterozygous missense mutations of the perforin gene. None had homozygous nonsense mutations. Our data suggest that nonsense perforin gene mutations yield early onset and missense mutations late onset in FHL2 cases.     

Critical role for perforin and Fas-dependent killing of dendritic cells in the control of inflammation

After stimulation of antigen-specific T cells, dendritic cell (DCs) are susceptible to killing by these activated T cells that involve perforin and Fas-dependent mechanisms. Fas-dependent DC apoptosis has been shown to limit DC accumulation and prevent the development of autoimmunity. However, a role for perforin in the maintenance of DC homeostasis for immune regulation remains to be determined. Here we show that perforin deficiency in mice, together with the deletion of Fas in DCs (perforin(-/-)DC-Fas(-/-)), led to DC accumulation, uncontrolled T-cell activation, and IFN-γ production by CD8+ T cells, resulting in the development of lethal hemophagocytic lymphohistiocytosis. Consistently, adoptive transfer of Fas(-/-) DCs induced over-activation and IFN-γ production in perforin(-/-) CD8+ T cells. Neutralization of IFN-γ prevented the spreading of inflammatory responses to different cell types and protected the survival of perforin(-/-)DC-Fas(-/-) mice. Our data suggest that perforin and Fas synergize in the maintenance of DC homeostasis to limit T cell activation, and prevent the initiation of an inflammatory cascade.     

Perforin gene analaysis in an Iranian family with familial hemophagocytic lymphohistiocytosis

Perforin gene (PRF1) mutations have been reported in 20-30% of patients with familial hemophagocytic lymphohistiocytosis (FHL), an immune disorder of infancy and early childhood. Cytotoxic T and natural killer (NK) cell activities are remarkably reduced or absent in FHL patients. We report the first cases of familial hemophagocytic lymphohistiocytosis in an Iranian family with two siblings. Exons 2 and 3 of the PRF1 gene were analyzed by polymerase chain reaction (PCR) amplification and direct sequencing. Perforin gene mutation(s) were detected in none of the cases. The result of our study indicates that not much evidence is present concerning a correlation between perforin gene defects and familial hemophagocytic lymphohistiocytosis etiology in these cases.     

Immunophenotypic analysis of Epstein-Barr virus (EBV)-infected CD8(+) T cells in a patient with EBV-associated hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a severe and often fatal condition characterized by uncontrolled activation of T cells and macrophages. In Epstein-Barr virus (EBV)-associated HLH (EBV-HLH), the pathogenic roles of ectopic EBV infection in the T-cell population and of clonal proliferation of EBV-infected T cells has been described. However, the immunophenotype of EBV-infected T cells has not been fully characterized. Here we describe a case of EBV-HLH presenting with a massive clonal proliferation of CD8(+) T cells with TCR VB14. Analysis of in situ hybridization for EBV-encoded small RNA1 showed that only CD8(+) T cells harbored EBV in this patient. The EBV-infected TCR VB14(+) CD8(+) T cells exhibited unique immunophenotypic features including lacked CD5 expression and a markedly bright expression of HLA-DR. After initiation of treatment with prednisolone, etoposide, and cyclosporin A, the percentage of infected cells declined progressively in parallel with other serum markers such as ferritin. These findings suggest that lacking expression of CD5 on CD8(+) T cells with specific TCR VB may serve as a useful marker of dysregulated T-cell activation and proliferation in EBV-HLH.     

Reduced perforin expression in systemic juvenile idiopathic arthritis is restored by autologous stem-cell transplantation

OBJECTIVES: Familial haemophagocytic lymphohistiocytosis (FHL) is a disorder characterized by deficient cytotoxic T-cell function and activated macrophages, owing to a defect in the perforin gene and absent perforin expression. Because symptoms of patients with systemic juvenile idiopathic arthritis (sJIA) are sometimes clinically very similar to those with FHL, we studied whether perforin expression in sJIA patients would be reduced also. METHODS: We determined the perforin expression levels on two subsets of CD8(+) cells (CD8(+)CD28(-)CD45RA(-) and CD8(+)CD28(-)CD45RA(+)) and natural killer (NK) cells from patients with sJIA under conventional treatment as well as before and after autologous stem-cell transplantation (ASCT). RESULTS: CD45RA(-) cytotoxic effector cells of sJIA patients (n=13) express significantly lower levels of perforin than polyarticular juvenile idiopathic arthritis (pJIA, n=9) patients [sJIA mean fluorescence intensity (MFI) 34.6; pJIA MFI 98.0] or control donors (MFI 124.6, n=5). A similar pattern was seen in the CD45RA(+) subset. Also NK cells from sJIA patients expressed significantly less intracellular perforin (sJIA MFI 398.4; controls MFI 972.4). In four patients with sJIA who were treated with ASCT, a clear increase in perforin expression was found at 12 months after ASCT in both cytotoxic effector cell subsets (CD45RA(-) subset before ASCT MFI 13.2; 12 months after ASCT MFI 172.3). CONCLUSION: We conclude that perforin expression can be severely reduced in sJIA. This finding may implicate defective cytotoxicity and haemophagocytosis and could thus explain why sJIA may be complicated by macrophage activation syndrome. ASCT leads to a reconstitution of the (T cell) immune system with a normal expression of perforin.     

A prospective evaluation of degranulation assays in the rapid diagnosis of familial hemophagocytic syndromes

Familial hemophagocytic lymphohistiocytosis (FHL) is a life-threatening disorder of immune regulation caused by defects in lymphocyte cytotoxicity. Rapid differentiation of primary, genetic forms from secondary forms of hemophagocytic lymphohistiocytosis (HLH) is crucial for treatment decisions. We prospectively evaluated the performance of degranulation assays based on surface up-regulation of CD107a on natural killer (NK) cells and cytotoxic T lymphocytes in a cohort of 494 patients referred for evaluation for suspected HLH. Seventy-five of 77 patients (97%) with FHL3-5 and 11 of 13 patients (85%) with Griscelli syndrome type 2 or Chediak-Higashi syndrome had abnormal resting NK-cell degranulation. In contrast, NK-cell degranulation was normal in 14 of 16 patients (88%) with X-linked lymphoproliferative disease and in 8 of 14 patients (57%) with FHL2, who were identified by diminished intracellular SLAM-associated protein (SAP), X-linked inhibitor of apoptosis protein (XIAP), and perforin expression, respectively. Among 66 patients with a clinical diagnosis of secondary HLH, 13 of 59 (22%) had abnormal resting NK-cell degranulation, whereas 0 of 43 had abnormal degranulation using IL-2-activated NK cells. Active disease or immunosuppressive therapy did not impair the assay performance. Overall, resting NK-cell degranulation below 5% provided a 96% sensitivity for a genetic degranulation disorder and a specificity of 88%. Therefore, degranulation assays allow a rapid and reliable classification of patients, benefiting treatment decisions.     

A Novel Perforin Gene Mutation in a Japanese Family with Hemophagocytic Lymphohistiocytosis

A 4-month-old girl with clinical features of hemophagocytic lymphohistiocytosis (HLH) was successfully treated with immunochemotherapy but died at the age of 1 year and 3 months, before hematopoietic stem cell transplantation could be performed. Her family history showed death during infancy of the eldest sister, suggesting a diagnosis of familial HLH (FHL). Direct sequencing of the DNA extracted from the patient's spleen tissue obtained at autopsy revealed a novel perforin gene mutation: a homozygous 1289G insertion (Asp430 frameshift and termination at amino acid residue 457), which has not previously been reported in FHL patients.     

Genetic subtypes of familial hemophagocytic lymphohistiocytosis: correlations with clinical features and cytotoxic T lymphocyte/natural killer cell functions

Mutations of the perforin (PRF1) and MUNC13-4 genes distinguish 2 forms of familial hemophagocytic lymphohistiocytosis (FHL2 and FHL3, respectively), but the clinical and biologic correlates of these genotypes remain in question. We studied the presenting features and cytotoxic T lymphocyte/natural killer (CTL/NK) cell functions of 35 patients for their relationship to distinct FHL subtypes. FHL2 (n = 11) had an earlier onset than either FHL3 (n = 8) or the non-FHL2/FHL3 subtype lacking a PRF1 or MUNC13-4 mutation (n = 16). Deficient NK cell activity persisted after chemotherapy in all cases of FHL2, whereas some patients with FHL3 or the non-FHL2/FHL3 subtype showed partial recovery of this activity during remission. Alloantigen-specific CTL-mediated cytotoxicity was deficient in FHL2 patients with PRF1 nonsense mutations, was very low in FHL3 patients, but was only moderately reduced in FHL2 patients with PRF1 missense mutations. These findings correlated well with Western blot analyses showing an absence of perforin in FHL2 cases with PRF1 nonsense mutations and of MUNC13-4 in FHL3 cases, whereas in FHL2 cases with PRF1 missense mutations, mature perforin was present in low amounts. These results suggest an association between the type of genetic mutation in FHL cases and the magnitude of CTL cytolytic activity and age at onset.     

Chronic active Epstein-Barr virus infection associated with mutations in perforin that impair its maturation

Chronic active Epstein-Barr virus infection (CAEBV) is a rare disease in which previously healthy persons develop severe, life-threatening illness. Mutations in the perforin gene have been found in familial hemophagocytic lymphohistiocytosis, which shares some features with CAEBV. We studied a patient who died at age 18, 10 years after the onset of CAEBV. The patient had high titers of antibodies to EBV, EBV RNA in lymph nodes, T-cell lymphoproliferative disease, and hemophagocytic lymphohistiocytosis. DNA sequencing showed novel mutations in both alleles of the perforin gene that resulted in amino acid changes in the protein. The quantity of the native form of perforin from the patient's stimulated peripheral blood mononuclear cells (PBMCs) was extremely low and immunoblotting showed accumulation of an uncleaved precursor form of perforin. Stimulated PBMCs from the patient were defective for Fas-independent cytotoxicity. These data imply that mutations in this patient resulted in reduced perforin-mediated cytotoxicity by his lymphocytes. This is the first case in which perforin mutations have been shown to result in accumulation of the uncleaved, immature form of perforin. Mutations in the perforin gene are associated with some cases of CAEBV with hemophagocytic lymphohistiocytosis.     

Fatal immune dysregulation due to a gain of glycosylation mutation in lymphocyte perforin

Mutations in the perforin gene (PRF1) are a common cause of the fatal immune dysregulation disorder, familial hemophagocytic lymphohistiocytosis (type 2 FHL, FHL2). Here we report a female infant born with biallelic PRF1 mutations: a novel substitution, D49N, and a previously identified in-frame deletion, K285del. We assessed the effects of each mutation on the cytotoxicity of human NK cells in which the expression of endogenous perforin was ablated with miR30-based short hairpin (sh) RNAs. Both mutations were detrimental for function, thereby explaining the clinically severe presentation and rapidly fatal outcome. We demonstrate that D49N exerts its deleterious effect by generating an additional (third) N-linked glycosylation site, resulting in protein misfolding and degradation in the killer cell. Our data provide a rationale for treating some cases of type 2 familial hemophagocytic lymphohistiocytosis, based on the pharmacologic inhibition or modification of glycosylation.     

Novel spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis in ethnic Omani patients

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive immune disorder, characterized by fever, hepatosplenomegaly, pancytopenia, hypertriglyceridemia, hypofibrinogenemia, markedly elevated levels of inflammatory cytokines, and impaired cytotoxic activity of lymphocytes. FHL is often fatal in early infancy. Histologic features include organ infiltration by activated macrophages and lymphocytes. Four genetic loci (FHL1, 2, 3, and 4) have been identified, of which FHL2 involves mutations in the perforin gene and is present in 20-50% of patients with FHL. We herein report the first comprehensive molecular analysis of 16 unrelated cases of FHL in ethnic Omanis. Using direct DNA sequencing analysis in 11 families, seven different mutations were identified in the coding region of the perforin gene, of which five were novel. Perforin gene defects do not seem to be involved in one-third of the cases of FHL in ethnic Omanis.     

Perforin expression in cytotoxic lymphocytes from patients with hemophagocytic lymphohistiocytosis and their family members.

Mutations in the perforin gene have been described in some patients with hemophagocytic lymphohistiocytosis (HLH), but the role of perforin defects in the pathogenesis of HLH remains unclear. Four-color flow cytometric analysis was used to establish normal patterns of perforin expression for control subjects of all ages, and patterns of perforin staining in cytotoxic lymphocytes (natural killer [NK] cells, CD8(+) T cells, CD56(+) T cells) from patients with HLH and their family members were studied. Eleven unrelated HLH patients and 19 family members were analyzed prospectively. Four of the 7 patients with primary HLH showed lack of intracellular perforin in all cytotoxic cell types. All 4 patients showed mutations in the perforin gene. Their parents, obligate carriers of perforin mutations, had abnormal perforin-staining patterns. Analysis of cytotoxic cells from the other 3 patients with primary HLH and remaining family members had normal percentages of perforin-positive cytotoxic cells. On the other hand, the 4 patients with Epstein-Barr virus-associated HLH typically had depressed numbers of NK cells but markedly increased proportions of CD8(+) T cells with perforin expression. Four-color flow cytometry provides diagnostic information that, in conjunction with evidence of reduced NK function, may speed the identification of life-threatening HLH in some families and direct further genetic studies of the syndrome.     

Review of hemophagocytic lymphohistiocytosis (HLH) in children with focus on Japanese experiences

Hemophagocytic lymphohistiocytosis (HLH) is characterized by fever and hepatosplenomegaly associated with pancytopenia, hypertriglyceridemia and hypofibrinogenemia. Increased levels of cytokines and impaired natural killer activity are biological markers of HLH. HLH can be classified into two distinct forms, including primary HLH, also referred to as familial hemophagocytic lymphohistiocytosis (FHL), and secondary HLH. Although FHL is an autosomal recessive disorder typically occurring in infancy, it is important to clarify that the disease may also occur in older patients. It is now considered that FHL is a disorder of T-cell function; moreover, clonal proliferation of T lymphocytes is observed in a few FHL patients, and cytotoxicity of these T lymphocytes for target cells is usually impaired. In 1999, perforin gene (PRF1) mutation was identified as a cause of 20-30% of FHL (FHL2) cases. In Japan, two specific mutations of PRF1 were also detected. Furthermore, in 2003, MUNC13-4 mutations were identified in some non-FHL2 patients (FHL3). Identification of other genes responsible for remaining cases is a major concern. Hematopoietic stem cell transplantation (HSCT) has been established as the only accepted curative therapy for FHL. Thus, appropriate diagnosis and prompt treatment with HSCT are necessary for FHL patients. Genetic analysis for PRF1 and MUNC13-4 and functional assay of cytotoxic T lymphocytes are recommended to be performed in each patient. In those patients displaying impaired cytotoxic function but lacking genetic defects, samples should be employed for identification of unknown genes. In the near future, an entire pathogenesis should be clarified in order to establish appropriate therapies including immunotherapy, HSCT and gene therapy.