Sandra Ammann

The syndrome of hemophagocytic lymphohistiocytosis in primary immunodeficiencies: implications for differential diagnosis and pathogenesis

Hemophagocytic lymphohistiocytosis is a hyperinflammatory syndrome defined by clinical and laboratory criteria. Current criteria were created to identify patients with familial hemophagocytic lmyphohistiocytosis in immediate need of immunosuppressive therapy. However, these criteria also identify patients with infection-associated hemophagocytic inflammatory states lacking genetic defects typically predisposing to hemophagocytic lymphohistiocytosis. These patients include those with primary immunodeficiencies, in whom the pathogenesis of the inflammatory syndrome may be distinctive and aggressive immunosuppression is contraindicated. To better characterize hemophagocytic inflammation associated with immunodeficiencies, we combined an international survey with a literature search and identified 63 patients with primary immunodeficiencies other than cytotoxicity defects or X-linked lymphoproliferative disorders, presenting with conditions fulfilling current criteria for hemophagocytic lymphohistiocytosis. Twelve patients had severe combined immunodeficiency with <100/μL T cells, 18 had partial T-cell deficiencies; episodes of hemophagocytic lymphohistiocytosis were mostly associated with viral infections. Twenty-two patients had chronic granulomatous disease with hemophagocytic episodes mainly associated with bacterial infections. Compared to patients with cytotoxicity defects, patients with T-cell deficiencies had lower levels of soluble CD25 and higher ferritin concentrations. Other criteria for hemophagocytoc lymphohistiocytosis were not discriminative. Thus: (i) a hemophagocytic inflammatory syndrome fulfilling criteria for hemophagocytic lymphohistiocytosis can be the initial manifestation of primary immunodeficiencies; (ii) this syndrome can develop despite severe deficiency of T and NK cells, implying that the pathophysiology is distinct and not appropriately described as “lympho”-histiocytosis in these patients; and (iii) current criteria for hemophagocytoc lymphohistiocytosis are insufficient to differentiate hemophagocytic inflammatory syndromes with different pathogeneses. This is important because of implications for therapy, in particular for protocols targeting T cells.

The risk of hemophagocytic lymphohistiocytosis in Hermansky-Pudlak syndrome type 2.

Genetic disorders of lymphocyte cytotoxicity predispose patients to hemophagocytic lymphohistiocytosis (HLH). Reduced lymphocyte cytotoxicity has been demonstrated in Hermansky-Pudlak syndrome type 2 (HPS2), but only a single patient was reported who developed HLH. Because that patient also carried a potentially contributing heterozygous RAB27A mutation, the risk for HLH in HPS2 remains unclear. We analyzed susceptibility to HLH in the pearl mouse model of HPS2. After infection with lymphocytic choriomeningitis virus, pearl mice developed all key features of HLH, linked to impaired virus control caused by a moderate defect in CTL cytotoxicity in vivo. However, in contrast to perforin-deficient mice, the disease was transient, and all mice fully recovered and controlled the infection. An additional heterozygous Rab27a mutation did not aggravate the cytotoxicity defect or disease parameters. In the largest survey of 22 HPS2 patients covering 234 patient years, we identified only 1 additional patient with HLH and 2 with incomplete transient HLH-like episodes, although cytotoxicity or degranulation was impaired in all 16 patients tested. HPS2 confers a risk for HLH that is lower than in Griscelli or Chediak-Higashi syndrome, probably because of a milder defect in cytotoxicity. Preemptive hematopoietic stem cell transplantation does not appear justified in HPS2.

Mutations in AP3D1 associated with immunodeficiency and seizures define a new type of Hermansky-Pudlak syndrome

Genetic disorders affecting biogenesis and transport of lysosome-related organelles are heterogeneous diseases frequently associated with albinism. We studied a patient with albinism, neutropenia, immunodeficiency, neurodevelopmental delay, generalized seizures, and impaired hearing but with no mutation in genes so far associated with albinism and immunodeficiency. Whole exome sequencing identified a homozygous mutation in AP3D1 that leads to destabilization of the adaptor protein 3 (AP3) complex. AP3 complex formation and the degranulation defect in patient T cells were restored by retroviral reconstitution. A previously described hypopigmented mouse mutant with an Ap3d1 null mutation (mocha strain) shares the neurologic phenotype with our patient and shows a platelet storage pool deficiency characteristic of Hermansky-Pudlak syndrome (HPS) that was not studied in our patient because of a lack of bleeding. HPS2 caused by mutations in AP3B1A leads to a highly overlapping phenotype without the neurologic symptoms. The AP3 complex exists in a ubiquitous and a neuronal form. AP3D1 codes for the AP3δ subunit of the complex, which is essential for both forms. In contrast, the AP3β3A subunit, affected in HPS2 patients, is substituted by AP3β3B in the neuron-specific heterotetramer. AP3δ deficiency thus causes a severe neurologic disorder with immunodeficiency and albinism that we propose to classify as HPS10.

A new functional assay for the diagnosis of X-linked inhibitor of apoptosis (XIAP) deficiency

X‐linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and a highly variable clinical presentation. Current diagnostic screening tests such as flow cytometry for XIAP expression or lymphocyte apoptosis assays have significant limitations. Based on recent evidence that XIAP is essential for nucleotide‐binding and oligomerization domains (NOD)1/2 signalling, we evaluated the use of a simple flow cytometric assay assessing tumour necrosis factor (TNF) production of monocytes in response to NOD2 stimulation by muramyl dipeptides (L18‐MDP) for the functional diagnosis of XIAP deficiency. We investigated 12 patients with XIAP deficiency, six female carriers and relevant disease controls. Irrespective of the diverse clinical phenotype, the extent of residual protein expression or the nature of the mutation, the TNF response was severely reduced in all patients. On average, L18‐MDP induced TNF production in 25% of monocytes from healthy donors or female carriers, while fewer than 6% of monocytes responded in affected patients. Notably, the assay clearly discriminated affected patients from disease controls with other immunodeficiencies accompanied by lymphoproliferation, hypogammaglobulinaemia or inflammatory bowel disease. Functional testing of the NOD2 signalling pathway is an easy, fast and reliable assay in the diagnostic evaluation of patients with suspected XIAP deficiency.

The minimum required level of donor chimerism in hereditary hemophagocytic lymphohistiocytosis

Reduced-intensity conditioning has improved survival after hematopoietic stem cell transplantation (HSCT) for hemophagocytic lymphohistiocytosis (HLH) at the cost of more frequent mixed chimerism. The minimum level of donor chimerism (DC) required to prevent HLH reactivation in humans remains to be determined. In a multicenter retrospective study, 103 patients transplanted for hereditary HLH (2000-2013) and DC permanently or transiently <75% (overall, CD3(+), CD56(+)) were analyzed regarding DC, specific immunologic function, occurrence of systemic reactivations (≥5/8 HLH criteria), partial systemic flares (<5 criteria and HLH-directed treatment), isolated central nervous system reactivations, and management. Recurrence was reported in 18 patients (systemic reactivation n = 11, partial flare n = 3, isolated central nervous system reactivation n = 4). Ten events occurred during profound immune suppression before day 180 (median DC, 10%; range, 1-100%; CD3(+) if available, otherwise overall DC), which renders a differentiation between secondary post-HSCT HLH and HLH related to the genetic defect difficult. Eight events occurred between 0.5 and 6.7 years post-HSCT (median DC, 13%; range, 0-30%). In 5 patients, overall and lineage-specific DC were ≤10% for >6 months (median, 5.1; range, 1.1-10 years) without reactivation. A second HSCT was performed in 18 patients (median, DC 4%; range, 0-19%). Death from reactivation occurred in 4 patients (22% of recurrences). Six patients died of transplant complications following a second HSCT (33% of second HSCT). We conclude that a DC >20%-30% is protective against late reactivation. Lower levels do not, however, inescapably result in recurrences. The decision for or against second HSCT must be based on a thorough risk assessment.

Primary and secondary hemophagocytic lymphohistiocytosis have different patterns of T-cell activation, differentiation and repertoire

Hemophagocytic lymphohistiocytosis (HLH) is a life‐threatening inflammatory syndrome characterized by hyperactivation of lymphocytes and histiocytes. T cells play a key role in HLH pathogenesis, but their differentiation pattern is not well characterized in patients with active HLH. We compared T‐cell activation patterns between patients with familial HLH (1°HLH), 2°HLH without apparent infectious trigger (2°HLH) and 2°HLH induced by a viral infection (2°V‐HLH). Polyclonal CD8+ T cells are highly activated in 1°HLH and 2°V‐HLH, but less in 2°HLH as assessed by HLA‐DR expression and marker combination with CD45RA, CCR7, CD127, PD‐1 and CD57. Absence of increased HLA‐DR expression on T cells excluded active 1° HLH with high sensitivity and specificity. A high proportion of polyclonal CD127−CD4+ T cells expressing HLA‐DR, CD57, and perforin is a signature of infants with 1°HLH, much less prominent in virus‐associated 2°HLH. The similar pattern and extent of CD8+ T‐cell activation compared to 2° V‐HLH is compatible with a viral trigger of 1°HLH. However, in most 1°HLH patients no triggering infection was documented and the unique activation of cytotoxic CD4+ T cells indicates that the overall T‐cell response in 1°HLH is different. This may reflect different pathways of pathogenesis of these two HLH variants.

Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperferritinemic systemic inflammatory disorders. Although profound cytotoxic impairment causes familial HLH (fHLH), the mechanisms driving non-fHLH and MAS are largely unknown. MAS occurs in patients with suspected rheumatic disease, but the mechanistic basis for its distinction is unclear. Recently, a syndrome of recurrent MAS with infantile enterocolitis caused by NLRC4 inflammasome hyperactivity highlighted the potential importance of interleukin-18 (IL-18). We tested this association in hyperferritinemic and autoinflammatory patients and found a dramatic correlation of MAS risk with chronic (sometimes lifelong) elevation of mature IL-18, particularly with IL-18 unbound by IL-18 binding protein, or free IL-18. In a mouse engineered to carry a disease-causing germ line NLRC4T337S mutation, we observed inflammasome-dependent, chronic IL-18 elevation. Surprisingly, this NLRC4T337S-induced systemic IL-18 elevation derived entirely from intestinal epithelia. NLRC4T337S intestines were histologically normal but showed increased epithelial turnover and upregulation of interferon-γ-induced genes. Assessing cellular and tissue expression, classical inflammasome components such as Il1b, Nlrp3, and Mefv predominated in neutrophils, whereas Nlrc4 and Il18 were distinctly epithelial. Demonstrating the importance of free IL-18, Il18 transgenic mice exhibited free IL-18 elevation and more severe experimental MAS. NLRC4T337S mice, whose free IL-18 levels were normal, did not. Thus, we describe a unique connection between MAS risk and chronic IL-18, identify epithelial inflammasome hyperactivity as a potential source, and demonstrate the pathogenicity of free IL-18. These data suggest an IL-18-driven pathway, complementary to the cytotoxic impairment of fHLH, with potential as a distinguishing biomarker and therapeutic target in MAS.

Symptomatic Males and Female Carriers in a Large Caucasian Kindred with XIAP Deficiency

PurposeX-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was originally described in male patients with X-linked lymphoproliferative syndrome type 2 (XLP2). Recent observations have highlighted a critical role of XIAP for the regulation of NOD2 signaling and are probably the molecular basis for increasingly recognized further immune dysregulatory symptoms of XIAP deficient patients, such as inflammatory bowel disease (IBD). We describe a large Caucasian family in which IBD and erythema nodosum (EN) also manifested in female carriers of XIAP mutations.MethodsClinical data and laboratory findings including flow cytometric analysis of XIAP protein expression and sequencing of the BIRC4 gene. NOD2 signaling was investigated by determination of TNFα production in monocytes upon L18-MDP stimulation in vitro.ResultsThe BIRC4 nonsense mutation p.P225SfsX226 was identified as the genetic cause of XIAP deficiency in our family. Surprisingly, clinical symptoms were not restricted to male patients, but also occurred in several female carriers. The most severely affected carrier demonstrated random X-inactivation, leading to a significant expression of mutated XIAP protein in monocytes, and consequently to impaired NOD2 responses in vitro.ConclusionOur report provides further evidence that clinical symptoms of XIAP deficiency are not restricted to male patients. Random X-inactivation may be associated with EN and mild IBD also in female carriers of BIRC4 mutations. Analysis of the X-inactivation pattern reflected by XIAP protein expression can identify such carriers and the analysis of NOD2 signaling by flow cytometry can confirm the functional significance. XIAP expression patterns should be investigated in female patients with a family history of EN and/or IBD.

First characterization of platelet secretion defect in patients with familial hemophagocytic lymphohistiocytosis type 3 (FHL-3)

To the editor: Familial hemophagocytic lymphohistiocytosis (FHL), a rare autosomal recessive disorder of lymphocyte cytotoxicity, is caused by mutations in genes encoding perforin (FHL-2) or proteins important for intracellular trafficking/exocytosis of perforin-containing lytic granules: Munc13-4

Effective Immunological Guidance of Genetic Analyses Including Exome Sequencing in Patients Evaluated for Hemophagocytic Lymphohistiocytosis

We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with “full” HLH and 79/111 patients with “incomplete/atypical” HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.