Evaluation of lymphocyte function by IFN-γ secretion capability assay in the diagnosis of lymphoma-associated hemophagocytic syndrome

Lymphoma-associated hemophagocytic syndrome (LAHS) is a highly life-threatening disease characterized by an uncontrolled immune disorder. Both under-recognition and delayed diagnosis may contribute to aggressive diseases, and a poorer prognosis. Identification of laboratory features specific for LAHS patients may allow for early detection and intervention with improved outcomes. In the present study, 120 lymphoma patients at first diagnosis were recruited and the function of lymphocytes was evaluated by IFN-γ secretion assay at first diagnosis and follow up. During the surveillance period, 20 patients who complicated with hemophagocytic lymphohistiocytosis (HLH) were classified as LAHS group, and 30 patients without infectious diseases during the course of treatment were classified as lymphoma control group. In addition, 20 non-malignant associated HLH patients recruited as HLH control group and 50 healthy control (HC) subjects were also included. The IFN-γ secretion capability of lymphocytes was compared between first diagnosis of lymphoma patients who was complicate with HLH or not in the disease progression. Our results showed that only NK cell activity was decreased in lymphoma control group, but the activities of NK, CD4+ and CD8+ T cells were all significantly decreased at the time of lymphoma diagnosis in those who would progress with HLH. During the course of treatment, lymphocyte function was relatively stable in lymphoma patients but became further decreased when suffering from complication of LAHS. The IFN-γ secretion capability of lymphocytes in LAHS and non-malignant associated HLH patients were all significantly decreased compared with HCs. So the occurrence of HLH was the key factor leading to the impaired activity of lymphocytes. These data suggest that decreased lymphocyte function might be used as a predictor of LAHS, which has critical clinical significance in diagnosis and further understanding the pathogenesis of the disease.     

Primary hemophagocytic syndromes point to a direct link between lymphocyte cytotoxicity and homeostasis

Summary: Hemophagocytic syndrome (HS) is a severe and often fatal syndrome resulting from potent and uncontrolled activation and proliferation of T‐lymphocytes, leading to excessive macrophage activation and multiple deleterious effects. The onset of HS characterizes several inherited disorders in humans. In each condition, the molecular defect impairs the granule‐dependent cytotoxic activity of lymphocytes, thus highlighting the determinant role of this function in driving the immune system to a state of equilibrium following infection. It has also been shown that some of the proteins required for lytic granule secretion are required for melanocyte function, leading to associated hypopigmentation in these conditions. This review focuses on several effectors of this secretory pathway, recently identified, because their defects cause these disorders, and discusses their role and molecular interactions in granule‐dependent cytotoxic activity.     

The role of cytotoxicity in lymphocyte homeostasis.

Several human inherited immune disorders lead to the same fatal lymphoproliferative syndrome, called the hemophagocytic syndrome. Through defective perforin expression or transport, these disorders highlight the determinant role of the secretory cytotoxic pathway in the regulation of the immune response and in lymphocyte homeostasis. In addition, new effectors of this secretory pathway have been identified.     

Macrophage activation syndrome in rheumatic disease: what is the role of the antigen presenting cell?

Macrophage Activation Syndrome (MAS), alternatively referred to as secondary hemophagocytic lymphohistiocytosis (HLH), is a complication of many rheumatic diseases, most commonly Systemic Juvenile Idiopathic Arthritis (SJIA). MAS consists of a fulminant picture of pan-cytopenia, hectic high fevers, hepatosplenomegaly, lymphadenopathy, rash, and central nervous systemic inflammation. It can arise from genetic defects in the cytotoxic effector response of CD8+ T-cells, resulting in an inability to terminate antigen presentation, which in turn leads to uncontrolled immune activation. However, in the case of most rheumatic diseases, no such defect in cytotoxic killing is present. Little is known about what the contributions from the antigen presenting cells are in the pathogenesis of MAS. In fact, macrophages may be playing a regulatory, anti-inflammatory in MAS. We review the proposed pathogenesis of MAS/HLH, what role macrophages may play in the disease, and the relationship of MAS to its most common associated rheumatic disease, SJIA.     

Genetic basis of hemophagocytic lymphohistiocytosis syndrome (Review).

The group of immune disorders which leads to the occurrence of hemophagocytic lymphohistiocytosis (HLH) syndrome presents a strange paradox in that patients with these conditions associate a dramatic immune response to infection with the failure to establish an effective immune response. During the last few years, significant progress was made in the characterization and the understanding of the molecular basis involved in these inherited immune disorders. The hemophagocytic lymphohistiocytosis syndrome which characterized the evolution of the Chediak-Higashi syndrome and the Griscelli disease results from defects affecting intracellular trafficking. A defective SH2 protein interacting with T lymphocyte intracellular signaling pathways is the cause of the X-linked lymphoproliferative disease, whereas at least three distinct genetic defects can lead to the familial hemophagocytic lymphohistiocytosis. The molecular characterization of these latter defects is in progress. This review summarizes the recent advances as well as their implications in the diagnosis and the understanding of the physiopathology of these disorders.     

Signaling pathways involved in the T‐cell‐mediated immunity against Epstein‐Barr virus: Lessons from genetic diseases

Primary immunodeficiencies (PIDs) provide researchers with unique models to understand in vivo immune responses in general and immunity to infections in particular. In humans, impaired immune control of Epstein‐Barr virus (EBV) infection is associated with the occurrence of several different immunopathologic conditions; these include non‐malignant and malignant B‐cell lymphoproliferative disorders, hemophagocytic lymphohistiocytosis (HLH), a severe inflammatory condition, and a chronic acute EBV infection of T cells. Studies of PIDs associated with a predisposition to develop severe, chronic EBV infections have led to the identification of key components of immunity to EBV – notably the central role of T‐cell expansion and its regulation in the pathophysiology of EBV‐associated diseases. On one hand, the defective expansion of EBV‐specific CD8 T cells results from mutations in genes involved in T‐cell activation (such as RASGRP1, MAGT1, and ITK), DNA metabolism (CTPS1) or co‐stimulatory pathways (CD70, CD27, and TNFSFR9 (also known as CD137/4‐1BB)) leads to impaired elimination of proliferating EBV‐infected B cells and the occurrence of lymphoma. On the other hand, protracted T‐cell expansion and activation after the defective killing of EBV‐infected B cells is caused by genetic defects in the components of the lytic granule exocytosis pathway or in the small adapter protein SH2D1A (also known as SAP), a key activator of T‐ and NK cell‐cytotoxicity. In this setting, the persistence of EBV‐infected cells results in HLH, a condition characterized by unleashed T‐cell and macrophage activation. Moreover, genetic defects causing selective vulnerability to EBV infection have highlighted the role of co‐receptor molecules (CD27, CD137, and SLAM‐R) selectively involved in immune responses against infected B cells via specific T‐B cell interactions.     

Clinical and Genetic Characteristics of XIAP Deficiency in Japan

Deficiency of X-linked inhibitor of apoptosis (XIAP) caused by XIAP/BIRC4 gene mutations is an inherited immune defect recognized as X-linked lymphoproliferative syndrome type 2. This disease is mainly observed in patients with hemophagocytic lymphohistiocytosis (HLH) often associated with Epstein-Barr virus infection. We described nine Japanese patients from six unrelated families with XIAP deficiency and studied XIAP protein expression, XIAP gene analysis, invariant natural killer T (iNKT) cell counts, and the cytotoxic activity of CD8(+) alloantigen-specific cytotoxic T lymphocytes. Of the nine patients, eight patients presented with symptoms in infancy or early childhood. Five patients presented with recurrent HLH, one of whom had severe HLH and died after cord blood transplantation. One patient presented with colitis, as did another patient's maternal uncle, who died of colitis at 4 years of age prior to diagnosis with XIAP deficiency. Interestingly, a 17-year-old patient was asymptomatic, while his younger brother suffered from recurrent HLH and EBV infection. Seven out of eight patients showed decreased XIAP protein expression. iNKT cells from patients with XIAP deficiency were significantly decreased as compared with age-matched healthy controls. These results in our Japanese cohort are compatible with previous studies, confirming the clinical characteristics of XIAP deficiency.     

Hemophagocytic lymphohistiocytosis: a hemophagocytic syndrome.

Hemophagocytic lymphohistiocytosis, terminology that designates a syndrome that may be familial or sporadic, with or without an associated viral infection, is presented as the prototype of a hemophagocytic syndrome, a condition in which there is uncontrolled activation of the cellular immune system. Diagnostic criteria include idiopathic fever, splenomegaly, cytopenias, hypertriglyceridemia, hypofibrinogenemia, and the presence of hemophagocytosis. The surgical and autopsy pathology features infiltrates composed of lymphocytes and ordinary, but activated, histiocytes and hemophagocytosis. The chronic hepatitis-like hepatic lesion is noted to be characteristic, if not unique, in this age group and setting. Current concepts of pathophysiology focus on the role of cytokines, particularly interleukin (IL)-1, IL-2, soluble IL-2 receptor, plasminogen activator, and prostaglandins. The clinicopathologic features of the syndrome can be accounted for by the uncontrolled and unopposed production and release of these mediators. Nosology places hemophagocytic lymphohistiocytosis in the position of the most important of the "benign" histiocytosis syndromes that involve ordinary histiocytes of the mononuclear phagocytic system in contrast to Langerhans cell histiocytosis (histiocytosis X) in which pathological dendritic histiocytes are operative. Features that distinguish hemophagocytic lymphohistiocytosis from other disorders, such as malignant histiocytosis, X-linked lymphoproliferative disorder, congenital immunodeficiency states, the accelerated phase of Chediak-Higashi syndrome, and cytophagic histiocytic panniculitis, which may be associated with a hemophagocytic syndrome, are presented.     

Hemophagocytic lymphohistiocytosis and primary immune deficiency disorders

Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled immune activation and is traditionally associated with inherited gene defects or acquired causes. In addition to abnormalities in cytotoxic granules and lysosomes, various primary immune deficiency disorders (PID) have been identified among patients suffering from HLH. Our purpose was twofold: to better characterize and detail the association between PID and HLH.     

Specyfika występowania limfohistiocytozy hemofagocytarnej w okresie wieku dorosłego

Hemophagocytic lymphohistiocytosis (HLH), also called hemophagocytic syndrome, is a clinical entity caused by an exaggerated inflammatory reaction. HLH can be triggered by various inherited or acquired factors. Uncontrolled activation of T lymphocytes and macrophages, together with an impaired cytotoxic function of NK cells, results in the massive cytokine release. Inherited HLH develops among children whilst HLH etiology in adults is usually acquired. The most common symptoms of HLH are unremitting fever, splenomegaly, and peripheral blood cytopenias. Laboratory tests disclose hyperferritinemia (often >10,000 μg/L), increased level of sIL-2R (>2400 U/L), hypertriglyceridemia, hypofibrinogenemia, coagulopathy, hyponatremia, and elevated liver transaminases and bilirubin. In 2004 The Histiocyte Society updated current diagnostic guidelines for HLH. In most cases, HLH is lethal if adequate therapy is not administered. The therapy of any HLH form is based on suppression of the hyperinflammation by destruction of activated CD8+ T-lymphocytes and macrophages, and treatment of any existing HLH triggers. Since HLH can be encountered by various medical specialists, basic knowledge of this entity and its diagnostic criteria should be familiar to all physicians. Treatment of HLH is difficult, long-lasting and often associated with a high morbidity. Early diagnosis and immediate introduction of adequate HLH therapy are crucial for the successful treatment of HLH.     

Pathogenese und Morphologie des hämophagozytischen Syndroms in der Milz

Hemophagocytic syndrome (macrophage activation syndrome) is a group of disorders characterized by clinical, histological and biological symptoms of T-lymphocytes/NK cell-driven macrophage stimulation with subsequent hemophagocytosis which can be observed in various organs, particularly in the bone marrow and spleen. The main clinical symptoms include cytopenia, multiple organ dysfunction, fever unresponsive to antibiotics, hepatosplenomegaly and rash. Besides rare inherited immune system anomalies (primary hemophagocytic syndrome), the most prevalent etiologies of hemophagocytic syndrome are infections (viral, bacterial and parasitic), cancers, lymphoproliferative disorders and systemic diseases. Hemophagocytic syndrome is treated with immunosuppressives, cytostatic drugs, plasmapheresis and intravenous immunoglobulins/anti-TNF antibodies and due to the multiorgan involvement and the associated high mortality, recognition of early serum changes, such as very high levels of ferritin is mandatory. The most relevant histological changes are the increase of histiocytes showing evidence of phagocytosis of erythroid cells, platelets and granulocytes as well as in the spleen a rarefaction of the white pulpa with lack of lymph follicles and abundant lymphocytes, plasma cells and plasma cell precursors in the pulpa cords.     

High frequency of QPY allele and linkage disequilibrium of granzyme-B in Epstein-Barr-virus-associated hemophagocytic lymphohistiocytosis

Mediation of Epstein-Barr virus (EBV)-specific cytotoxicity in T lymphocyte via the perforin/granzyme pathway has been demonstrated; therefore, a study involving cytolytic molecules was essential for the clarification of hemophagocytic lymphohistiocytosis (HLH) pathogenesis. This investigation, which analysed the frequency of three allelic mutations of granzyme-B (55Q/R, 95P/A and 247Y/H) in patients with EBV-HLH and infectious mononucleosis, identified the high prevalence of the QPY haplotype in EBV-HLH patients in comparison with healthy controls. A > G polymorphism was also detected in intron 5; furthermore, nearly complete linkage disequilibrium was observed among these polymorphisms. The recessive role of the QPY haplotype of granzyme-B might be responsible for the pathogenesis of EBV-HLH. Cytotoxicity and DNA fragmentation of cytotoxic T lymphocytes did not differ among patients characterized by the QPY/QPY, RAH/RAH and QPY/RAH genotypes. This finding suggested that DNA fragmentation in target cells is mediated not only by granzyme-B but also by other molecules, including other granzymes or Fas.     

Granulomatous inflammation and hypogammaglobulinemia: Clinical conundrum of familial hemophagocytic lymphohistiocytosis type 5

Familial hemophagocytic lymphohistiocytosis (HLH) is an inherited disorder characterized by systemic hyperinflammation caused by an uncontrolled immune response mediated by T-lymphocytes, natural killer (NK) cells, and macrophages. Most children with familial HLH present within first 2 years of life and can have fatal disease unless hematopoietic stem cell transplant (HSCT) is performed (1). However, few patients may have late presentation and prolonged survival. With increasing awareness and facilities to identify HLH these disorders are being identified beyond infancy (2–4). Clinical and laboratory features are often similar to other primary immune deficiency diseases and pose diagnostic challenges (4–6). We report two patients who presented beyond the first decade of life with HLH, granulomatous inflammation, hypogammaglobulinemia, reduced B cells and were diagnosed to have familial HLH type 5 due to defect in STXBP2 gene.     

Interleukin 1 and interferon-gamma: cytokines that provide reciprocal regulation of macrophage and T cell function

Interactions between macrophages and T cells are symbiotic, since optimal functions of both cell types require interchange of soluble mediators. Upon activation, macrophages release interleukin-1 (alias lymphocyte activating factor, leukocytic endogenous mediator, and endogenous pyrogen), a family of molecules with multivarious biological effects, ranging from induction of fever and the acute phase response to lymphocyte activation and concomitant release of interleukin-2. Interleukin-2 induces activation and replication of several subsets of precursor lymphocytes, including cytotoxic T cells, lymphokine-activated killer (LAK) cells, and natural killer (NK) cells, and enhances their cytotoxic activity for tumor cells. Both interleukin-2 and leukotrienes enhance production of immune interferon (interferon-gamma) by activated T cells. Aside from antiviral activity, interferon-gamma produces a number of immunomodulatory effects, including macrophage activation (Ia induction, antimicrobial effector function, and activation of oxidative metabolism) and augmentation of NK function. Expression of Ia on accessory cell membranes is required for the initiation of many antigen-specific, T-dependent immune responses. Interferon-gamma also synergizes with a variety of microbial agents to augment macrophage tumoricidal function and enhance interleukin-1 secretion. The production of interferon-gamma appears to have a critical role in feeding back the cascade of interleukins in a loop of amplification. Both interleukin-1 and interferon-gamma modulate release of arachidonate metabolites in various cells. This cascade of cytokines, in collaboration with arachidonate oxygenation products, regulates immunity and sets the stage for many of the events underlying inflammation. Various anti-inflammatory drugs and immunopotentiators appear to act by modulating cytokine pathways.     

Profiles of immune infiltration in abdominal aortic aneurysm and their associated marker genes: a gene expression-based study

Immune-mediated inflammation plays a key role in the pathology of abdominal aortic aneurysm (AAA). We aimed to use a computational approach to profile the immune infiltration patterns and related core genes in AAA samples based on the overexpression of gene signatures. The microarray datasets of AAA and normal abdominal tissues were acquired from gene expression omnibus (GEO) database. We evaluated the composition of immune infiltrates through microenvironment cell populations (MCP)-counter. Weighted gene correlation network analysis (WGCNA) was employed to construct the co-expression network and extract gene information in the most relevant module. Functional and pathway enrichment analysis was performed and immune infiltration related core genes were screened. AAA tissues had a higher level of infiltration by cytotoxic lymphocytes, NK cells, T cells, fibroblasts, myeloid dendritic cells, and neutrophils than normal aorta. The red module was strongly correlated with the infiltrating levels of T cells and cytotoxic lymphocytes. Gene ontology (GO) and pathway analyses revealed that genes in the most relevant module were mainly enriched in T cell activation, regulation of lymphocyte activation, cytokine-cytokine receptor interaction, and chemokine signaling pathway, etc. The expression of GZMK, CCL5, GZMA, CD2, and EOMES showed significant correlations with cytotoxic lymphocytes, while CD247, CD2, CD6, RASGRP1, and CD48 expression were positively associated with T cell infiltration. In conclusion, we comprehensively analyzed profiles of infiltrated immune cells in AAA tissues and their associated marker genes. Our data may provide a novel clue to indicate the underlying molecular mechanisms of AAA formation in terms of immune infiltration.     

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.     

B lymphocytes mediate Fas-dependent cytotoxicity in MRL/lpr mice

The Fas/Fas ligand (FasL) pathway is one of the two major effector mechanisms of T cell-mediated cytotoxicity. To prevent nonspecific killing by lymphoid cells, FasL expression on the cell surface of immune effector cells is strictly regulated. However, MRL/lpr autoimmune-prone mice massively overexpress FasL on their T lymphocytes, which render them able to kill Fas+ targets in vitro and in vivo. It is surprising that we show in the present work that B lymphocytes purified from MRL/lpr spleen cells express FasL to the same extent as T cells at the mRNA and protein level. These B cells are potent cytotoxic effectors against Fas+ but not Fas- targets. The B lymphocyte effectors were used ex vivo without any in vitro activation by B cell stimuli. Furthermore, we found that MRL/lpr B lymphocytes have the same cytotoxic potential as natural killer cells, which have been characterized as potent, Fas-mediated, cytotoxic effectors. The level of membrane-anchored FasL increases with the size of the B cell and cell-surface activation marker CD69 expression, indicating that the expression of FasL is up-regulated in parallel with the activation state of the B cell. The activated B cell population contained the major cytotoxic activity, and a minor part was associated with CD138/Syndecan-1+ plasma cells.     

The Reed-Sternberg cell/lymphocyte interaction: ultrastructure and characteristics of binding.

Autologous T lymphocytes form broad, unspecialized, noninvaginating contacts with Reed-Sternberg cells in vitro. These differ from contacts between cytotoxic lymphocytes and their targets and from the uropodal type of lymphocyte adherence described in many antigen- (or mitogen-) dependent systems but show some resemblance to antigen-independent lymphocyte/macrophage contacts. Adherence is not confined to a specific T-cell subset; most adherent cells are negative for ANAE, Fc gamma, and Fc mu receptors. A minority have Fc mu or Fc gamma receptors or show ANAE staining. Adherence is dependent on divalent cations and intact surface proteins but is independent of temperature, cell metabolism, and intact microtubules and does not appear to be mediated by Fc gamma receptors or IgG. These characteristics distinguish it from immune T-cell/target-cell binding and from antigen-independent T-cell/macrophage or T-cell/B-lymphoblast binding. Contrary to previous suggestions, this interaction is not related to a cytotoxic attack. The lack of similarities with other lymphocyte adherence systems leads the authors to suggest that a unique receptor system is involved.     

MIP-1alpha expression in tissues from patients with hemophagocytic syndrome

Hemophagocytic syndrome (HPS) is a clinicopathologic syndrome that can be precipitated by a variety of causes and is characterized by a systemic activation of macrophages, which are induced to undergo phagocytosis. Chemokines play an important role in the inflammatory cell recruitment into tissues. We examined the expression of chemokines and cytokines in tissues exhibiting histologic evidence of HPS in a variety of settings: peripheral T-cell lymphoma, three patients; nasal T/NK cell lymphoma, one patient; subcutaneous panniculitis-like T-cell lymphoma, one patient; and chronic EBV infection, one patient. Compared with control tissues, we found elevated macrophage inflammatory protein-1alpha (MIP-1alpha) and interferon-gamma (IFN-gamma) expression, but not macrophage-derived chemotactic factor (MDC) or TNF-alpha, in tissues of patients with HPS irrespective of the cause or setting. MIP-1alpha can promote macrophage chemotaxis and IFN-gamma promotes macrophage activation. Elevated expression of IP-10 and monokine induced by IFN-gamma (Mig) was also detected in tissues exhibiting features of HPS, providing an explanation for the occurrence of chemoattraction of T-cells and NK cells. Immunohistochemical analysis of tissues with evidence of phagocytic activity in that site showed MIP-1alpha characteristically localized to endothelial cells of blood vessels and splenic sinuses, lymphocytes, and macrophages. These results provide evidence for MIP-1alpha chemokine expression in tissues from patients with HPS and suggest that MIP-1alpha may play an important role in the pathogenesis of the hemophagocytic syndrome.