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

Abstract 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.

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

Blood ◽

10.1182/blood-2007-02-074468 ◽

2007 ◽

Vol 110 (6) ◽

pp. 1906-1915 ◽

Cited By ~ 205

Author(s):

Yenan T. Bryceson ◽

Eva Rudd ◽

Chengyun Zheng ◽

Josefine Edner ◽

Daoxin Ma ◽

...

Keyword(s):

Nk Cells ◽

Hemophagocytic Lymphohistiocytosis ◽

Nk Cell ◽

Severe Disease ◽

Target Cells ◽

Loss Of Function ◽

Cytotoxic Lymphocyte ◽

Familial Hemophagocytic Lymphohistiocytosis ◽

Genes Encoding ◽

Syntaxin 11

Abstract 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.

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Analysis of natural killer–cell function in familial hemophagocytic lymphohistiocytosis (FHL): defective CD107a surface expression heralds Munc13-4 defect and discriminates between genetic subtypes of the disease

Blood ◽

10.1182/blood-2006-04-015693 ◽

2006 ◽

Vol 108 (7) ◽

pp. 2316-2323 ◽

Cited By ~ 122

Author(s):

Stefania Marcenaro ◽

Federico Gallo ◽

Stefania Martini ◽

Alessandra Santoro ◽

Gillian M. Griffiths ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Hemophagocytic Lymphohistiocytosis ◽

Cytokine Production ◽

Nk Cell ◽

Killer Cell ◽

Target Cells ◽

Familial Hemophagocytic Lymphohistiocytosis ◽

Control Subjects ◽

Healthy Control

Abstract Natural killer (NK) cells from patients with familial hemophagocytic lymphohistiocytosis because of PRF1 (FHL2, n = 5) or MUNC13-4 (FHL3, n = 8) mutations were cultured in IL-2 prior to their use in various functional assays. Here, we report on the surface CD107a expression as a novel rapid tool for identification of patients with Munc13-4 defect. On target interaction and degranulation, FHL3 NK cells displayed low levels of surface CD107a staining, in contrast to healthy control subjects or perforin-deficient NK cells. B-EBV cell lines and dendritic cell targets reveal the FHL3 NK-cell defect, whereas highly susceptible tumor targets were partially lysed by FHL3 NK cells expressing only trace amounts of Munc13-4 protein. Perforin-deficient NK cells were completely devoid of any ability to lyse target cells. Cytokine production induced by mAb-crosslinking of triggering receptors was comparable in patients and healthy control subjects. However, when cytokine production was induced by coculture with 721.221 B-EBV cells, FHL NK cells resulted in high producers, whereas control cells were almost ineffective. This could reflect survival versus elimination of B-EBV cells (ie, the source of NK-cell stimulation) in patients versus healthy control subjects, thus mimicking the pathophysiologic scenario of FHL.

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Chronic active EB virus infection with hemophagocytic lymphohistiocytosis and progression to aggressive NK cell leukemia: a case report and review of the literature

10.21203/rs.3.rs-855964/v1 ◽

2021 ◽

Author(s):

Li-min Gao ◽

Hui-fang Li ◽

Sha Zhao ◽

Wen-Yan Zhang ◽

Qiang Li ◽

...

Keyword(s):

Nk Cells ◽

Hemophagocytic Lymphohistiocytosis ◽

Hemophagocytic Syndrome ◽

Nk Cell ◽

Review Of The Literature ◽

Cell Leukemia ◽

Hematopoietic Stem ◽

Ebv Infection ◽

To Receive ◽

Timely Treatment

Abstract BackgroundThe clinical trend of EBV infected patients has been always the focus of attention. It is extremely difficult to identify which patients may resolve spontaneously or proceed to chronic stage or proceed rapidly.Case descriptionWe report a rare case of chronic active EBV infection (CAEBV) with hemophagocytic lymphohistiocytosis progressed to childhood aggressive NK cell leukemia (ANKL) and cured by allogeneic hematopoietic stem cell transplantation.ConclusionsThe EBV infection of T/NK cells can lead to CAEBV, and if the T/NK cells carry HLH related gene mutation, it is easy to accompany with the hemophagocytic syndrome, and this type of CAEBV may rapidly progress to ANKL or other neoplastic diseases. Therefore, we should reinforce the knowledge on this kind of patients to receive appropriate and timely treatment.

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Efficacy of moderately dosed etoposide in macrophage activation syndrome - hemophagocytic lymphohistiocytosis (MAS-HLH)

The Journal of Rheumatology ◽

10.3899/jrheum.200941 ◽

2021 ◽

pp. jrheum.200941

Author(s):

AnnaCarin Horne ◽

Tatiana von Bahr Greenwood ◽

Samuel C.C. Chiang ◽

Marie Meeths ◽

Caroline Björklund ◽

...

Keyword(s):

Lupus Erythematosus ◽

Hemophagocytic Lymphohistiocytosis ◽

Macrophage Activation Syndrome ◽

Macrophage Activation ◽

Nk Cell ◽

Cell Activity ◽

Neutropenic Sepsis ◽

Lymphocyte Cytotoxicity ◽

Activation Syndrome

Objective Macrophage activation syndrome (MAS) constitutes one subtype of the hyperinflammatory syndrome hemophagocytic lymphohistiocytosis (HLH), and the term MAS-HLH was recently proposed for HLH with underlying autoimmune/autoinflammatory conditions. The mortality of MAS-HLH has been estimated to 5-10%. Here we report our experiences with moderately dosed etoposide in severe MAS-HLH, administered with the objective to effectively reduce severe hyperinflammatory activity with limited side effects. Methods In addition to conventional anti-inflammatory treatment, moderately dosed etoposide was administered to seven children affected by rapidly progressing MAS-HLH with central nervous system (n=5) and/or pulmonary (n=5) involvement. Three had underlying systemic onset juvenile idiopathic arthritis (sJIA), two atypical sJIA (no arthritis at diagnosis), and two systemic lupus erythematosus. We performed lymphocyte cytotoxicity analyses in all seven and genetic analyses in six. Results All children promptly responded to moderately dosed etoposide (50-100 mg/m2 once weekly), added to conventional MAS-HLH treatment which was considered insufficient. The mean accumulated etoposide dose was 671 mg/m2 (range 300-1,050 mg/m2), as compared to 1,500 mg/m2 recommended the first 8 weeks of the HLH-94/HLH-2004 protocols. One child developed neutropenic fever and another neutropenic sepsis (neutrophils 0.3x109/L at therapy onset). Five/seven children had low percentages (<5%) circulating NK-cells prior to or in association with diagnosis; NK-cell activity was pathologically low in two/five children studied. Disease-causing variants in HLH-associated genes were not found. All children were alive at latest follow-up (2-9 years after onset); neurological symptoms had normalized in four/five affected children. Conclusion Moderately dosed etoposide may be beneficial in severe and/or refractory MAS-HLH.

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1011. Sepsis and Secondary Hemophagocytic Lymphohistiocytosis

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.848 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S301-S301 ◽

Cited By ~ 1

Author(s):

Farhan Fazal ◽

Naveet Wig ◽

Manish Soneja ◽

Dipendra K Mitra ◽

Sk Panda ◽

...

Keyword(s):

Diagnostic Criteria ◽

Hemophagocytic Lymphohistiocytosis ◽

Nk Cell ◽

Immunosuppressive Treatment ◽

Cell Activity ◽

Unknown Etiology ◽

Laboratory Features ◽

Life Threatening ◽

Non Hodgkins Lymphoma ◽

Staphylococcal Aureus

Abstract Background Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory condition diagnosed by HLH 2004 criteria. This criterion has common clinical and laboratory features with sepsis and tropical fevers, but there is marked difference in management and outcome of these two entities. The study is conducted to know whether there is any difference in the clinico-laboratory features, management, and outcome of sepsis with or without secondary HLH. Methods This is a prospective observational study where patients presenting with sepsis and bicytopenia are included. The patients underwent relevant investigations according to 2004 HLH diagnostic criteria. The patients are divided into sepsis with or without HLH. The underlying etiology, treatment, and outcome of the two groups are analysed. Results Fifty sepsis patients are included in the study, out of which 28 fulfilled the HLH diagnostic criteria which comprised of 18 men and 10 women. The etiology were bacterial (three enteric fever, three tuberculosis, two scrub typhus, one Staphylococcal aureus), viral (one dengue fever, two HIV, two encephalitis), fungal (one aspergillosis, one mucormycosis, two others), parasites (three malaria, one leishmania) malignancy (two hodgkin lymphoma, one non-Hodgkins lymphoma), and unknown etiology in six patients, with >1 etiology in three patients (Figure 2). The percentage of each criterion fulfilled in both groups is given in Figure 1, showing an increased occurrence of splenomegaly, low NK cell activity, hypertriglyceridemia in HLH patients. Steroids along with supportive treatment was given to 53% and etoposide was added in 7%. Treatment for underlying etiology alone without immunosuppressive treatment was given in 39%. The mortality in those with HLH vs. without HLH was 42% and 31%, respectively. The median duration of hospital stay was 18 and 36 days in HLH and without HLH group, respectively. Conclusion HLH should be suspected in sepsis patients with bicytopenia specially in tropical fevers. There is increased mortality if the sepsis patients fulfil HLH criteria. Early diagnosis and management is of paramount importance. Disclosures All authors: No reported disclosures.

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X-Linked Lymphoproliferative Disease

Journal of Experimental Medicine ◽

10.1084/jem.192.3.337 ◽

2000 ◽

Vol 192 (3) ◽

pp. 337-346 ◽

Cited By ~ 351

Author(s):

Silvia Parolini ◽

Cristina Bottino ◽

Michela Falco ◽

Raffaella Augugliaro ◽

Silvia Giliani ◽

...

Keyword(s):

B Cell ◽

Nk Cells ◽

Cell Lines ◽

Nk Cell ◽

Hla Class I ◽

Lymphoproliferative Disease ◽

Class I ◽

Src Homology 2 ◽

Src Homology ◽

Src Homology 2 Domain

2B4 is a surface molecule involved in activation of the natural killer (NK) cell–mediated cytotoxicity. It binds a protein termed Src homology 2 domain–containing protein (SH2D1A) or signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), which in turn has been proposed to function as a regulator of the 2B4-associated signal transduction pathway. In this study, we analyzed patients with X-linked lymphoproliferative disease (XLP), a severe inherited immunodeficiency characterized by critical mutations in the SH2D1A gene and by the inability to control Epstein-Barr virus (EBV) infections. We show that, in these patients, 2B4 not only fails to transduce triggering signals, but also mediates a sharp inhibition of the NK-mediated cytolysis. Other receptors involved in NK cell triggering, including CD16, NKp46, NKp44, and NKp30, displayed a normal functional capability. However, their activating function was inhibited upon engagement of 2B4 molecules. CD48, the natural ligand of 2B4, is highly expressed on the surface of EBV+ B cell lines. Remarkably, NK cells from XLP patients could not kill EBV+ B cell lines. This failure was found to be the consequence of inhibitory signals generated by the interaction between 2B4 and CD48, as the antibody-mediated disruption of the 2B4–CD48 interaction restored lysis of EBV+ target cells lacking human histocompatibility leukocyte antigen (HLA) class I molecules. In the case of autologous or allogeneic (HLA class I+) EBV+ lymphoblastoid cell lines, restoration of lysis was achieved only by the simultaneous disruption of 2B4–CD48 and NK receptor–HLA class I interactions. Molecular analysis revealed that 2B4 molecules isolated from either XLP or normal NK cells were identical. As expected, in XLP-NK cells, 2B4 did not associate with SH2D1A, whereas similar to 2B4 molecules isolated from normal NK cells, it did associate with Src homology 2 domain–containing phosphatase 1.

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Failure of the beige genotype (bg/bg) to alter the morphology of uterine granulated metrial gland cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156523 ◽

1989 ◽

Vol 47 ◽

pp. 908-909

Author(s):

S. Yamashiro ◽

T. Bast ◽

B.A. Croy

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Severe Combined Immunodeficiency ◽

Cell Lineage ◽

Outer Zone ◽

Late Gestation ◽

Secretory Cells ◽

Morphological Marker ◽

Metrial Gland ◽

Chediak Higashi Syndrome

Granulated metrial gland (GMG) cells are bone marrow-derived cells that differentiate from small, lymphoid-like precursor cells to become large granulated lymphocyte-like cells during murine pregnancy. The cells are localized in the mesometrial triangle and decidua basalis by day 8 of gestation, migrate through the mature placenta and become degenerate in late gestation. The function and lineage of GMG cells are unknown but some experiments suggest that they may be NK cells. Mice of genotype scid/scid have a severe combined immunodeficiency and lack functional B and T lymphocytes. However, both NK cells and GMG cells have been demonstrated in scid/scid mice. To determine whether these two populations are of the same lineage we developed mice of genotype scid/scid,bg/bg, providing a morphological marker, the Chediak-Higashi giant granule phenotype, to NK cells and have asked whether GMG cells share this lymphomyeloid phenotype.Small pieces of uterine tissues including endo-myometriura from scid/scid and scid/scid.bg/bg at 12th day of gestation were routinely processed for transmission electron microscopy. Ultrastrueturally, large, oval GMG cells were easily recognizable in both scid/scid (Fig. 1) and scid/bg (Fig. 2) uteri due to their cytological features. Nuclei of these cells contained fine chromatin and prominent nucleolus. The cytoplasm was abundant and contained large amounts of free ribosomes and granular endoplasmic reticulum, well developed Golgi complex and prominent membrane-bound granules of variable shapes and sizes (Figs. 1 & 2). The granules were generally oval to spherical and consisted of two zones. The central homogeneous zone, comprising of about 90% of the granule, was less electron opaque than crescent-shaped, outer zone. The crescent-shaped zone contained minute vacuoles and particles (Figs. 1 & 2 insets). The GMG cells exhibited features commonly seen in secretory cells, which were consistent with previous reports. The inclusion characteristic in Chediak-Higashi syndrome was not observed in the GMG cells of the scid/scid.bg/bg (Fig. 2). These observations are suggestive of GMG cells not sharing the NK cell lineage pathway.

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Herpesvirus Evasion of Natural Killer Cells

Journal of Virology ◽

10.1128/jvi.02105-17 ◽

2018 ◽

Vol 92 (11) ◽

Cited By ~ 26

Author(s):

Steffi De Pelsmaeker ◽

Nicolas Romero ◽

Massimo Vitale ◽

Herman W. Favoreel

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Viral Infections ◽

Nk Cell ◽

Cell Activity ◽

Adaptive Immune Response ◽

Nk Cell Activity ◽

Disease Symptoms ◽

Adaptive Immune ◽

Life Threatening

ABSTRACTNatural killer (NK) cells play an important role in the host response against viral infections and cancer development. They are able to kill virus-infected and tumor cells, and they produce different important cytokines that stimulate the antiviral and antitumor adaptive immune response, particularly interferon gamma. NK cells are of particular importance in herpesvirus infections, which is illustrated by systemic and life-threatening herpesvirus disease symptoms in patients with deficiencies in NK cell activity and by the myriad of reports describing herpesvirus NK cell evasion strategies. The latter is particularly obvious for cytomegaloviruses, but increasing evidence indicates that most, if not all, members of the herpesvirus family suppress NK cell activity to some extent. This review discusses the different NK cell evasion strategies described for herpesviruses and how this knowledge may translate to clinical applications.

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Evaluation of purified natural killer cell functions in familial hemophagocytic lymphohistiocytosis

LymphoSign Journal ◽

10.14785/lpsn-2015-0007 ◽

2015 ◽

Vol 2 (4) ◽

pp. 207-212 ◽

Cited By ~ 1

Author(s):

Benyamin Rosental ◽

Avishai Shemesh ◽

Michal Yaron-Mendelson ◽

Lauren C. Klein ◽

Yona Kodman ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Hemophagocytic Lymphohistiocytosis ◽

Nk Cell ◽

Killer Cell ◽

Positive Family History ◽

False Negative ◽

Specific Lysis ◽

Familial Hemophagocytic Lymphohistiocytosis ◽

Cell Functions

Background: Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, genetic, immune dysregulation disorder of aberrant hyperactivation of lymphocytes causing inflammation and hemophagocytosis. We report on a 3-month old male who was evaluated for the possibility of FHL because of a positive family history. The patient was asymptomatic; however, levels of the soluble interleukin 2 (IL-2) receptor were elevated and the quantity and function of the natural killer (NK) cells were severely decreased. Methods: Purification of NK cells and evaluation of the cytotoxicity and IFNγ/TNFα secretion of NK cells after IL-2 activation relative to the patient's family members. Results: The patient's NK specific lysis was enhanced compared with his mother, and it was slightly higher than his sister. The IFNγ and TNFα secretion by the patient's NK cells after challenge with target 721 cells or anti-natural cytotoxicity receptors (NKp30 and NKp44) antibodies showed levels that are close to the mother's and sister's NK secretion levels. Owing to a low yield of NK cells from the patient's father the results for his NK cells are incomplete. The patient did not undergo HSCT and continued to be followed. He is now 7 years old and thriving without signs of FHL. His last examination was in August 2012 for functionality of isolated NK cells. The results showed normal cytotoxicity, cytokine secretion, and CD107a up-regulation to the NK cell surface. Conclusion: We propose that NK function assessment in patients with presumed FHL should be performed on isolated NK cell populations. This practice may reduce the number of false-negative results in NK function assays. Statement of novelty: In this case report we show that functional assessment of unpurified NK cells could lead to a false-negative assessment in 1 of the parameters in FHL. Assessment of NK function without NK purification may lead to an erroneous diagnosis of poor NK function.

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Gntb-A, a Novel Sh2d1a-Associated Surface Molecule Contributing to the Inability of Natural Killer Cells to Kill Epstein-Barr Virus–Infected B Cells in X-Linked Lymphoproliferative Disease

Journal of Experimental Medicine ◽

10.1084/jem.194.3.235 ◽

2001 ◽

Vol 194 (3) ◽

pp. 235-246 ◽

Cited By ~ 218

Author(s):

Cristina Bottino ◽

Michela Falco ◽

Silvia Parolini ◽

Emanuela Marcenaro ◽

Raffaella Augugliaro ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Epstein Barr Virus ◽

Cell Activation ◽

Nk Cell ◽

Lymphoproliferative Disease ◽

Sh2 Domain ◽

Target Cells ◽

Barr Virus ◽

Epstein Barr

In humans, natural killer (NK) cell function is regulated by a series of receptors and coreceptors with either triggering or inhibitory activity. Here we describe a novel 60-kD glycoprotein, termed NTB-A, that is expressed by all human NK, T, and B lymphocytes. Monoclonal antibody (mAb)-mediated cross-linking of NTB-A results in the induction of NK-mediated cytotoxicity. Similar to 2B4 (CD244) functioning as a coreceptor in the NK cell activation, NTB-A also triggers cytolytic activity only in NK cells expressing high surface densities of natural cytotoxicity receptors. This suggests that also NTB-A may function as a coreceptor in the process of NK cell activation. Molecular cloning of the cDNA coding for NTB-A molecule revealed a novel member of the immunoglobulin superfamily belonging to the CD2 subfamily. NTB-A is characterized, in its extracellular portion, by a distal V-type and a proximal C2-type domain and by a cytoplasmic portion containing three tyrosine-based motifs. NTB-A undergoes tyrosine phosphorylation and associates with the Src homology 2 domain–containing protein (SH2D1A) as well as with SH2 domain–containing phosphatases (SHPs). Importantly, analysis of NK cells derived from patients with X-linked lymphoproliferative disease (XLP) showed that the lack of SH2D1A protein profoundly affects the function not only of 2B4 but also of NTB-A. Thus, in XLP-NK cells, NTB-A mediates inhibitory rather than activating signals. These inhibitory signals are induced by the interaction of NTB-A with still undefined ligands expressed on Epstein-Barr virus (EBV)-infected target cells. Moreover, mAb-mediated masking of NTB-A can partially revert this inhibitory effect while a maximal recovery of target cell lysis can be obtained when both 2B4 and NTB-A are simultaneously masked. Thus, the altered function of NTB-A appears to play an important role in the inability of XLP-NK cells to kill EBV-infected target cells.

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