Diagnosis of Chediak Higashi disease in a 67-year old woman.

Chediak-Higashi disease is a rare disease caused by bi-allelic mutations in the lysosomal trafficking regulator gene, LYST. Individuals typically present in early childhood with partial oculocutaneous albinism, a bleeding diathesis, recurrent infections secondary to immune dysfunction, and risk of developing hemophagocytic lymphohistiocytosis (HLH). Without intervention, mortality is high in the first decade of life. However, some individuals with milder phenotypes have attenuated hematologic and immunologic presentations, and lower risk of HLH. Both classic and milder phenotypes develop progressive neurodegeneration in early adulthood. Here we present a remarkable patient diagnosed with Chediak-Higashi disease at age 67, many decades after the diagnosis is usually established. Diagnosis was suspected by observing the pathognomonic granules within leukocytes, and confirmed by identification of bi-allelic mutations in LYST, reduced LYST mRNA expression, enlarged lysosomes within fibroblasts, and decreased NK cell lytic activity. This case further expands the phenotype of Chediak-Higashi disease and highlights the need for increased awareness. Individuals with milder phenotypes may escape early diagnosis, but identification is important for close monitoring of potential complications, and to further our understanding of the function of LYST.

NK cell defects in X-linked pigmentary reticulate disorder.

X-linked reticulate pigmentary disorder (XLPDR, Mendelian Inheritance in Man #301220) is a rare syndrome characterized by recurrent infections and sterile multiorgan inflammation. The syndrome is caused by an intronic mutation in POLA1, the gene encoding the catalytic subunit of DNA polymerase-α (Pol-α), which is responsible for Okazaki fragment synthesis during DNA replication. Reduced POLA1 expression in this condition triggers spontaneous type I interferon expression, which can be linked to the autoinflammatory manifestations of the disease. However, the history of recurrent infections in this syndrome is as yet unexplained. Here we report that patients with XLPDR have reduced NK cell cytotoxic activity and decreased numbers of NK cells, particularly differentiated, stage V, cells (CD3-CD56dim). This phenotype is reminiscent of hypomorphic mutations in MCM4, which encodes a component of the minichromosome maintenance (MCM) helicase complex that is functionally linked to Pol-α during the DNA replication process. We find that POLA1 deficiency leads to MCM4 depletion and that both can impair NK cell natural cytotoxicity and show that this is due to a defect in lytic granule polarization. Altogether, our study provides mechanistic connections between Pol-α and the MCM complex and demonstrates their relevance in NK cell function.

Liposome Preparation for the Analysis of Lipid-Receptor Interaction and Efferocytosis.

Efficient phagocytosis of apoptotic cells (efferocytosis) is essential for immune homeostasis. Phospholipids exposed on the surface of apoptotic cells, such as phosphatidylserine, supply important "eat-me" signals. Liposomes are lipid bilayer vesicles that can be generated from one or several types of phospholipids of interest. Thus, these vesicles offer versatility, flexibility, and, importantly, a three-dimensional structure for studying the interaction between lipids and their receptors as well as the lipid-receptor interaction-mediated signaling events controlling efferocytosis by cells like professional phagocytes. Here, we describe methods to prepare liposomes, perform liposome-based lipid-receptor binding assays, use liposomes to block efferocytosis, and utilize liposome-coated beads as apoptotic cell surrogates for phagocytosis. © 2018 by John Wiley & Sons, Inc.

An actin cytoskeletal barrier inhibits lytic granule release from natural killer cells in patients with Chediak-Higashi syndrome.

BACKGROUND: Chediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the lysosomal trafficking regulator gene (LYST), resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis caused by impaired function of cytotoxic lymphocytes, including natural killer (NK) cells. OBJECTIVE: We sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS. METHODS: We generated a human cell model of CHS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system and super-resolution microscopy to visualize F-actin and lytic granules in normal and LYST-deficient NK cells. RESULTS: Loss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of the CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis and impaired integrity of endolysosomal compartments. The large granules had an acidic pH and normal activity of lysosomal enzymes and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunologic synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunologic synapse or decreasing the lytic granule size restored the ability of LYST-deficient NK cells to degranulate and kill target cells. CONCLUSION: The cortical actin and granule size play significant roles in NK cell cytotoxic function. We present evidence that the periodicity of subsynaptic actin is an important factor limiting the release of large lytic granules from NK cells from patients with CHS and could be a novel target for pharmaceutical intervention.

Differences in Granule Morphology yet Equally Impaired Exocytosis among Cytotoxic T Cells and NK Cells from Chediak-Higashi Syndrome Patients.

Chediak-Higashi syndrome (CHS) is caused by autosomal recessive mutations in LYST, resulting in enlarged lysosomal compartments in multiple cell types. CHS patients display oculocutaneous albinism and may develop life-threatening hemophagocytic lymphohistiocytosis (HLH). While NK cell-mediated cytotoxicity has been reported to be uniformly defective, variable defects in T cell-mediated cytotoxicity has been observed. The latter has been linked to the degree of HLH susceptibility. Since the discrepancies in NK cell- and T cell-mediated cellular cytotoxicity might result from differences in regulation of cytotoxic granule release, we here evaluated perforin-containing secretory lysosome size and number in freshly isolated lymphocytes from CHS patients and furthermore compared their exocytic capacities. Whereas NK cells from CHS patients generally contained a single, gigantic perforin-containing granule, cytotoxic T cells predominantly contained several smaller granules. Nonetheless, in a cohort of 21 CHS patients, cytotoxic T cell and NK cell granule exocytosis were similarly impaired upon activating receptor stimulation. Mechanistically, polarization of cytotoxic granules was defective in cytotoxic lymphocytes from CHS patients, with EEA1, a marker of early endosomes, mislocalizing to lysosomal structures. The results leads to the conclusion that lysosome enlargement corresponds to loss of distinct organelle identity in the endocytic pathway, which on a subcellular level more adversely affects NK cells than T cells. Hence, vesicular size or numbers do not per se dictate the impairment of lysosomal exocytosis in the two cell types studied.

Dendritic cells expressing immunoreceptor CD300f are critical for controlling chronic gut inflammation.

Proinflammatory cytokine overproduction and excessive cell death, coupled with impaired clearance of apoptotic cells, have been implicated as causes of failure to resolve gut inflammation in inflammatory bowel diseases. Here we have found that dendritic cells expressing the apoptotic cell-recognizing receptor CD300f play a crucial role in regulating gut inflammatory responses in a murine model of colonic inflammation. CD300f-deficient mice failed to resolve dextran sulfate sodium-induced colonic inflammation as a result of defects in dendritic cell function that were associated with abnormal accumulation of apoptotic cells in the gut. CD300f-deficient dendritic cells displayed hyperactive phagocytosis of apoptotic cells, which stimulated excessive TNF-α secretion predominantly from dendritic cells. This, in turn, induced secondary IFN-γ overproduction by colonic T cells, leading to prolonged gut inflammation. Our data highlight a previously unappreciated role for dendritic cells in controlling gut homeostasis and show that CD300f-dependent regulation of apoptotic cell uptake is essential for suppressing overactive dendritic cell-mediated inflammatory responses, thereby controlling the development of chronic gut inflammation.

Natural killer cell activity and dysfunction in Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) encompasses disorders with abnormal function of lysosomes and lysosome-related organelles, and some patients who develop immunodeficiency. The basic mechanisms contributing to immune dysfunction in HPS are ill-defined. We analysed natural killer (NK) cells from patients diagnosed with HPS-1, HPS-2, HPS-4, and an unreported HPS subtype. NK cells from an HPS-2 and an unreported HPS subtype share a similar cellular phenotype with defective granule release and cytotoxicity, but differ in cytokine exocytosis. Defining NK cell activity in several types of HPS provides insights into cellular defects of the disorder and understanding of mechanisms contributing to HPS pathogenesis.

Lipopolysaccharide-Induced CD300b Receptor Binding to Toll-like Receptor 4 Alters Signaling to Drive Cytokine Responses that Enhance Septic Shock.

Receptor CD300b is implicated in regulating the immune response to bacterial infection by an unknown mechanism. Here, we identified CD300b as a lipopolysaccharide (LPS)-binding receptor and determined the mechanism underlying CD300b augmentation of septic shock. In vivo depletion and adoptive transfer studies identified CD300b-expressing macrophages as the key cell type augmenting sepsis. We showed that CD300b, and its adaptor DAP12, associated with Toll-like receptor 4 (TLR4) upon LPS binding, thereby enhancing TLR4-adaptor MyD88- and TRIF-dependent signaling that resulted in an elevated pro-inflammatory cytokine storm. LPS engagement of the CD300b-TLR4 complex led to the recruitment and activation of spleen tyrosine kinase (Syk) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). This resulted in an inhibition of the ERK1/2 protein kinase- and NF-κB transcription factor-mediated signaling pathways, which subsequently led to a reduced interleukin-10 (IL-10) production. Collectively, our data describe a mechanism of TLR4 signaling regulated by CD300b in myeloid cells in response to LPS.

DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis.

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.

Chediak-Higashi syndrome: Lysosomal trafficking regulator domains regulate exocytosis of lytic granules but not cytokine secretion by natural killer cells.

BACKGROUND: Mutations in lysosomal trafficking regulator (LYST) cause Chediak-Higashi syndrome (CHS), a rare immunodeficiency with impaired cytotoxic lymphocyte function, mainly that of natural killer (NK) cells. Our understanding of NK cell function deficiency in patients with CHS and how LYST regulates lytic granule exocytosis is very limited. OBJECTIVE: We sought to delineate cellular defects associated with LYST mutations responsible for the impaired NK cell function seen in patients with CHS. METHODS: We analyzed NK cells from patients with CHS with missense mutations in the LYST ARM/HEAT (armadillo/huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) or BEACH (beige and Chediak-Higashi) domains. RESULTS: NK cells from patients with CHS displayed severely reduced cytotoxicity. Mutations in the ARM/HEAT domain led to a reduced number of perforin-containing granules, which were significantly increased in size but able to polarize to the immunologic synapse; however, they were unable to properly fuse with the plasma membrane. Mutations in the BEACH domain resulted in formation of normal or slightly enlarged granules that had markedly impaired polarization to the IS but could be exocytosed on reaching the immunologic synapse. Perforin-containing granules in NK cells from patients with CHS did not acquire certain lysosomal markers (lysosome-associated membrane protein 1/2) but were positive for markers of transport vesicles (cation-independent mannose 6-phosphate receptor), late endosomes (Ras-associated binding protein 27a), and, to some extent, early endosomes (early endosome antigen 1), indicating a lack of integrity in the endolysosomal compartments. NK cells from patients with CHS had normal cytokine compartments and cytokine secretion. CONCLUSION: LYST is involved in regulation of multiple aspects of NK cell lytic activity, ranging from governance of lytic granule size to control of their polarization and exocytosis, as well as regulation of endolysosomal compartment identity. LYST functions in the regulated exocytosis but not in the constitutive secretion pathway.

Emerging role of CD300 receptors in regulating myeloid cell efferocytosis.

Engulfment of apoptotic cells is predominantly executed by phagocytes via the recognition of "eat me" signals like phosphatidylserine (PS). Various PS-specific receptors exist on phagocytes, including Tyro3, Axl, and MerTK receptor tyrosine kinases (TAMs), T-cell immunoglobulin and mucin domain containing 1 and 4 (TIM1/4), and the newly identified CD300 family. The aim of the present auto-commentary is to highlight recent findings regarding the Cd300lf and Cd300lb receptors and their emerging roles in the development of autoimmune disease.

p85α recruitment by the CD300f phosphatidylserine receptor mediates apoptotic cell clearance required for autoimmunity suppression.

Apoptotic cell (AC) clearance is essential for immune homeostasis. Here we show that mouse CD300f (CLM-1) recognizes outer membrane-exposed phosphatidylserine, and regulates the phagocytosis of ACs. CD300f accumulates in phagocytic cups at AC contact sites. Phosphorylation within CD300f cytoplasmic tail tyrosine-based motifs initiates signals that positively or negatively regulate AC phagocytosis. Y276 phosphorylation is necessary for enhanced CD300f-mediated phagocytosis through the recruitment of the p85α regulatory subunit of phosphatidylinositol-3-kinase (PI3K). CD300f-PI3K association leads to activation of downstream Rac/Cdc42 GTPase and mediates changes of F-actin that drive AC engulfment. Importantly, primary macrophages from CD300f-deficient mice have impaired phagocytosis of ACs. The biological consequence of CD300f deficiency is predisposition to autoimmune disease development, as FcγRIIB-deficient mice develop a systemic lupus erythematosus-like disease at a markedly accelerated rate if CD300f is absent. In this report we identify the mechanism and role of CD300f in AC phagocytosis and maintenance of immune homeostasis.

Evidence for defective Rab GTPase-dependent cargo traffic in immune disorders.

A fully functional immune system is essential to protect the body against pathogens and other diseases, including cancer. Vesicular trafficking provides the correct localization of proteins within all cell types, but this process is most exquisitely controlled and coordinated in immune cells because of their specialized organelles and their requirement to respond to selected stimuli. More than 60 Rab GTPases play important roles in protein trafficking, but only five Rab-encoding genes have been associated with inherited human disorders, and only one of these (Rab27a) causes an immune defect. Mutations in RAB27A cause Griscelli Syndrome type 2 (GS2), an autosomal recessive disorder of pigmentation and severe immune deficiency. In lymphocytes, Munc13-4 is an effector of Rab27a, and mutations in the gene encoding this protein (UNC13D) cause Familial Hemophagocytic Lymphohistiocytosis Type 3 (FHL3). The immunological features of GS2 and FHL3 include neutropenia, thrombocytopenia, and immunodeficiency due to impaired function of cytotoxic lymphocytes. The small number of disorders caused by mutations in genes encoding Rabs could be due to their essential functions, where defects in these genes could be lethal. However, with the increasing use of next generation sequencing technologies, more mutations in genes encoding Rabs may be identified in the near future.

Membrane-type 6 matrix metalloproteinase regulates the activation-induced downmodulation of CD16 in human primary NK cells.

CD16 (FcγRIIIa), the low-affinity receptor for IgG, expressed by the majority of human NK cells, is a potent activating receptor that facilitates Ab-dependent cell-mediated cytotoxicity (ADCC). ADCC dysfunction has been linked to cancer progression and poor prognosis for chronic infections, such as HIV; thus, understanding how CD16 expression is regulated by NK cells has clinical relevance. Importantly, CD16 cell-surface expression is downmodulated following NK cell activation and, in particular, exposure to stimulatory cytokines (IL-2 or IL-15), likely owing to the action of matrix metalloproteinases (MMPs). In this article, we identify membrane-type 6 (MT6) MMP (also known as MMP25) as a proteinase responsible for CD16 downmodulation. IL-2-induced upregulation of MT6/MMP25 cell-surface expression correlates with CD16 downmodulation. MT6/MMP25, sequestered in intracellular compartments in unstimulated NK cells, translocates to the cell surface after stimulation; moreover, it polarizes to the effector-target cell interface of the CD16-mediated immunological synapse. siRNA-mediated disruption of MT6/MMP25 expression enhances the ADCC capacity of NK cells, emphasizing the important functional role of MT6/MMP25 in the regulation of ADCC activity. Thus, this study uncovers a previously unknown role of MT6/MMP25 in human NK cells, and suggests that inhibition of MT6/MMP25 activity could improve ADCC efficacy of therapeutically administered NK cells that require IL-2 for culture and expansion.

LAMP1/CD107a is required for efficient perforin delivery to lytic granules and NK-cell cytotoxicity.

Secretory lysosomes of natural killer (NK) cells, containing perforin and granzymes, are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell apoptosis. Lysosome-associated membrane protein (LAMP) 1/CD107a is used as a marker for NK-cell degranulation, but its role in NK-cell biology is unknown. We show that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNA interference (RNAi) cells fail to deliver granzyme B to target cells. Reduction of LAMP1 expression affects the movement of lytic granules and results in decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi network-derived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity.

Human Hsp40 proteins, DNAJA1 and DNAJA2, as potential targets of the immune response triggered by bacterial DnaJ in rheumatoid arthritis.

Hsp40 proteins of bacterial and human origin are suspected to be involved in the pathogenesis of rheumatoid arthritis (RA). It has been shown that sera of RA patients contain increased levels of antibodies directed to bacterial and human Hsp40s. The aim of this work was to explore immunological similarities between the bacterial (DnaJ) and human (DNAJA1 and DNAJA2) Hsp40 proteins in relation to their possible involvement in the RA. Using polyclonal antibodies directed against a full-length DnaJ or its domains, against DNAJA1 and DNAJA2, as well as monoclonal anti-DnaJ antibodies, we found immunological similarities between the bacterial and human Hsp40s. Both ELISA and Western blotting showed that these similarities were not restricted to the conserved J domains but were also present in the C-terminal variable regions. We also found a positive correlation between the levels of the anti-DnaJ and anti-DNAJA1 antibodies in the sera of RA patients. This finding supports the molecular mimicry hypothesis that human Hsp40 could be the targets of antibodies originally directed against bacterial DnaJ in RA.

Human NK cell lytic granules and regulation of their exocytosis.

Natural killer (NK) cells form a subset of lymphocytes that play a key role in immuno-surveillance and host defense against cancer and viral infections. They recognize stressed cells through a variety of germline-encoded activating cell surface receptors and utilize their cytotoxic ability to eliminate abnormal cells. Killing of target cells is a complex, multi-stage process that concludes in the directed secretion of lytic granules, containing perforin and granzymes, at the immunological synapse. Upon delivery to a target cell, perforin mediates generation of pores in membranes of target cells, allowing granzymes to access target cell cytoplasm and induce apoptosis. Therefore, lytic granules of NK cells are indispensable for normal NK cell cytolytic function. Indeed, defects in lytic granule secretion lead or are related to serious and often fatal diseases, such as familial hemophagocytic lymphohistiocytosis (FHL) type 2-5 or Griscelli syndrome type 2. A number of reports highlight the role of several proteins involved in lytic granule release and NK cell-mediated killing of tumor cells. This review focuses on lytic granules of human NK cells and the advancements in understanding the mechanisms controlling their exocytosis.

Toso, a functional IgM receptor, is regulated by IL-2 in T and NK cells.

We find that the cell surface receptor Toso is dramatically downregulated by in vitro stimulation of human T and NK cells with IL-2 in a STAT5-dependent manner. The fact that IL-2 is known to prime NK and T cells for Fas/TNF-mediated activation-induced cell death (AICD) fits nicely with the original and recent descriptions of Toso as an inhibitor of Fas/TNF-induced apoptosis. In support of this possibility, effector memory T cells express markedly lower levels of Toso than those of naive T cells, indicating that activation in vivo correlates with the downregulation of Toso. Moreover, in vitro activation of memory T cells through TCR dramatically downregulates Toso expression compared with that of naive CD4 T cells. However, overexpression of Toso in human NK cells and Jurkat T cells does not inhibit Fas-mediated apoptosis, and, in agreement with other recent reports, Toso clearly functions as an IgM receptor. Unlike CD16, Toso expression by NK cells does not convey cytotoxic potential, but its ligation does trigger intracellular signaling in NK cells. In summary, our data indicate that Toso is a functional IgM receptor that is capable of activating signaling molecules, is regulated by IL-2, and is not inherently an antiapoptotic molecule.

Cutting edge: mouse CD300f (CMRF-35-like molecule-1) recognizes outer membrane-exposed phosphatidylserine and can promote phagocytosis.

Reportedly, CD300f negatively regulates interactions between dendritic and T cells and acts as an anti-inflammatory molecule in a multiple sclerosis mouse model. We found that a CD300f/Fc chimeric protein specifically binds to apoptotic/dead splenocytes and to apoptotic cells from starved or irradiated lymphocytic cell lines, an observation extended to insect cells. CD300f also binds PMA/ionomycin-activated splenocytes and Ag-stimulated T cells, an interaction inhibited by Annexin V. By ELISA, cosedimentation, and surface plasmon resonance using phospholipid-containing liposomes, we show that CD300f preferentially binds phosphatidylserine and requires a metal ion. Exogenous expression of CD300f in cell lines results in enhanced phagocytosis of apoptotic cells. We conclude that expression of CD300f conveys additional capacity to recognize phosphatidylserine to myeloid cells. The result of this recognition may vary with the overall qualitative and quantitative receptor content, as well as signaling capacity of the expressing effector cell, but enhanced phagocytosis is one measurable outcome.