Current and future directions of pathologic analysis in hemophagocytic syndrome

Hemophagocytic syndrome is a rare disorder with dysfunction of cytotoxic T lymphocyte (CTL) or NK cell activity, leading to excessive production of inflammatory cytokines and various clinical symptoms. HLH can be classified as either primary or secondary form; primary HLH includes familial hemophagocytic lymphohistiocytosis (FHL) and several immune deficiencies. All affected genes are involved in the transport and membrane fusion, or exocytosis of perforin/granzyme in lytic granules. Making a rapid screening of FHL with flow cytometry followed by genetic analysis is mandatory for the appropriate treatment of this fatal disease. Whereas, pathogenesis of secondary HLH is still unknown; several genetic backgrounds to affect on the pathway of T-cell activity will be associated with secondary HLH. With perforin- or Munc-deficient mouse model that develop HLH-like symptoms after virus infection, CD8+ T cells and interferon-gamma have been proven to be necessary for the HLH development. These data will provide new targets for specific therapeutic intervention of HLH in the future.     

Natural killer (NK) cell response to virus infections in mice with severe combined immunodeficiency. The stimulation of NK cells and the NK cell-dependent control of virus infections occur independently of T and B cell function

The activation, proliferation, and antiviral properties of natural killer (NK) cells were examined in severe combined immunodeficiency (SCID) mice to determine the influence of mature T or B cells on virus-induced NK cell functions and to more conclusively determine the antiviral properties of prototypical CD3- NK cells. NK cells were activated to high levels of cytotoxicity 3 d after infection of mice with lymphocytic choriomeningitis virus (LCMV) or murine cytomegalovirus (MCMV). Analyses of spleen leukocytes from LCMV-infected mice by a variety of techniques indicated that the NK cells proliferated and increased in number during infection. Propidium iodide staining of the DNA of cycling cells revealed that the great majority of proliferating spleen leukocytes 3 d after LCMV infection was of the NK cell phenotype (CD3-, Ig-, Mac-1+, CZ1+, 50% Thy-1+), in contrast to uninfected mice, whose proliferating cells were predominantly of other lineages. Analyses of the NK cell responses over a 2 wk period in control CB17 mice infected with MCMV indicated a sharp rise in serum interferon (IFN) and spleen NK cell activity early (days 3-5) in infection, followed by sharp declines at later stages. In SCID mice the IFN levels continued to rise over a 10-d period, whereas the NK cell response peaked on day 3-5 and gradually tapered. In contrast to the immunocompetent CB17 mice, SCID mice did not clear the MCMV infection and eventually succumbed. SCID mice, again in contrast to immunocompetent CB17 mice, also failed to clear infections with LCMV and Pichinde virus (PV); these mice, infected as adults, did not die but instead developed long-term persistent infections. Depletion of the NK cells in vivo with antiserum to asialo GM1 rendered both SCID and CB17 control mice much more sensitive to MCMV infection, as shown by titers of virus in organs and by survival curves. In contrast, similar depletions of NK cells did not enhance the titers of the NK cell-resistant virus, LCMV. Two variants of PV, one sensitive to NK cells and the other selected for resistance to NK cells by in vivo passage, were also tested in NK cell-depleted SCID mice. The NK-sensitive PV replicated to higher titers in NK cell-depleted SCID mice, whereas the titers of the NK cell-resistant PV were the same, whether or not the mice had NK cells. These experiments support the concept that CD3- prototypical NK cells mediate resistance to NK cell-sensitive viruses via a mechanism independent of antiviral or "natural" antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adoptive transfer studies demonstrating the antiviral effect of natural killer cells in vivo

We carried out adoptive transfer studies to determine the role of natural killer (NK) cells in resistance to murine cytomegalovirus (MCMV) and lymphocytic choriomeningitis virus (LCMV). We transferred leukocytes from adult mice into suckling mice 1 d before injecting them with virus. Resistance was measured by enhancement of survival and reduction of virus multiplication in the spleens of recipient mice. The phenotype of the cell population capable of mediating resistance to MCMV was that of a nylon wool-nonadherent, asialo GM1+, NK 1.2+, Ly-5+, Thy-1-, Ia-, low density lymphocyte; this is the phenotype of an NK cell. Cloned NK cells, but not cloned T cells, provided resistance to MCMV in suckling mice. Cloned NK cells also provided resistance to MCMV in irradiated adult mice, and antibody to asialo GM1, which depletes NK cell activity in vivo, enhanced the synthesis of MCMV in athymic nude mice. Neither adult leukocytes nor cloned NK cells influenced LCMV synthesis in suckling mice. We conclude that a general property of NK cells may be to provide natural resistance to virus infections, and that NK cells can protect mice from MCMV but not from LCMV.     

Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-D^k and the MCMV m04 protein. Using H2 chimeras between H2-D^b and -D^k, we demonstrate that the H2-D^k peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Δm04) virus infection. MA/My mice, which express Ly49P and H2-D^k, are resistant to MCMV; however, infection with Δm04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Δm04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatability complex class I–restricted recognition of virally infected cells by an activating NK cell receptor.Humans (1) and mice (2) lacking NK cells are vulnerable to certain viral infections. NK cells patrol for aberrant cells, which they eliminate through cytotoxic activity and secretion of cytokines (3). NK cell activation depends on a complex array of inhibitory and activating receptors. Inhibitory receptors recognizing MHC class I molecules, including human killer cell immunoglobulin-like receptors (KIR) and mouse Ly49 receptors, play a predominant role. Both receptor families contain activating receptors, but the ligands are not well characterized. Viruses have devised numerous strategies to modulate the function of NK cells and have influenced the evolution of NK cell receptors.The identification of mouse genes that protect from mouse cytomegalovirus (MCMV) infection has provided insights into the NK cell–mediated control of viral proliferation. In C57BL/6 mice, NK cells expressing the activating Ly49H receptor that binds directly to m157, which is a MCMV-encoded cell surface glycoprotein (3), control viral replication early after infection (4). The essential role of Ly49H was confirmed by the transfer of MCMV resistance to genetically susceptible mice by Ly49h transgenesis (5), whereas absence of Ly49h in C57BL/6 mice abolishes resistance (6). Moreover, C57BL/6 mice are susceptible to MCMV mutants lacking m157 (Δm157) (7). Collectively, these data demonstrated the importance of activating NK cell receptor–specific recognition of a virus-encoded ligand in host resistance.Despite lacking Ly49h, MA/My mice are resistant to MCMV (8, 9), which depends on the specific combination of MA/My alleles at Ly49 and MHC loci. The activating receptor Ly49P in MA/My mice recognizes MCMV-infected cells, and Ly49P-dependent activation is abrogated by an anti–H2-D^k antibody (9). These results suggested that MA/My resistance is conferred by NK cell–mediated recognition of infected cells by a mechanism involving Ly49P, H2-D^k, and an additional molecule expressed during MCMV infection.CMVs possess numerous genes dedicated to manipulating the host immune response. Three MCMV gene products alter host MHC class I expression: m04, m06, and m152 (10). m152 retains peptide-loaded MHC class I in the endoplasmic reticulum cis-Golgi intermediate compartment (11). m04 and m06 carry cytoplasmic (CT) motifs involved in cargo-sorting pathways; m06 redirects MHC class I to the late endosome–lysosome pathway for degradation, thus preventing antigen presentation (12). m04 associates with MHC class I in the ER, and these complexes travel to the cell surface (13, 14); however, m04 does not down-regulate MHC class I.Here, we found that Ly49P recognition depends on the following: H2-D^k expression on the infected cell, the peptide-binding groove of H2-D^k, and m04. Infection of MA/My mice with a m04-deletion MCMV mutant resulted in increased susceptibility, suggesting that Ly49P-mediated NK cell recognition of H2-D^k complexed with m04 on the infected cell is necessary to attenuate virus replication.     

EBAG9 tempers lymphocyte killing activity

The cytotoxic activity of lymphocytes is crucial for immune surveillance and homeostasis. Several independent, naturally occurring genetic models characterized by defects in granule trafficking or exocytosis have helped to decipher the multiple steps and molecules that regulate the cytotoxic process. The study by Rüder and colleagues in this issue of the JCI shows that an engineered absence of EBAG9, previously reported as a tumor-associated antigen, enhances cytotoxic activity of CTLs but not NK cells, likely acting on the endosomal-lysosomal trafficking of the cytotoxic effectors (see the related article beginning on page 2184). This finding adds a new piece to the puzzle of complex mechanisms that tightly regulate the capacity of the cytotoxic response and suggests a new target to negatively modulate CTL responsiveness. CTLs and NK cells (collectively known as “cytotoxic lymphocytes”) are major players in the body’s defense against viral infection and cancer via their ability to seek out and kill infected or tumorigenic cells. Although CTLs are activated by specific antigen recognition, the cytotoxic activity of NK cells is initiated by specific activating receptors or combinations thereof and is inhibited by self MHC class I recognition. Once cytotoxic cells have recognized their targets and formed a conjugate, the trafficking of granule components, including perforin and granzymes, to the immunological synapse (IS) between the cell and its target, leads to the delivery of perforin and granzymes into the target cells and subsequent target destruction through apoptosis (1, 2). Studies of natural or engineered mutants involving cytotoxic function tell us a great deal about the in vivo function of this pathway. So far, only genetic diseases leading to the loss of cytotoxic function have been reported (3). These studies have revealed the tremendous importance of this cytotoxic pathway in immune homeostasis. Congenital defects that lead to either impaired perforin function or its dysregulated release result in the specific severe condition hemophagocytic lymphohistiocytosis (HLH) syndrome. This syndrome stems from the inability of activated cytotoxic cells to clear antigen-presenting targets. The failure to clear antigen causes unremitting polyclonal CD8⁺ T cell expansion, activation, and infiltration of visceral organs associated with macrophage activation, with the deleterious release of multiple inflammatory cytokines including IFN-γ (3, 4). In addition, the studies of these natural mutants both in humans and in mice have contributed to the characterization of critical effectors of the cytotoxic machinery and their function. They also provided evidence of the exquisite regulation of this process, which occurs in successive steps following cell activation. For instance, studies of Griscelli syndrome type 2, a rare condition characterized by partial albinism and occurrence of an HLH syndrome (5), and familial HLH type 3 (FHL3) (6) have shown the critical role of two proteins, the small GTPase Rab27a and the priming factor Munc13-4, at a late step of the secretory pathway. Rab27a regulates the tethering of mature cytotoxic granules that have polarized at the IS, whereas Munc13-4 primes the docked cytotoxic granules before their fusion with the plasma membrane at the IS. Other proteins, for which their defect impairs lymphocyte cytotoxic activity, likely regulate an upstream step in the cytotoxic pathway. They include adapter protein 3 (AP3), which is deficient in Hermansky-Pudlak syndrome type 2 (HPS2) (7, 8), and the lysosomal trafficking regulator (LYST) protein, which is deficient in Chediak-Higashi syndrome (9, 10). Their respective defects include missorting of transmembrane proteins to lysosomes and an increase in the size of cytotoxic granules. Although the precise functions of these effectors in the cytotoxic pathway remain poorly understood, both proteins likely regulate lysosomal biogenesis and membrane fusion along the cytotoxic pathway. The proteins mentioned above are all required for proper cytotoxic activity of lymphocytes. In contrast, Rüder and colleagues report in this issue of the JCI that estrogen receptor–binding fragment–associated antigen 9 (EBAG9) acts as a negative regulator of cytotoxic function (11). Through the generation of Ebag9-deficient mice, they show that loss of EBAG9 enhances the release of cytotoxic granules in vitro as well as the cytotoxic capacity of CTLs from deficient mice in vivo. The increase in the sorting of cytotoxic effector molecules to cytotoxic granules associated with EBAG9 deficiency, together with the demonstration that EBAG9 interacts with the γ2-adaptin adapter protein, indicate that EBAG9 plays a pivotal role in the sorting of endosomal-lysosomal proteins. Therefore, the work presented by Rüder and colleagues introduces a new player involved in the tuning of the cytotoxic function of lymphocytes. However, it remains to be determined what the precise molecular mechanism is that underlies the observed phenotype; there is no evidence of immunopathogenesis in EBAG9-deficient mice. In the future, it may be worth studying the consequences of lymphocytic choriomeningitis virus (LCMV) infection in EBAG9-deficient mice, as in this setting mice impaired in cytotoxic lymphocyte function demonstrate a clear immune disease, i.e., HLH (12). An “opposite” phenotype, with limitation of the immune response to pathogen, might be seen, as we discuss below. The trafficking of lysosomal proteins in other cells, such as mast cells, may also be increased in EBAG9-deficient mice, potentially exacerbating an anaphylactic reaction.     

中枢神经系统受累的儿童噬血细胞性淋巴组织增生症临床分析

目的分析儿童噬血细胞性淋巴组织增生症(hemophagocytic lymphohistiocytosis, HLH)患儿中枢神经系统受累的临床特征和预后。方法回顾性分析13例出现中枢神经系统受累的HLH患儿的临床及影像学资料,并应用Kaplan-Meier法分析5年生存率。结果 13例HLH患儿中癫痫发作7例,意识障碍3例,面神经麻痹1例,烦躁1例,吞咽困难和构音障碍1例;1例有恶性血液病家族史;3例MUNC13-4基因突变,1例编码perforin-1基因突变;4例感染EB病毒;13例患儿均行脑CT和MRI检查,7例影像学表现正常,6例异常;7例癫痫发作患者中2例服用抗癫痫药物治疗,5例未服用抗癫痫药物情况下癫痫停止发作;13例患儿死亡4例,5年生存率为69%。结论中枢神经系统受累的HLH患者死亡风险较高,脑CT/MRI影像检查可表现异常,对提高HLH诊断有一定价值。     

Recombinant mouse cytomegalovirus expressing a ligand for the NKG2D receptor is attenuated and has improved vaccine properties

Human CMV (HCMV) is a major cause of morbidity and mortality in both congenitally infected and immunocompromised individuals. Development of an effective HCMV vaccine would help protect these vulnerable groups. NK group 2, member D (NKG2D) is a potent activating receptor expressed by cells of the innate and adaptive immune systems. Its importance in HCMV immune surveillance is indicated by the elaborative evasion mechanisms evolved by the virus to avoid NKG2D. In order to study this signaling pathway, we engineered a recombinant mouse CMV expressing the high-affinity NKG2D ligand RAE-1γ (RAE-1γMCMV). Expression of RAE-1γ by MCMV resulted in profound virus attenuation in vivo and lower latent viral DNA loads. RAE-1γMCMV infection was efficiently controlled by immunodeficient hosts, including mice lacking type I interferon receptors or immunosuppressed by sublethal γ-irradiation. Features of MCMV infection in neonates were also diminished. Despite tight innate immune control, RAE-1γMCMV infection elicited strong and long-lasting protective immunity. Maternal RAE-1γMCMV immunization protected neonatal mice from MCMV disease via placental transfer of antiviral Abs. Despite strong selective pressure, the RAE-1γ transgene did not exhibit sequence variation following infection. Together, our results indicate that use of a recombinant virus encoding the ligand for an activating NK cell receptor could be a powerful approach to developing a safe and immunogenic HCMV vaccine.     

Murine cytomegalovirus is regulated by a discrete subset of natural killer cells reactive with monoclonal antibody to Ly49H.

Antiviral roles of natural killer (NK) cell subsets were examined in C57BL/6 mice infected with murine cytomegalovirus (MCMV) and other viruses, including lymphocytic choriomeningitis virus (LCMV), vaccinia virus (VV), and mouse hepatitis virus (MHV). Each virus vigorously induced an NK cell infiltrate into the peritoneal cavity and liver, causing some redistributions of NK cell subsets defined by monoclonal antibody (mAb) directed against Ly49A, C/I, D, and G2. Striking results were seen with a mAb (1F8) reactive with the positively signaling molecule Ly49H, present in MCMV-resistant C57BL/6 mice. mAb 1F8 also stains Ly49 C and I, but exclusion of those reactivities with mAb 5E6, which recognizes Ly49 C and I, indicated that Ly49H(+) cells infiltrated the peritoneal cavity and liver and were particularly effective at synthesizing interferon gamma. Depletion of 1F8(+) but not 5E6(+) cells in vivo by mAb injections enhanced MCMV titers by 20-1,000-fold in the spleen and approximately fivefold in the liver. Titers of LCMV or VV were not enhanced. These anti-MCMV effects were attributed to prototypical NK1.1(+)CD3(-) NK cells and not to NK1.1(+)CD3(+) "NK/T" cells. This is the first evidence that control of a virus infection in vivo is mediated by a distinct NK cell subset.     

Pathogenesis of of cytomegalovirus infection. I. Activation of virus from bone marrow-derived lymphocytes by in vitro allogenic reaction

After infection in utero or at birth with a cell culture adapted strain of mouse cytomegalovirus (MCMV), several mouse strains developed a latent virus infection in the presence of specific antiviral antibodies. Up to 5 mo after infection, MCMV could be activated and recovered from spleen lymphocytes of the infected animals that were co-cultivated with histoincompatible (H-2 foreign) mouse embryo cells from uninfected animals. In contrast, co-cultivation of lymphoid cells from infected mice with mouse embryo cells from syngeneic, histocompatible (H-2 similar) donors did not activate MCMV. Similarly, MCMV was not recovered from sonicated lymphoid cells. Virus was activated by treating viable lymphoid cells with lipopolysaccharide, a B-cell mitogen, but was not activated by a variety of other mitogens such as phytohemagglutinin, concanavalin A, or pokeweed mitogen. Subsequent purification of lymphoid cells from the infected mice by a variety of techniques indicated that MCMV was harbored in the B-lymphocyte population.     

Requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration

The presence of natural killer (NK) cells contributes to early defense against murine cytomegalovirus (MCMV) infection. Although NK cells can mediate in vivo protection against MCMV, the mechanism by which they do so has not been defined. The studies presented here evaluate cytokine production by NK cells activated during MCMV infection and the role of NK cell-produced cytokines in early in vivo antiviral defenses. Experiments with normal C57BL/6, T cell-deficient C57BL/6 nude, and severe combined immunodeficient mice lacking T and B cells demonstrated that both interferon gamma (IFN-gamma) and tumor necrosis factor (TNF) production were induced at early times after infection with MCMV. Conditioned media samples prepared with cells from these mice, on day 2 after infection, produced 11-43 pg/million cells of IFN-gamma and 12-19 pg/million cells of TNF as evaluated by specific protein enzyme-linked immunosorbent assays. Studies in the NK- and T cell-deficient mouse line, E26, in mice that had been depleted in vivo of NK cells by treatment with antibodies eliminating NK cells, anti-asialo ganglio-N- tetraosylceramide or anti-NK1.1, and with populations of cells that had been depleted of NK cells by complement treatment with the anti-NK cell antibody, SW3A4, demonstrated that NK cells were solely responsible for the IFN-gamma but were not required for TNF production. The in vivo absence of NK cells was accompanied by increased viral hepatitis and viral replication in both immunocompetent and immunodeficient mice, as well as decreased survival time of immunodeficient mice. In vivo treatments with antibodies neutralizing IFN-gamma demonstrated that this factor contributed to the NK cell-mediated antiviral defense and reduced the measured parameters of viral defense to levels indistinguishable from those observed in NK cell-deficient mice. These effects appeared to be independent of cytolytic activity, as NK cells isolated from anti-IFN-gamma-treated mice mediated killing at levels comparable to those observed in control-treated mice. The consequences of interleukin 12 (IL-12) administration, a known potent inducer of IFN- gamma production by NK cells, were evaluated in MCMV-infected mice. Low IL-12 doses, i.e., 1 ng/d, increased NK cell cytotoxicity and IFN-gamma production up to twofold and resulted in improved antiviral status; virus-induced hepatitis was decreased as much as fivefold, and viral burdens were decreased to levels below detection.(ABSTRACT TRUNCATED AT 400 WORDS)     

家族性噬血细胞性淋巴组织细胞增生症1例并文献复习

目的加强对家族性噬血细胞性淋巴组织细胞增生症（familially hemophagocytic lymphohistiocytosis,FHL）的认识。方法报道确诊为FHL的新病例1例,结合国内外报道的FHL的病例,对该病的临床特点进行汇总分析。结果FHL2常与PRF1基因突变相关,约20%~40%的患者存在穿孔素基因突变。结论对于有阳性家族史,基因诊断明确,应尽早行化疗或者造血干细胞移植。若无家族史,未发现与继发性HLH相关的原发病因,可考虑行基因筛查以明确是否存在FHL的可能。     

The cytomegalovirus m155 gene product subverts natural killer cell antiviral protection by disruption of H60-NKG2D interactions

Natural killer (NK) cells are an important early mediator of host immunity to murine cytomegalovirus (MCMV) infection. However, MCMV has evolved mechanisms to elude recognition and clearance by NK cells. We have identified an MCMV immune evasion protein that impairs NKG2D-mediated NK cell antiviral activity. Infection of BALB/c 3T3 cells with the Smith strain of MCMV resulted in strong down-regulation of H60, a high affinity ligand for NKG2D, from the surface of virus-infected cells. The MCMV m155 protein specifically down-regulated H60 without affecting expression of the other known NKG2D ligands, RAE-1 and MULT-1. Treatment with the proteasome inhibitors lactacystin or epoxomicin reversed m155 down-regulation of H60. An MCMV mutant virus lacking m155 was severely attenuated in BALB/c mice; however, treatment with neutralizing anti-NKG2D monoclonal antibody or with NK-depleting anti-asialo GM1 antisera restored virulence of the mutant virus. Thus, down-regulation of H60 by m155 is a powerful mechanism of inhibiting NKG2D-mediated antiviral function.     

Failed CTL/NK cell killing and cytokine hypersecretion are directly linked through prolonged synapse time

Failure of cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells to kill target cells by perforin (Prf)/granzyme (Gzm)-induced apoptosis causes severe immune dysregulation. In familial hemophagocytic lymphohistiocytosis, Prf-deficient infants suffer a fatal “cytokine storm” resulting from macrophage overactivation, but the link to failed target cell death is not understood. We show that prolonged target cell survival greatly amplifies the quanta of inflammatory cytokines secreted by CTLs/NK cells and that interferon-γ (IFN-γ) directly invokes the activation and secondary overproduction of proinflammatory IL-6 from naive macrophages. Furthermore, using live cell microscopy to visualize hundreds of synapses formed between wild-type, Prf-null, or GzmA/B-null CTLs/NK cells and their targets in real time, we show that hypersecretion of IL-2, TNF, IFN-γ, and various chemokines is linked to failed disengagement of Prf- or Gzm-deficient lymphocytes from their targets, with mean synapse time increased fivefold, from ∼8 to >40 min. Surprisingly, the signal for detachment arose from the dying target cell and was caspase dependent, as delaying target cell death with various forms of caspase blockade also prevented their disengagement from fully competent CTLs/NK cells and caused cytokine hypersecretion. Our findings provide the cellular mechanism through which failed killing by lymphocytes causes systemic inflammation involving recruitment and activation of myeloid cells.CTLs and NK cells are essential eliminators of cancerous and virus-infected cells. After immunological synapse (IS) formation, these “killer cells” release perforin (Prf) and granzymes (Gzms) from their specialized secretory vesicles (Jenkins and Griffiths, 2010). Prf transiently forms pores on the target cell membrane, enabling diffusion of proapoptotic serine protease Gzms into the cytosol (Lopez et al., 2013a,b), to trigger caspase activation via both the extrinsic and intrinsic (mitochondrial) pathways. In our recent study, target cell death was thus initiated within 2–3 min of Prf pore formation (Lopez et al., 2013a). After detaching, a CTL/NK cell can rapidly attack other target cells, and “serial killing” of up to 10 cells can be observed for NK cells in vitro within 6 h (Choi and Mitchison, 2013).Prf-dependent cytotoxicity is critical for human immune homeostasis: infants with biallelic PRF1 gene mutations develop a fatal immune dysregulation syndrome, type 2 familial hemophagocytic lymphohistiocytosis (FHL2; Stepp et al., 1999). This hyperinflammatory state reflects release of the proinflammatory cytokine IFN-γ by CTLs/NK cells after their failure to shut down the antigen-driven phase of the immune response and copious IL-1β, IL-6, and TNF that then emanate from the myeloid compartment. Intractable fever, pancytopenia, multiorgan failure, and death result unless patients receive cytotoxic agents or, ultimately, bone marrow transplantation (Janka, 2012). Prf1 knockout mice also develop a fatal FHL-like state after challenge with certain antigenic or viral stimuli (Kägi et al., 1994; Jordan et al., 2004; van Dommelen et al., 2006). In other congenital forms of FHL, PRF1 expression is normal, but the trafficking, docking, or exocytosis of cytotoxic granules is impaired and Prf is not delivered to the IS (Sieni et al., 2014). Linking failed killing by lymphoid cells with fatal hyperinflammation, mediated principally by myeloid cells (particularly macrophages), remains a central unanswered question.In the current study, we discovered that failure of Prf/Gzm cytotoxicity by human or mouse CTLs/NK cells dramatically extends the life of the IS, leading to repetitive calcium signaling and their pronounced hypersecretion of inflammatory cytokines and chemokines. In turn, this inflammatory “cocktail” was capable of activating naive macrophages and evoking IL-6 secretion. By blocking caspase processing in the target cell, we further demonstrated that disengagement of CTLs/NK cells from the target was specifically dependent on target cell death, revealing that the dying cell provides a caspase-dependent signal for detachment. Our study provides a mechanistic explanation for the immunopathology of FHL and links fatal myeloid cell activation with marked delay or failure of target cell death mediated by lymphocytes. Furthermore, our finding that corruption of apoptotic pathways in tumor target cells attacked by CTLs/NK cells can influence the resultant inflammatory milieu has implications for our understanding of the immune response to cancer and the mode of action of immune-based therapies that aim to augment lymphocytotoxicity.     

Mode of regulation of natural killer cell activity by interferon

Whereas xenogeneic tumors such as baby hamster kidney or HeLa cells grow in nude mice, the same cells persistently infected with a variety of viruses are rejected. Spleen cells from normal nude mice were found to be induced to produce interferon and to exert natural killer (NK) activity on virus persistently infected (PI) tumor cells, and not on uninfected parental cells in vitro. The phenotype of the interferon-producing cells and the NK effector cells was found to be the same namely, Qa 5(+), Ly 5(+), ganglio-N- tetraosylceramide, with 35 percent of the NK cells also expressing Thy 1.2. NK activity against virus PI tumor cell lines could be nonspecifically augmented both in vivo and in vitro by prior contact with virus PI tumor cells. It was unambiguously demonstrated with chemically homogeneous mouse interferon that interferon, and not a contaminant, was responsible for the augmentation of NK activity in vitro. Studies on the mode of interferon action in augmenting NK activity revealed that the target cell for interferon action was serologically distinct from the NK effector cell. Anti-Ly 5 + complement (C)-treated spleen cells were depleted of NK activity and the ability to produce interferon, but, upon incubation with interferon for 1-3 h, regained both NK activity and susceptibility to anti-Ly 5 + C. Treatment with anti-Qa 5 + C eliminated NK activity, which could not be restored by the addition of interferon. We conclude that interferon produced by Ly 5(+) cells in response to virus PI tumor cells acts on Ly 5(-) precursor cells and induces their differentiation into functional Ly 5(+) NK effector cells.     

NK细胞活性与血清可溶性CD25水平检测在继发性噬血细胞性淋巴组织细胞增多症诊断中的意义

本研究探讨继发性噬血细胞性淋巴组织细胞增多症（secondary hemophagocytic lymphohistiocytosis，HLH）患者外周血NK细胞活性、血清可溶性CD25（sCD25）水平的检测在早期诊断中的意义。收集2005年6月至2008年5月继发性HLH疑似病例38例，25名正常健康人员作为对照，采用LDH释放法检测外周血NK细胞活性，ELISA法检测血sCD25的水平，38例疑似病例根据HLH-2004诊断标准分为排除组和诊断组，比较各组NK细胞活性及sCD25水平的差异，同时比较确诊组确诊前后各项诊断标准的符合情况。结果表明：38例疑似患者中有22例最终确诊为继发性HLH，其NK细胞活性均明显低于正常对照组，差异有显著统计学意义（P〈0．001），血清sCD25水平明显高于正常对照组，差异亦有统计学意义（p〈0．05），且确诊组患者的NK细胞活性和血清sCD25水平在疾病早期100％出现异常。结论：NK细胞活性及血清sCD25水平的检测对于继发性HLH的早期诊断可能具有重要的意义。     

儿童脓毒症合并噬血细胞综合征诊断学特征分析

目的探讨儿童脓毒症合并噬血细胞综合征的诊断学特征。 方法回顾性分析2016年1月至2020年7月广东省妇幼保健院儿科重症监护室收治并确诊的37例脓毒症合并噬血细胞综合征患儿的临床症状及实验室检测结果。 结果37例脓毒症合并噬血细胞综合征患儿最常见的临床症状为发热(100.00%)，峰值38 ℃以上，其余主要特征包括肝大(32.43%)、皮疹(32.43%)、浅表淋巴结肿大(27.03%)和三凹征(21.62%)。实验室检查中，37例患儿均出现了不同程度的丙氨酸转氨酶、D-二聚体及血清铁蛋白水平升高，血红蛋白水平及血小板计数降低29例(78.38%)，B淋巴细胞比例升高23例(62.16%)，NK细胞比例降低24例(64.86%)，天冬氨酸转氨酶升高34例(91.89%)，骨髓中均发现噬血细胞。29例(78.38%)患儿病原体检测阳性；EB病毒检出率最高(48.65%)；真菌以白念珠菌为主(18.92%)。单一原发感染部位以呼吸道感染为主(37.84%)。经治疗，治愈出院15例，转院治疗8例，放弃治疗12例，死亡2例。 结论脓毒症合并噬血细胞综合征患儿的临床表现复杂多样，实验室及病原体检测中易出现高铁血红蛋白血症和(或)EB病毒感染，早期诊断对救治患儿的生命有重大作用。     

Successful treatment of murine cytomegalovirus disease does not prevent latent virus infection

9-(1,3-Dihydroxy-2-propoxymethyl)guanine (DHPG), a nucleoside analogue, inhibits the replication of human and murine cytomegalovirus (MCMV) in cell culture. We studied the effects of treatment with DHPG on acute MCMV infection in mice and assessed the impact of drug therapy on the eventual development of latent viral infection. In virus-susceptible Balb/c mice, DHPG treatment limited dissemination of virus infection and prevented death. In sublethal infection of both Balb/c and virus-resistant C3H/St mice, DHPG prevented recovery of infectious virus from visceral organs, including the spleen. Despite these effects of drug treatment on virus replication during acute infection, latent MCMV could be reactivated in vivo by immunosuppression and in vitro by spleen explantation in virtually all mice. These results indicate that successful treatment of MCMV infection and marked suppression of viral replication do not prevent establishment of viral latency.     

可溶性IL-2受体及NK细胞活性在噬血细胞性淋巴组织细胞增多症中的意义

本研究通过检测噬血细胞性淋巴组织细胞增多症(HLH)患者外周血中可溶性IL-2受体(sCD25)及NK细胞活性,探讨它们在HLH中的意义。应用酶联免疫吸附法检测20例HLH患者、15例正常对照者、20例急性髓系白血病(AML)患者及20例系统性红斑狼疮(SLE)患者外周血血清sCD25水平;用流式细胞术CD107a标记法及传统的LDH释放法检测HLH组及正常对照组外周血NK细胞活性。结果显示:HLH组血清sCD25水平较正常对照组、AML组及SLE组均明显升高(P<0.001);HLH组NK细胞活性明显低于正常对照组(P<0.05);将流式细胞术CD107a标记法与传统的LDH释放法检测NK细胞活性进行相关分析,该两种检测方法之间具有显著相关性(r=0.73,P<0.05)。结论:血清sCD25及外周血NK细胞活性检测是HLH重要的辅助诊断指标,流式细胞术CD107a标记法检测NK细胞活性简单、稳定、重复性高,有助于HLH的临床诊断。