COPA mutations impair ER-Golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis.

Unbiased genetic studies have uncovered surprising molecular mechanisms in human cellular immunity and autoimmunity. We performed whole-exome sequencing and targeted sequencing in five families with an apparent mendelian syndrome of autoimmunity characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease. We identified four unique deleterious variants in the COPA gene (encoding coatomer subunit α) affecting the same functional domain. Hypothesizing that mutant COPA leads to defective intracellular transport via coat protein complex I (COPI), we show that COPA variants impair binding to proteins targeted for retrograde Golgi-to-ER transport. Additionally, expression of mutant COPA results in ER stress and the upregulation of cytokines priming for a T helper type 17 (TH17) response. Patient-derived CD4(+) T cells also demonstrate significant skewing toward a TH17 phenotype that is implicated in autoimmunity. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease.

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

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

Hemophagocytic lymphohistiocytosis in syntaxin-11-deficient mice: T-cell exhaustion limits fatal disease.

Syntaxin-11 (Stx11), an atypical member of the SNARE protein family, is part of the cytolytic machinery of T and NK cells and involved in the fusion of lytic granules with the plasmamembrane. Functional loss of syntaxin-11 in humans causes defective degranulation and impaired cytolytic activity of T and NK cells. Furthermore, patients with STX11 deficiency develop familial hemophagocytic lymphohistiocytosis type 4 (FHL4), a life-threatening disease of severe hyperinflammation. We established Stx11-deficient mice as an animal model for FHL4. Stx11-deficient mice exhibited severely reduced degranulation and cytolytic activity of CTL and NK cells and developed all clinical symptoms of hemophagocytic lymphohistiocytosis (HLH) after infection with lymphocytic choriomeningitis virus (LCMV). The HLH phenotype was further characterized by hyperactive CD8 T cells and continuous IFN-γ production. However, in contrast to perforin-deficient mice, which represent a model for FHL2, progression of HLH was not fatal. Survival of Stx11-deficient mice was determined by exhaustion of antigen-specific T cells, characterized by expression of inhibitory receptors, sequential loss of effector functions, and finally T-cell deletion. Blockade of inhibitory receptors on T cells in Stx11-deficient mice converted nonfatal disease course into fatal HLH, identifying T-cell exhaustion as an important factor for determination of disease severity in HLH.

Graded defects in cytotoxicity determine severity of hemophagocytic lymphohistiocytosis in humans and mice.

Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease of hyperinflammation resulting from immune dysregulation due to inherited defects in the cytolytic machinery of natural killer and T cells. In humans, mutations in seven genes encoding proteins involved in cytolytic effector functions have so far been identified that predispose to HLH. However, although most affected patients develop HLH eventually, disease onset and severity are highly variable. Due to the genetic heterogeneity and variable time and nature of disease triggers, the immunological basis of these variations in HLH progression is incompletely understood. Several murine models of primary HLH have been established allowing to study HLH pathogenesis under more defined conditions. Here we directly compare the clinical HLH phenotype in six HLH-prone mouse strains with defects in the granule-dependent cytotoxic pathway. A severity gradient of HLH manifestations could be identified that is defined by the genetically determined residual lytic activity of cytotoxic T lymphocytes (CTL) and their ability to control lymphocytic choriomeningitis virus, which was used as a trigger for disease induction. Importantly, analysis of cohorts of HLH patients with severe bi-allelic mutations in the corresponding genes yielded a similar severity gradient in human HLH as reflected by the age at disease onset. Our findings define HLH as a threshold disease determined by subtle differences in the residual lytic activity of CTL.

Subtle differences in CTL cytotoxicity determine susceptibility to hemophagocytic lymphohistiocytosis in mice and humans with Chediak-Higashi syndrome.

Perforin-mediated cytotoxicity is important for controlling viral infections, but also for limiting immune reactions. Failure of this cytotoxic pathway leads to hemophagocytic lymphohistiocytosis (HLH), a life-threatening disorder of uncontrolled T-cell and macrophage activation. We studied susceptibility to HLH in 2 mouse strains (souris and beige(J)) and a cohort of patients with partial defects in perforin secretion resulting from different mutations in the LYST gene. Although both strains lacked NK-cell cytotoxicity, only souris mice developed all clinical and histopathologic signs of HLH after infection with lymphocytic choriomeningitis virus. The 2 strains showed subtle differences in CTL cytotoxicity in vitro that had a large impact on virus control in vivo. Whereas beige(J) CTLs eliminated lymphocytic choriomeningitis virus infection, souris CTLs failed to control the virus, which was associated with the development of HLH. In LYST-mutant patients with Chediak-Higashi syndrome, CTL cytotoxicity was reduced in patients with early-onset HLH, whereas it was retained in patients who later or never developed HLH. Thus, the risk of HLH development is set by a threshold that is determined by subtle differences in CTL cytotoxicity. Differences in the cytotoxic capacity of CTLs may be predictive for the risk of Chediak-Higashi syndrome patients to develop HLH.

Major histocompatibility complex-dependent cytotoxic T lymphocyte repertoire and functional avidity contribute to strain-specific disease susceptibility after murine respiratory syncytial virus infection.

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vß8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vß9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vß8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.

ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis.

Lymphocytes mediate cytotoxicity by polarized release of the contents of cytotoxic granules toward their target cells. Here, we have studied the role of the calcium release-activated calcium channel ORAI1 in human lymphocyte cytotoxicity. Natural killer (NK) cells obtained from an ORAI1-deficient patient displayed defective store-operated Ca(2+) entry (SOCE) and severely defective cytotoxic granule exocytosis leading to impaired target cell lysis. Similar findings were obtained using NK cells from a stromal interaction molecule 1-deficient patient. The defect occurred at a late stage of the signaling process, because activation of leukocyte functional antigen (LFA)-1 and cytotoxic granule polarization were not impaired. Moreover, pharmacological inhibition of SOCE interfered with degranulation and target cell lysis by freshly isolated NK cells and CD8(+) effector T cells from healthy donors. In addition to effects on lymphocyte cytotoxicity, synthesis of the chemokine macrophage inflammatory protein-1ß and the cytokines TNF-α and IFN-γ on target cell recognition was impaired in ORAI1-deficient NK cells, as previously described for T cells. By contrast, NK cell cytokine production induced by combinations of IL-12, IL-15, and IL-18 was not impaired by ORAI1 deficiency. Taken together, these results identify a critical role for ORAI1-mediated Ca(2+) influx in granule exocytosis for lymphocyte cytotoxicity as well as for cytokine production induced by target cell recognition.

Lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections.

Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-alpha, IFN-beta, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-lambda) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-lambda plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-lambda receptor defect. Careful analysis revealed that expression of functional IFN-lambda receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-lambda contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.

Influence of a single viral epitope on T cell response and disease after infection of mice with respiratory syncytial virus.

CTL are important for virus clearance but also contribute to immunopathology after the infection of BALB/c mice with respiratory syncytial virus (RSV). The pulmonary immune response to RSV is dominated by a CTL population directed against the CTL epitope M2-1 82-90. Infection with a virus carrying an M2-1 N89A mutation introduced by reverse genetics failed to activate this immunodominant CTL population, leading to a significant decrease in the overall antiviral CTL response. There was no compensatory increase in responses to the mutated epitope, to the subdominant epitope F 85-93, or to yet undefined minor epitopes in the N or the P protein. However, there was some increase in the response to the subdominant epitope M2-1 127-135, which is located in the same protein and presented by the same H-2Kd MHC molecule. Infection with the mutant virus reversed the oligoclonality of the T cell response elicited by the wild-type virus. These changes in the pattern and composition of the antiviral CTL response only slightly impaired virus clearance but significantly reduced RSV-induced weight loss. These data illustrate how T cell epitope mutations can influence the virus-host relationship and determine disease after an acute respiratory virus infection.

Qualitative and quantitative risk assessment for human salmonellosis due to multi-resistant Salmonella Typhimurium DT104 from consumption of Danish dry-cured pork sausages.

Salmonella Typhimurium DT104 (DT104) is unwanted in products for human consumption due to its antibiotic resistance and ability to cause disease. We intended to set up an improved monitoring and management program to aid in deciding when to use pork contaminated with DT104 for production of sausages without jeopardizing consumer safety. We started by carrying out two assessments of the risk for human health associated with consumption of sausages produced by: (1) Danish pork from average slaughter days; (2) imported pork (IMP) with average prevalence of DT104. The assessments showed that, if Salmonella is present, it is usually in lower numbers (< or =50 per 400 cm(2) surface). Additionally, during processing, the numbers will be reduced by at least 2 log-units. In Danish (DK) pork, DT104 constitutes 0.2-1.0% of the Salmonella isolates reported, while in imported pork (IMP), 18%. We estimated that out of one million, 25 g servings of DK dry-cured sausages, up to two DT104 bacteria could be found in each of 245 servings. Out of one million servings of 25 g IMP dry-cured sausages, up to two DT104 bacteria would occur in each of 19,260 servings.