KIR2DL4-HLAG interaction at human NK cell-oligodendrocyte interfaces regulates IFN-γ-mediated effects.

Interactions between germline-encoded natural killer (NK) cell receptors and their respective ligands on tumorigenic or virus-infected cells determine NK cell cytotoxic activity and/or cytokine secretion. NK cell cytokine responses can be augmented in and can potentially contribute to multiple sclerosis (MS), an inflammatory disease of the central nervous system focused upon the oligodendrocytes (OLs). To investigate mechanisms by which NK cells may contribute to MS pathogenesis, we developed an in vitro human model of OL-NK cell interaction. We found that activated, but not resting human NK cells form conjugates with, and mediate cytotoxicity against, human oligodendrocytes. NK cells, when in conjugate with OLs, rapidly synthesize and polarize IFN-γ toward the OLs. IFN-γ is capable of reducing myelin oligodendrocyte and myelin associated glycoproteins (MOG and MAG) content. This activity is independent of MHC class-I mediated inhibition via KIR2DL1, but dependent upon the interaction between NK cell-expressed KIR2DL4 and its oligodendrocyte-expressed ligand, HLA-G. NK cells from patients with MS express higher levels of IFN-γ following conjugation to OLs, more actively promote in vitro reduction of MOG and MAG and have higher frequencies of the KIR2DL4 positive population. These data collectively suggest a mechanism by which NK cells can promote pathogenic effects upon OLs.

Latent therapeutic demand model for the immunoglobulin replacement therapy of primary immune deficiency disorders in the USA.

BACKGROUND AND OBJECTIVES: Our research aim is to model latent therapeutic demand (LTD) for the immunoglobulin replacement therapy (IgGRT) of primary immune deficiency disorders (PIDDs) in the USA. Given the high level of variability of IgGRT use and major differences among American and European practices in the management of patients with PIDDs, we develop a USA-specific LTD model for common variable immune deficiency (CVID), hyper IGM syndrome, severe combined immune deficiency, Wiskott-Aldrich syndrome and X-linked agammaglobulinemia (XLA). METHODS AND MATERIALS: We use decision analysis methods to model the underlying IgGRT demand for PIDDs by assessing USA-specific epidemiology and treatment. Data for the epidemiology and treatment variables were obtained from the medical literature, USIDNET and Immune Deficiency Foundation. The uncertainty surrounding the variables was modelled using probability distributions and evaluated using Monte Carlo simulation. RESULTS: The mean treatment dose from USIDNET and European Society for Immunodeficiencies (ESID) was significantly different for treating CVID, and the number of annual infusions from USIDNET and ESID was significantly different for treating CVID and XLA. The mean and standard deviation of LTD for all PIDDs is 105·1 ± 88·5 g per 1000 population, with CVID contributing the most to LTD. CONCLUSION: Estimating country-specific LTD is important to ensure an adequate supply of IgGRT and an optimal treatment for patients with PIDDs and for improving national healthcare policymaking and production planning.

Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS: We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS: Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS: Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Current treatment options with immunoglobulin G for the individualization of care in patients with primary immunodeficiency disease.

Primary antibody deficiencies require lifelong replacement therapy with immunoglobulin (Ig)G to reduce the incidence and severity of infections. Both subcutaneous and intravenous routes of administering IgG can be effective and well tolerated. Treatment regimens can be individualized to provide optimal medical and quality-of-life outcomes in infants, children, adults and elderly people. Frequency, dose, route of administration, home or infusion-centre administration, and the use of self- or health-professional-administered infusion can be tailored to suit individual patient needs and circumstances. Patient education is needed to understand the disease and the importance of continuous therapy. Both the subcutaneous and intravenous routes have advantages and disadvantages, which should be considered in selecting each patient's treatment regimen. The subcutaneous route is attractive to many patients because of a reduced incidence of systemic adverse events, flexibility in scheduling and its comparative ease of administration, at home or in a clinic. Self-infusion regimens, however, require independence and self-reliance, good compliance on the part of the patient/parent and the confidence of the physician and the nurse. Intravenous administration in a clinic setting may be more appropriate in patients with reduced manual dexterity, reluctance to self-administer or a lack of self-reliance, and intravenous administration at home for those with good venous access who prefer less frequent treatments. Both therapy approaches have been demonstrated to provide protection from infections and improve health-related quality of life. Data supporting current options in IgG replacement are presented, and considerations in choosing between the two routes of therapy are discussed.

Vaccine-associated varicella and rubella infections in severe combined immunodeficiency with isolated CD4 lymphocytopenia and mutations in IL7R detected by tandem whole exome sequencing and chromosomal microarray.

In areas without newborn screening for severe combined immunodeficiency (SCID), disease-defining infections may lead to diagnosis, and in some cases, may not be identified prior to the first year of life. We describe a female infant who presented with disseminated vaccine-acquired varicella (VZV) and vaccine-acquired rubella infections at 13 months of age. Immunological evaluations demonstrated neutropenia, isolated CD4 lymphocytopenia, the presence of CD8(+) T cells, poor lymphocyte proliferation, hypergammaglobulinaemia and poor specific antibody production to VZV infection and routine immunizations. A combination of whole exome sequencing and custom-designed chromosomal microarray with exon coverage of primary immunodeficiency genes detected compound heterozygous mutations (one single nucleotide variant and one intragenic copy number variant involving one exon) within the IL7R gene. Mosaicism for wild-type allele (20-30%) was detected in pretransplant blood and buccal DNA and maternal engraftment (5-10%) demonstrated in pretransplant blood DNA. This may be responsible for the patient's unusual immunological phenotype compared to classical interleukin (IL)-7Rα deficiency. Disseminated VZV was controlled with anti-viral and immune-based therapy, and umbilical cord blood stem cell transplantation was successful. Retrospectively performed T cell receptor excision circle (TREC) analyses completed on neonatal Guthrie cards identified absent TREC. This case emphasizes the danger of live viral vaccination in severe combined immunodeficiency (SCID) patients and the importance of newborn screening to identify patients prior to high-risk exposures. It also illustrates the value of aggressive pathogen identification and treatment, the influence newborn screening can have on morbidity and mortality and the significant impact of newer genomic diagnostic tools in identifying the underlying genetic aetiology for SCID patients.

Burden of copy number variation in common variable immunodeficiency.

Common variable immunodeficiency (CVID) has been associated recently with a dramatic increase in total copy number variation burden, the cause of which is unclear. In order to explore further the origin and clinical relevance of this finding, we quantified the total genomic copy number variation (CNV) burden in affected patients and evaluated clinical details in relationship to total CNV burden. No correlation was found between total CNV burden and either patient age or time elapsed since symptom onset, and higher total burden did not correlate with incidence of malignancy or other subphenotypes. These findings suggest that the increased CNV burden is static and intrinsic to CVID as a disease.

Evaluation of correlation between dose and clinical outcomes in subcutaneous immunoglobulin replacement therapy.

The importance of serum immunoglobulin (Ig)G concentration in IgG replacement therapy for primary immunodeficiency diseases is established in certain settings. Generally, IgG is infused via the intravenous (IVIG) or subcutaneous (SCIG) route. For IVIG infusion, published data demonstrate that higher IgG doses and trough levels provide patients with improved protection from infection. The same conclusions are not yet accepted for SCIG; data from two recent Phase III studies and a recent post-hoc analysis, however, suggest the same correlation between higher SCIG dose and serum IgG concentration and decreased incidence of infection seen with IVIG. Other measures of clinical efficacy have not been considered similarly. Thus, combined analyses of these and other published SCIG studies were performed; a full comparison of the 13 studies was, however, limited by non-standardized definitions and reporting. Despite these limitations, our analyses indicate that certain clinical outcomes improve at higher SCIG doses and associated higher serum IgG concentrations, and suggest that there might be opportunity to improve patient outcomes via SCIG dose adjustment.

Comparison of American and European practices in the management of patients with primary immunodeficiencies.

Primary immunodeficiency diseases (PIDs) comprise a heterogeneous group of rare disorders. This study was devised in order to compare management of these diseases in the northern hemisphere, given the variability of practice among clinicians in North America. The members of two international societies for clinical immunologists were asked about their management protocols in relation to their PID practice. An anonymous internet questionnaire, used previously for a survey of the American Academy of Allergy, Asthma and Immunology (AAAAI), was offered to all full members of the European Society for Immunodeficiency (ESID). The replies were analysed in three groups, according to the proportion of PID patients in the practice of each respondent; this resulted in two groups from North America and one from Europe. The 123 responses from ESID members (23·7%) were, in the majority, very similar to those of AAAAI respondents, with > 10% of their practice devoted to primary immunodeficiency. There were major differences between the responses of these two groups and those of the general AAAAI respondents whose clinical practice was composed of < 10% of PID patients. These differences included the routine use of intravenous immunoglobulin therapy (IVIg) for particular types of PIDs, initial levels of IVIg doses, dosing intervals, routine use of prophylactic antibiotics, perceptions of the usefulness of subcutaneous immunoglobulin therapy (SCIg) and of the risk to patients' health of policies adopted by health-care funders. Differences in practice were identified and are discussed in terms of methods of health-care provision, which suggest future studies for ensuring continuation of appropriate levels of immunoglobulin replacement therapies.

Antibody targeting of anaplastic lymphoma kinase induces cytotoxicity of human neuroblastoma.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies-as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.

Challenges in the use of allogeneic hematopoietic SCT for ectodermal dysplasia with immune deficiency.

Genetic mutations of proteins regulating nuclear factor of kappa-light polypeptide gene enhancer in B lymphocyte (NF-kappaB) activation result in heritable diseases of development and immunity. Hypomorphic, X-linked mutations in the IKBKG gene (NF-kappaB essential modulator (NEMO) protein), and hypermorphic, autosomal dominant mutations in the IKBA gene (inhibitor of NF-kappaB (IkappaB)-alpha protein), are associated with a phenotype of immune deficiency and often ectodermal dysplasia (ED-ID). ED-ID predisposes patients to recurrent and life-threatening infections and is typically fatal within the first few years of life. Allogeneic hematopoietic SCT (HSCT) may correct the immune deficiency associated with NEMO or IkappaBalpha mutations, but there is very little published data. We gathered clinical data on three ED-ID patients that had undergone HSCT. Conditioning regimens were variable, as were the stem cell sources. All three patients experienced engraftment difficulties as well as post transplant complications. These cases suggest that patients with immune deficiencies caused by NEMO or IkappaBalpha mutations may have intrinsic barriers to successful engraftment, which require further investigation.

Cell penetrating peptide inhibitors of nuclear factor-kappa B.

The nuclear factor kappa B (NF-kappaB) transcription factors are activated by a range of stimuli including pro-inflammatory cytokines. Active NF-kappaB regulates the expression of genes involved in inflammation and cell survival and aberrant NF-kappaB activity plays pathological roles in certain types of cancer and diseases characterized by chronic inflammation. NF-kappaB signaling is an attractive target for the development of novel anti-inflammatory or anti-cancer drugs and we discuss here how the method of peptide transduction has been used to specifically target NF-kappaB. Peptide transduction relies on the ability of certain small cell-penetrating peptides (CPPs) to enter cells, and a panel of CPP-linked inhibitors (CPP-Is) has been developed to directly inhibit NF-kappaB signaling. Remarkably, several of these NF-kappaB-targeting CPP-Is are effective in vivo and therefore offer exciting potential in the clinical setting.

Monosomy 1p36 uncovers a role for OX40 in survival of activated CD4+ T cells.

Monosomy 1p36 is a subtelomeric deletion syndrome associated with congenital anomalies presumably due to haploinsufficiency of multiple genes. Although immunodeficiency has not been reported, genes encoding costimulatory molecules of the TNF receptor superfamily (TNFRSF) are within 1p36 and may be affected. In one patient with monosomy 1p36, comparative genome hybridization and fluorescence in- situ hybridization confirmed that TNFRSF member OX40 was included within the subtelomeric deletion. T cells from this patient had decreased OX40 expression after stimulation. Specific, ex vivo T cell activation through OX40 revealed enhanced proliferation, and reduced viability of patient CD4+ T cells, providing evidence for the association of monosomy 1p36 with reduced OX40 expression, and decreased OX40-induced T cell survival. These results support a role for OX40 in human immunity, and calls attention to the potential for haploinsufficiency deletions of TNFRSF costimulatory molecules in monosomy 1p36.

The Wiskott-Aldrich syndrome.

The Wiskott-Aldrich Syndrome (WAS) is an inherited immunodeficiency caused by a variety of mutations in the gene encoding the WAS protein (WASp). WASp is expressed in hematopoetic cells and facilitates the reorganization of the actin cytoskeleton in response to many important cell stimuli. Extensive study of WAS and more recently WASp has given great insight into the relevance of this molecule and related molecules to both basic cell biology and human immune defenses.

Decreased natural killer (NK) cell function in chronic NK cell lymphocytosis associated with decreased surface expression of CD11b.

Chronic natural killer cell lymphocytosis (CNKL) is characterized by greatly increased numbers of natural killer (NK) cells and patients with this disease may survive for long periods. This is in contrast to patients with leukemic proliferations of NK cells who can have a rapidly progressive clinical course. We identified a pediatric patient who was largely healthy who had CNKL and we sought to determine if the expanded CD16(+)CD3(-) population in this patient functions differently than classical NK cells. Cytotoxic activity against NK cell-sensitive K562 target cells was present, but lower than that in control donors when calculated as lytic units per CD16(+)CD3(-) cell. This cytolytic activity was inducible in patient samples by IL-2/IL-12 stimulation proportionately to that induced in samples from control donors. Intracellular perforin was also present and induced in patient CD16(+)CD3(-) cells similarly to controls. Other presumed NK cell activities, such as IL-2/IL-12 induced IFN-gamma expression and initiation of apoptosis evidenced by annexin V binding after CD16 crosslinking were present in patient samples. Patient CD16(+)CD3(-) cells, however, differed from classical NK cells, as the majority did not express CD56, CD57, CD8, or CD11b. Most convincingly, there was a 5 log decrease in CD11b expression in patient CD16(+)CD3(-) cells compared to control as determined by mean channel fluorescence. These observed differences may explain the relatively benign phenotype of this disorder.

Early murine cytomegalovirus (MCMV) infection induces liver natural killer (NK) cell inflammation and protection through macrophage inflammatory protein 1alpha (MIP-1alpha)-dependent pathways.

Natural killer (NK) cells mediate defense against early murine cytomegalovirus (MCMV) infections in liver. The chemokine, macrophage inflammatory protein 1alpha (MIP-1alpha), can promote inflammatory responses. Our studies evaluated contributions of NK cells to early MCMV-induced liver inflammation and MIP-1alpha requirements for inflammation and delivery of antiviral defenses. NK cells were shown to be responsible for focal inflammation, and to be induced to migrate at high levels, in MCMV-infected livers. MIP-1alpha gene expression was elevated at coinciding times, and mice deficient in MIP-1alpha function were dramatically inhibited in both inflammatory and protective liver responses. The results precisely define MIP-1alpha-dependent steps required to achieve NK cell inflammation during, and mechanisms promoting defense against, viral infections in tissues.

Mechanisms for virus-induced liver disease: tumor necrosis factor-mediated pathology independent of natural killer and T cells during murine cytomegalovirus infection.

The contribution of endogenous NK cells and cytokines to virus-induced liver pathology was evaluated during murine cytomegalovirus infections of mice. In immunocompetent C57BL/6 mice, the virus induced a self-limited liver disease characterized by hepatitis, with focal inflammation, and large grossly visible subcapsular necrotic foci. The inflammatory foci were most numerous and contained the greatest number of cells 3 days after infection; they colocalized with areas of viral antigen expression. The largest number of necrotic foci was found 2 days after infection. Overall hepatic damage, assessed as increased expression of liver enzymes in serum, accompanied the development of inflammatory and necrotic foci. Experiments with neutralizing antibodies demonstrated that although virus-induced tumor necrosis factor (TNF) can have antiviral effects, it also mediated significant liver pathology. TNF was required for development of hepatic necrotic foci and increased levels of liver enzymes in serum but not for increased numbers of inflammatory foci. The necrotic foci and liver enzyme indications of pathology occurred independently of NK and T cells, because mice rendered NK-cell deficient by treatment with antibodies, T- and B-cell-deficient Rag-/- mice, and NK- and T-cell-deficient E26 mice all manifested both parameters of disease. Development of necrotic foci and maximally increased levels of liver enzymes in serum also were TNF dependent in NK-cell-deficient mice. Moreover, in the immunodeficient E26 mice, virus-induced liver disease was progressive, with eventual death of the host, and neutralization of TNF significantly increased longevity. These results establish conditions separating hepatitis from significant liver damage and demonstrate a cytokine-mediated component to viral pathogenesis.

Interferon-alpha/beta inhibition of interleukin 12 and interferon-gamma production in vitro and endogenously during viral infection.

Interferon (IFN)-alpha/beta-mediated negative regulation of interleukin 12 (IL-12) and IFN-gamma proteins is reported here. Both IFN-alpha and IFN-beta inhibited fixed Staphylococcus aureus Cowan strain induction of IL-12 and IFN-gamma production by mouse splenic leukocytes in culture. Extended studies with IFN-alpha demonstrated that inhibition was at the level of biologically active IL-12 p70. Effects were selective, as induction of tumor necrosis factor was unaffected and induction of IL-6 was enhanced. Neutralization of IFN-alpha/beta expressed endogenously during infections with murine cytomegalovirus (MCMV) enhanced early IL-12 and IFN-gamma protein production. Furthermore, during infections of mice with lymphocytic choriomeningitis virus (LCMV), this treatment revealed a previously undetected early IL-12 and IFN-gamma protein expression, and mice deficient in IFN-alpha/beta receptor function, but not control mice, also expressed endogenous LCMV-induced IL-12. The effects of IFN-alpha/beta neutralization on production of IL-12 and IFN-gamma during the viral infections were detected in both serum samples and medium conditioned with splenic leukocytes isolated from infected animals. In vitro studies demonstrated that splenic leukocytes isolated from LCMV-infected mice were primed to produce IL-12 in response to stimulation with Staphylococcus aureus Cowan strain, but that this responsiveness was sensitive to added IFN-alpha. Moreover, endogenous IFN-alpha/beta induced by LCMV inhibited in vivo lipopolysaccharide stimulation of IL-12 production. These results demonstrate a new pathway for regulating cytokine responses, and suggest a mechanism for inhibition of IL-12-dependent immune responses during viral infections.