Clinical consequences of defects in the IL-12-dependent interferon-gamma (IFN-γ) pathway

Following infection with intracellular pathogens like mycobacteria, listeria, toxoplasma and leishmania, mononuclear phagocytes and related antigen-presenting cells (APC), i.e. dendritic cells, secrete the heterodimeric cytokine IL-12. IL-12 comprises two disulphide-linked subunits, p40 and p35, which together form the biologically active p70 hetrodimer molecule (reviewed in [1,2]). IL-12 production by macrophages and dendritic cells can also be enhanced by a T cell-dependent pathway through interaction of CD40 on the surface of the APC with CD40-ligand expressed on activated T cells. The IL-12 receptor (IL-12R), which is expressed by both natural killer (NK) cells at certain stages of development and by activated T cells, is made up of two chains called IL-12Rβ1 and IL-12Rβ2, respectively. Both receptor chains have extracellular, transmembrane and intracellular segments. Each of these receptor proteins can only bind to IL-12 with low affinity, but when co-expressed can bind IL-12 with high affinity, initiating a physiological response to this cytokine [3]. A schematic representation of the IL-12 receptor-mediated intracellular signalling pathway is illustrated in Fig. 1. Binding of IL-12 to the IL-12Rβ1 and β2 chains induces phosphorylation of the kinases Tyk2 and Jak2, which associate with the cytoplasmic tails of the β1 and β2 chains, respectively. Subsequently the signal transducing protein STAT4 binds to the cytoplasmic tail of the β2 receptor and in turn becomes phosphorylated. It then dissociates from the receptor, dimerizes and translocates to the nucleus, where it acts as a transcriptional activator by binding to specific DNA response elements (RE) in the promoter regions of IL-12 inducible genes [27,31]. Binding of IL-12 to activated CD4 T cells partitions them to develop and differentiate along the so-called Th1 pathway, crucially important for cell-mediated immunity against intracellular pathogens. Furthermore, acting at picomolar and subpicomolar levels on T cells and NK cells, IL-12 induces high-level production of the cytokine IFN-γ[2]. IFN-γ plays a central role in the resistance of mammalian hosts to pathogens, particularly bacteria and parasites capable of intramacrophage survival (reviewed in [4,5]). The main cells producing IFN-γ are activated Th1 cells, activated CD8 cytotoxic cells of the TC1 phenotype, and activated NK cells. Biologically active IFN-γ is a homodimer, which has a range of pleiotropic effects on a number of cell types, with the ability to modulate the function of over 200 genes. It is one of the principal macrophage-activating cytokines, and mice with disrupted IFN-γ or IFN-γ receptor (IFN-γR) genes show increased susceptibility to intracellular pathogens including Leishmania major, Listeria monocytogenes, mycobacteria and certain viruses, e.g. vaccinia virus. IFN-γ interacts with a specific cell surface receptor, which is widely expressed on most nucleated cells. The IFN-γR consists of two transmembrane proteins, namely IFN-γR1 which is a ligand-binding chain, and IFN-γR2, which is required for signal transduction. A schematic representation of the IFN-γ receptor-mediated intracellular signalling pathway is shown in Fig. 2. A schematic representation of the IFN-γ receptor-mediated intracellular signalling pathways (modified from [4,5]). Two IFN-γR1 chains dimerize on binding the IFN-γ homodimer and subsequently associate with two IFN-γR2 chains. Receptor subunit assembly leads to the trans-phosphorylation and activation of Jak1 and Jak2 kinases, which associate with the IFN-γR1 and IFN-γR2 chains, respectively. These phosphorylate a tyrosine residue on the intracytoplasmic domain of IFN-γR1, forming a paired set of docking sites for STAT1. Two STAT1 molecules then bind to these docking sites and subsequently become phosphorylated. Tyrosine phosphorylated STAT1 molecules then lose their affinity for IFN-γR1 chain docking sites, dissociate from the receptor and form homodimeric complexes. Phosphorylated STAT1 homodimers translocate to the nucleus and bind to specific sequences in the promoter region of early IFN-γ inducible genes, switching on gene transcription. Reprinted, with permission, from the Annual Review of Immunology, Vol. 15, © 1997 by Annual Reviews www.annualReviews.org Key actions of IFN-γ include increased expression of MHC class I and class II proteins which enhance antigen processing and presentation, activation of mononuclear phagocytes through a multiplicity of effects, influencing IgG heavy-chain switching and modulating the production of cytokines such as IL-12, tumour necrosis factor-alpha (TNF-α) and IFN-γ itself. Following IFN-γR ligation the receptor–ligand complexes recycle into an acidified subcellular compartment, where they dissociate. Free IFN-γ is then degraded by lysosomal enzymes. The uncoupled IFN-γR1 receptors eventually relocate to the cell surface via an intracellular pool. An amino acid motif present on the intracellular domain of the IFN-γR1 close to the Jak1 association site is required for normal recycling of this receptor (Fig. 2). The above background information helps in the understanding of the clinical, pathological and immunological features of defects in the IL-12-dependent, high-output IFN-γ pathway and the laboratory methods required for identifying these defects. The initial breakthrough arose from studies on a Maltese kindred who developed infection with poorly pathogenic non-tuberculous mycobacteria (NTM) [6]. Despite anti-mycobacterial chemotherapy three of the four affected patients died and the survivor had persistent infection. Peripheral blood mononuclear cells (PBMC) from the affected individuals failed to show up-regulation of endotoxin-induced TNF-α production on the addition of recombinant IFN-γ. Subsequently, a genome-wide search using microsatellite markers identified a region on 6q, where the affected children were homozygous for eight markers [7]. Investigations concentrating on the IFN-γR1 gene, which maps to 6q 23:24, revealed that the affected individuals had a point mutation at nucleotide 395 of this gene. This introduced a premature stop codon in the coding region for the extracellular domain of this receptor, preventing its expression, with complete abrogation of responses to IFN-γ[7]. Parallel work on idiopathic bacille Calmette–Guérin (BCG) infections in the absence of recognized immunodeficiency states [8] led to the simultaneous identification of a homozygous recessive mutation of the same gene as the cause of fatal disseminated BCG infection during infancy [9]. Subsequent investigation of patients with increased susceptibility to poorly pathogenic mycobacterial infections in the absence of known causes of primary and secondary immunodeficiency has led to the identification of three categories of gene defects. These comprise mutations in the IL12p40 subunit, IL-12Rβ1 as well as IFN-γR chains 1 and 2 [10]. Complete IFN-γR1 deficiency is caused by mutations resulting in the generation of premature stop codons in the DNA coding the extracellular domain of this protein. This prevents surface expression of the receptor, complete absence of binding of IFN-γ to the cell surface and consequent abrogation of the normal physiological responses to this cytokine [9,11–13]. These mutations have been seen in unrelated families from different ethnic backgrounds, each kindred having a unique mutation. A recessive mutation generating a premature stop codon in the extracellular domain of the IFN-γR2 chain resulted in the absence of STAT1 phosphorylation in response to IFN-γ, despite the normal expression of IFN-γR1 chains on the cells of the affected individual [14]. One patient has been described with complete absence of IFN-γR function because of compound heterozygous mutations in the IFN-γR1 chain [13]. A second type of complete IFN-γ receptor deficiency has recently been described which manifests itself not as a lack of receptor expression but by the failure of IFN-γ to bind to its receptor [15]. Partial IFN-γR1 deficiency resulting from a homozygous recessive missense mutation causing an isoleucine to threonine substitution at position 87 in the extracellular domain of the receptor (designated as I87T) has been described [16]. This probably reduced but did not abrogate binding of IFN-γ to the receptor. Cells from these patients showed signalling responses to high, but not low, in vitro concentrations of IFN-γ. A similar response phenotype has also been recently reported in a patient with partial IFN-γR2 deficiency [17]. This recessive mutation resulted from a nucleotide substitution in the IFN-γR2 gene causing an amino acid substitution in the extracellular region of the encoded receptor. The commonest individual category of IFN-γR1 mutations comprise the heterozygous autosomal dominant mutation seen in nine individuals from three unrelated families and nine sporadic cases [18]. The mutation was either a one base-pair deletion (one kindred) or a four base-pair deletion (all others) at nucleotide 818 of the IFN-γR1. Thus there were 12 independent mutation events at a single mutation site indicating that this is a small deletion hot-spot in the human genome. The mutated allele is translated into stable mRNA, which encodes a truncated form of IFN-γR1 (illustrated in Fig. 3), that lacks the intracytoplasmic domain and thus has no Jak1 binding, recycling and STAT1 docking motifs. Due to these collective actions truncated receptors exert a dominant negative effect. The receptor deficiency however, is partial as weak signalling is detectable in vitro using supra-physiological levels of IFN-γ, and surprisingly clinical responses were obtained with IFN-γ augmentation of anti-mycobacterial chemotherapy (see below). The excessive accumulation of the abnormal receptors on the cell surface result in a 0·5–1 log increase in surface staining with MoAbs directed against IFN-γR1, observable by flow cytometry, which is diagnostically useful in screening for this defect. A schematic representation of the IFN-γ receptor-mediated intracellular signalling pathways and its defective function within patients with heterozygous dominant negative IFN-γR1 deficiency. The truncated R1 chain paired with the wild-type R1 chain is able to bind the IFN-γ homodimer but is incapable of inducing signalling. Furthermore, unlike receptors comprising paired wild-type IFN-γR1 chains, these abnormal receptor complexes do not recycle following ligand binding, resulting in their accumulation at the cell surface. A child belonging to consanguineous Pakistani parents who presented with recurrent disseminated BCG infection (following neonatal immunization) and Salmonella enteritis septicaemia was found to have a 4·4-kb deletion spanning two coding exons of the IL-12p40 subunit [19]. The parents, the maternal grandmother and a healthy sibling were heterozygous for this defect. Monocytes and dendritic cells were unable to secrete IL-12 p40, or the biologically active IL-12 p70 cytokine, when appropriately activated. As a result, antigen-driven IFN-γ production by blood lymphocytes was markedly impaired, but could be reconstituted by the addition of exogenous recombinant IL-12. The child also showed a clinical response to IFN-γ complementation of anti-microbial chemotherapy, resulting in a complete cure of the BCG and salmonella infections, which has been sustained for 4 years after cessation of therapy. In two additional reports of impaired IL-12 production, defects in the structural genes of IL-12 p40 and p35 have not been identified. The first report described a kindred with recurrent disseminated Mycobacterium avium infection, where the patient's cells had reduced, although not completely ablated, IL-12 p70 production [20]. The patient's lymphocytes had reduced in vitro phytohaemagglutinin (PHA)-driven IFN-γ production which could be enhanced by exogenous recombinant IL-12. The affected members of this kindred were all male and appeared to show abnormal regulation of IL-12 production, while the females showed an intermediate phenotype, implying putative X-linked inheritance of this defect. The second report documents a 3-year-old female with IL-12 deficiency associated with recurrent episodes of severe recurrent pneumococcal sepsis [21]. In this latter patient antibody production, including specific antibody levels to protein and polysaccharide antigens, was normal. The patient's mononuclear cells were however incapable of producing detectable IL-12 p70 or p40 when appropriately stimulated (with Staphylococcus aureus Cowan strain-1 (SAC)) even after supplementation with exogenous recombinant IFN-γ. Seven patients from six unrelated kindreds, affected by idiopathic severe disseminated mycobacterial and salmonella infection, were found to have homozygous recessive mutations of IL-12Rβ1[22,23]. The mutations were unique to each kindred and affected the extracellular domain of the IL-12Rβ1 receptor. These null mutations prevented cell surface expression of the receptor on activated T cells, as detected by flow cytometry with a specific MoAb. These patients showed defective in vitro antigen-driven IFN-γ production that could not be augmented by exogenous recombinant IL-12. In one of these patients potentially fatal disseminated M. avium infection was cured by the augmentation of anti-mycobacterial chemotherapy with IFN-γ, suggesting that, as in the IL-12-deficient child, impaired IFN-γ secretion was probably responsible for the disease susceptibility of these patients. All individuals with such defects so far reported presented with persistent salmonella and/or mycobacterial infections, with two exceptions. In one instance two young asymptomatic children have been identified with partial IFN-γR1 deficiency who have not been vaccinated with BCG [18]. In the second instance, a young adult sibling of an IL-12Rβ1-deficient patient was identified with the same genetic and functional defect. She had curable salmonella septicaemia as an infant but has not developed mycobacterial infection. She also had no reported problems following BCG vaccination in early infancy [23]. Fourteen patients with complete IFN-γR deficiency have developed mycobacterial infection in early childhood or infancy with a uniformly poor prognosis [24]. The infections were caused either by BCG following vaccination or by chance infection with poorly pathogenic environmental NTM. Mycobacterium avium-intracellulare is the commonest of the latter opportunistic pathogens, while there have been a smaller number of reports of infection caused by other atypical mycobacteria including M. chelonei, M. fortuitum, M. smegmatis, M. abscessus and M. kansasii[7,9,12,13]. All patients with complete IFN-γR deficiency vaccinated with BCG have developed severe disseminated disease. In patients with complete IFN-γR mycobacterial are of i.e. are poorly and comprise of macrophages which are In the of these infections have been despite anti-microbial chemotherapy, even when with IFN-γ therapy. In patients with partial IFN-γR BCG infection is associated with and the infection is curable with anti-mycobacterial chemotherapy Two patients with partial dominant IFN-γR deficiency and two with IL-12Rβ1 deficiency have BCG vaccination with no effects In the IL-12 child, the disseminated BCG infection, although was curable with IFN-γ supplementation of chemotherapy [19]. In IL-12Rβ1 two patients who developed disseminated with were cured with anti-mycobacterial chemotherapy [23]. three patients with disseminated M. avium infections were also cured with anti-mycobacterial chemotherapy In kindred with IL-12Rβ1 one died of disseminated M. avium infection despite chemotherapy, while who developed disseminated M. avium infection at the of failed to to anti-mycobacterial chemotherapy but was cured by the addition of In patients with the dominant partial IFN-γR1 mutation of with poorly pathogenic mycobacterial infections with anti-mycobacterial most cases required of with disease Two females from one kindred who with M. avium and did not to chemotherapy and were cured only by the addition of IFN-γ therapy. In a kindred one patient died of disseminated M. avium infection, despite years of chemotherapy with a range of anti-mycobacterial had disseminated and recurrent M. avium infection, which was cured by of anti-mycobacterial Both these patients had disease with poor the of the second patient had M. avium infection, with and was cured with 2 years of This that even in related individuals with the same and probably the same of mycobacterial there is in and to other immunological The of two with dominant negative IFN-γR1 mutation described in the above had recurrent disseminated M. infection and despite chemotherapy, at the of it is that on these mutations to no from was for genetic a of the patients with defects of the cytokine pathway also developed salmonella the to salmonella or or salmonella In two other patients have had disease to or These infections have been curable with anti-microbial chemotherapy, although have been the of the child with complete IL-12 p40 who was heterozygous for this had persistent infection caused by the of 2 and 4 years which was only cured with [19]. has subsequently infections in patients include to and persistent In these patients have not in developed infections caused by extracellular bacteria or causing infections, infections or of these patients have had infections caused by viruses, including and a report has the of severe infections in four patients with IFN-γR deficiency from mycobacterial disease The pathogens and or with and and type when for these patients the of severe infections be the of cytokine pathway to be in cases of severe infections with such Two of the clinical in these patients two patients with partial dominant negative IFN-γR1 deficiency with were as [18]. in one patient cells, but had atypical pathological initial no mycobacteria were detected by or by The patients did not to and chemotherapy for of and were subsequently found to have infection caused by BCG and M. respectively. Both patients failed to to anti-mycobacterial chemotherapy, but were cured when their was augmented with IFN-γ in infections can be to in patients with cytokine in two patients who had complete IFN-γR1 deficiency and were with M. and M. fortuitum, initial showed with no [6]. In for mycobacteria were infection only when the patients were with on the that they were from an disease. In patient with IFN-γR1 M. infection was with after and in children with persistent and markers with or defects of the cytokine pathway be in the with for poorly pathogenic mycobacterial signalling by the Th1 cytokine or its is in cytokine these of a unique to the functional of the in It is that immunity is by Th1 cytokines, such as IFN-γ, and that cytokines and are responsible for An increase of with clinical and of have been within patients with or clinical on patients with cytokine pathway revealed that of the patients showed of severe such as or and only such as levels were also within the normal range in patients in the clinical phenotype that normal of responses can in the absence of a functional Th1 These have important for understanding the of disease and for the development of to for patients with severe of patients from poorly pathogenic mycobacterial infections for receptor pathway IFN-γ IFN-γ phytohaemagglutinin for for avium for Mycobacterium avium-intracellulare for bacille Calmette–Guérin (BCG) to of IL-12 Staphylococcus aureus Cowan of patients from poorly pathogenic mycobacterial infections for receptor pathway IFN-γ IFN-γ phytohaemagglutinin for for avium for Mycobacterium avium-intracellulare for bacille Calmette–Guérin (BCG) to of IL-12 Staphylococcus aureus Cowan These defects be and are most to be found with disseminated or recurrent infection to poorly pathogenic mycobacteria or environmental with infections caused by salmonella salmonella infections in these patients are persistent and recurrent despite therapy. In the following categories of patients defects in the cytokine be in the but are to be with disease caused by and with M. infection who are have but develop either recurrent or disseminated disease. with severe infection, to (see with persistent and and despite the absence of detectable mycobacterial infection (see with to or with atypical pathological on the in the of but by information in mice with defects of the cytokine pathway, this be in patients with other infections of impaired cell-mediated i.e. severe A of the cytokine pathway is the of its on each other to This is most by who showed that patients with complete IFN-γR defects have reduced in vitro production of IFN-γ, IL-12, investigation of this pathway of all its to up a that of a defect. on information from patients to the following be to cytokine pathway in The of IFN-γ production is required following in vitro with as well as antigens, e.g. Mycobacterium avium-intracellulare all the cytokine patients reduced in vitro IFN-γ production It is then important to this in IFN-γ production can be by supplementation with exogenous recombinant IL-12 IL-12 production be using both mononuclear cell as well as stimulated with aureus IL-12 p70 and p40 can be using TNF-α from and also be using with or IFN-γ supplementation the of impaired regulation of IL-12 production, IL-12 responses of patient be using aureus or with exogenous with defective regulation of IL-12 production to aureus with IFN-γ but not with aureus [20]. of IFN-γR1 can be by flow cytometry using MoAbs or expression the of a mutation in the extracellular domain of the IFN-γR1 chain, while enhanced expression heterozygous IFN-γR1 mutation. IFN-γR2 expression can be by flow cytometry although the is not IFN-γR despite normal surface expression, it is important to functional responses to exogenous IFN-γ by of the (see or by up-regulation of MHC class I or CD40 on or MHC class II molecules on cell from from patients of signal by following specific cytokine cell are proteins by and with to or with an antibody capable of phosphorylated signalling is phosphorylated STAT1 be detected within the These methods are described in the response required for of phosphorylated STAT1 by IFN-γ, it is to complete and partial of receptor flow cytometry be to IFN-γR function by of intracellular phosphorylated STAT1 within cells using a specific of IL-12Rβ1 and expression is by flow cytometry following staining of T cell with a against IL-12Rβ1 or an is for expression the is The of signalling pathways can be using for the of the of phosphorylated STAT4 to a of patients with idiopathic disseminated poorly pathogenic infection have been identified to have mutations of the cytokine described identified to in the cytokine pathway are in the in (see below). the has only been in identifying a gene in the Maltese described by [7]. 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