Interferon-γ and interleukin-12 pathway defects and human disease

Abstract A genetic component to human mycobacterial disease susceptibility has long been postulated. Over the past five years, mutations in the interferon-γ (IFNγ) receptor, IL-12 receptor β1 (IL-12Rβ1), and IL-12 p40 genes have been recognized. These mutations are associated with heightened susceptibility to disease caused by intracellular pathogens including nontuberculous mycobacteria, vaccine-associated bacille Calmette Guerin (BCG), Salmonella species, and some viruses. We describe the genotype-phenotype correlations in IFNγ receptor, IL-12Rβ1, and IL-12 p40 deficiency, and discuss how study of these diseases has enhanced knowledge of human host defense against mycobacteria and other intracellular pathogens.


Introduction
Infections with intracellular bacteria such as mycobacteria remain an important cause of human morbidity and mortality worldwide. Immunologic protection against such organisms depends on cell mediated immunity, the major eector of which is the IFNgactivated macrophage. The importance of IFNg pathways in host defense against intracellular bacteria was initially made clear through the experimental study of knockout mice. More recently, the identi®cation and characterization of humans with mutations in IFNg receptor proteins, IL-12 receptor b1, or IL-12 p40 has con®rmed the importance of these pathways in human host defense.

IFNg and the IFNg receptor
IFNg was ®rst identi®ed on the basis of its in vitro antiviral activity [1]. It is produced predominantly by T cells and NK cells in response to a variety of in¯ammatory or immune stimuli, and in general, it stimulates the development and function of immune eector cells. IL-12 and IL-18 secreted by macrophages and dendritic cells are thought to be the primary inducers of IFNg production in an in¯ammatory reaction [2±5] (Fig. 1).
IFNg receptors are expressed on almost all nucleated cells, and show species speci®city in their ability to bind IFNg [6]. The functional IFNg receptor is composed of two 90 kDa IFNgR1 (formerly a or ligand-binding chain, or CD119w) proteins and two 62 kDa IFNgR2 (formerly b or signal transducing chain, or accessory factor-1) proteins [7]. The human IFNGR1 gene contains seven exons, and is located on chromosome 6 [8]. The extracellular portion of IFNgR1 contains the IFNg ligand-binding domain; the intracellular portion contains domains necessary for signal transduction and receptor recycling [6,7] (Fig. 2). The IFNGR2 gene also contains seven exons, and is located on human chromosome 21 [9,10]. The extracellular domain of IFNgR2 interacts with the IFNgR1/IFNg complex, but does not itself play a major role in ligand binding [7]. The intracellular IFNgR2 domain is necessary for signal transduction [11] (Fig. 2). In the absence of stimulation, IFNgR1 and IFNgR2 are not strongly associated with each other. However, inactive Janus kinase 1 (JAK1) is bound to the four amino acid sequence ( 266 LPKS 269 ) in the membrane proximal IFNgR1 intracellular domain [7], and inactive JAK2 is bound to a proline- Fig. 1. IFNg and IL-12 production and response pathways. In response to infection with mycobacteria, macrophages produce cytokines including IL-12. IL-12 and IL-18 synergistically stimulate production of IFNg by CD4 + T cells and NK cells. IFNg ligand binds to its receptor on the macrophage cell surface and activates the macrophage resulting in enhanced TNFa production, production of cytokines and chemokines, upregulation of MHC class II expression, enhanced antigen processing, and production of reactive oxygen and (in mice) nitrogen intermediates. rich sequence ( 263 PPSIPLQIEEYL 274 ) in the IFNgR2 intracellular domain [12].
IFNg binds as a homodimer to two IFNgR1 proteins, thereby facilitating the binding of two IFNgR2 proteins to the IFNgR1/IFNg complex [12±16] (Fig. 3). Within this complex the IFNgR1 and IFNgR2 intracellular domains, with their constitutively associated JAKs, are brought into proximity. Ligand binding results in reciprocal transphosphorylation of the JAKs, and subsequent phosphorylation of IFNgR1 Y 440 [7,17]. Through its SH2 domain, one latent STAT1 recognizes and binds to each tyrosine phosphorylated IFNgR1 ( 440 YDKPH 444 ) site [18]. Receptor associated STAT1 proteins are subsequently tyrosine phosphorylated and, so activated, form homodimers that translocate to the nucleus where they bind to IFNg activation sequences (GAS) of IFNg-inducible genes [19±23]. The intracellular IFNgR1 motif 270 LI 271 is important for directing receptor tracking through the cell, including recycling of the receptor o of the cell surface after ligand binding [6,24].
Three observations indicate a biological role for intracellular IFNg. First, IFNg delivered by liposomes has been shown to activate murine macrophages to a tumoricidal state [25]. Second, microinjected IFNg can induce Ia expression on murine macrophages [26]. Finally, secretion-defective human IFNg expressed in murine ®broblasts induces an antiviral state in those cells [27]. Recently it was demonstrated that a polybasic nuclear localization sequence in the carboxyl termi-nus of IFNg is required for nuclear translocation and biological activity of this cytokine [28]. Both human and murine IFNg have been shown to interact with the cytoplasmic domain of their species-matched IFNgR1. A plausible role for IFNg is that of an intracellular chaperone that facilitates nuclear translocation of STAT1 which itself lacks a nuclear translocation sequence [28,29].
IFNg activates transcription of a large number of genes that play roles in antiviral activity, apoptosis, antigen processing, MHC protein expression, and type 1 T helper cell (TH1) development. IFNg also activates macrophages to kill or restrict growth of microbial targets; this function appears to be important in host defense against mycobacteria. In mice, IFNg-induced generation of reactive nitrogen intermediates is one mechanism of Mycoplasma tuberculosis killing [30,31].  However, reactive nitrogen intermediates have not been shown conclusively to play a major role in mycobacterial killing by human cells. Despite our understanding of IFNg signal transduction, the pathways by which this cytokine activates mycobactericidal macrophage activities in humans are poorly understood.

IL-12 and the IL-12 receptor
IL-12 is a heterodimeric cytokine produced primarily by antigen presenting cells. It enhances proliferation and cytolytic activity of natural killer (NK) and T cells, and stimulates their IFNg production [32]. IL-12 plays a key role in promoting TH1 responses and subsequent cell mediated immunity [33±35]. Production of IL-12 stimulated by microbial lipoproteins, including a 19-kD M. tuberculosis lipoprotein, is mediated by Tolllike receptors [36]. IL-12 is composed of two disul®delinked subunits, p35 and p40, which are encoded by unrelated genes on human chromosomes 3 and 5, respectively [37±39]. Functional IL-12 receptors are expressed primarily on activated T and NK cells [40]. Two IL-12 receptor subunits, IL-12Rb1 and IL-12Rb2, have been cloned from human and mouse T cells [41±43]. Coexpression of these two subunits is required for high anity binding of IL-12 [43]. Expression of IL-12Rb2 is tightly controlled and may be an important mechanism for regulation of IL-12 responsiveness [44].

Knock-out murine models
The importance of IFNg pathways in host defense has been demonstrated in mice with targeted disruptions of the IFNg, IFNGR1, or IFNGR2 genes [45]. After experimental inoculation, IFNg and IFNGR1 knockout mice have increased susceptibility to experimental challenge with a wide spectrum of infectious agents, including mycobacteria [46±49], bacteria [50,51], parasites [52±54], and viruses [55±58]. In contrast to wild-type (WT) mice, IFNg and IFNGR1 knockout mice develop neither mature granulomas nor protective immunity after experimental infection with Mycobacterium bovis bacille Calmette-Guerin (BCG) [48,49]; IFNg knockout mice develop neither mature granulomas nor protective immunity after experimental infection with M. tuberculosis Erdman strain [46,47]. IFNg knockout mice experimentally infected with certain Mycobacterium avium strains develop higher tissue levels of bacteria than WT mice [59], and they do not develop protective immunity to some attenuated strains of Salmonella typhimurium [60,61]. Of note, neither IFNGR1 nor IFNg knockout mice develop spontaneous infection with environmental nontuberculous mycobacteria (NTM), even when housed in non-sterile facilities [62]. Mice with targeted disruptions of the IFNGR2 gene have been less well characterized, but have been shown to be highly susceptible to sublethal challenge with Listeria monocytogenes [63].
Direct comparison between IFNg and IFNGR1 knockout mice has been problematic because the mouse strains have had dierent genetic backgrounds.
Recently the responses to HSV1 or vaccinia virus challenges were compared in IFNg and IFNGR1 knockout mice derived from the same genetic background [64]. Mortality from challenge with either virus was signi®cantly greater in IFNGR1 knockout mice than IFNg knockout mice. The mechanism underlying these dierences has not been established, and it is not yet known if results of mycobacterial challenge would be dierent in IFNGR1 versus IFNg knockout mice.
Mice with disrupted genes for IL-12 p40 or IL-12Rb1 have also been described. Compared with WT mice, IL-12 p40 knockout mice are more susceptible to experimental infection with BCG [65,66], M. tuberculosis [67], and virulent M. avium [68], and they fail to form mature granulomas in response to BCG and M. tuberculosis [65±67]. It appears that IFNg or IFNGR1 knockout mice are more susceptible than IL-12 p40 or IL-12Rb1 knockout mice to experimental infection with BCG and M. tuberculosis, but no study has directly compared the mycobacterial susceptibilities of these knockout models. Overall, knockout mice are good models for studying some aspects of mycobacterial immunity and pathogenesis. However, dierences in the role of reactive nitrogen intermediates [30,31], and in manifestations of mycobacterial infection (e.g. experimental tuberculosis in wild-type mice is a chronic, ultimately fatal pulmonary disease) somewhat limit the extrapolation to humans of results obtained in knockout mice.

Human IFNg receptor de®ciencies
The existence of a genetic component to human mycobacterial disease susceptibility has long been postulated. Dierences in susceptibility to M. tuberculosis infection among dierent racial groups [69] and in twins [70], and manifestations of leprosy [71] support this hypothesis. Also in support of this idea is a tragic incident in which a single virulent viable M. tuberculosis strain was inadvertently used to immunize infants [72,73]. Responses to the vaccine ranged from death to recovery, arguing for a genetic basis for resistance to tuberculosis.
Elucidation of the critical role of IFNg receptor genes in control of nontuberculous mycobacterial (NTM) infection began with the identi®cation of kin-dreds in whom aected individuals had severe infection with poorly virulent environmental mycobacteria, in the absence of a known immunode®ciency [74±76]. Parental consanguinity in some of these kindreds suggested a Mendelian disorder of autosomal recessive inheritance [74]. Immunologic investigation of four related Maltese children who had disseminated NTM infections showed diminished TNFa production in response to stimulation with IFNg plus endotoxin in a whole blood assay [77]. A subsequent genome-wide search using microsatellite analysis identi®ed a region on chromosome 6q for which all aected children in this family were homozygous [78]. IFNGR1 was known to map to that chromosomal region, and was further investigated. Patient leukocytes lacked expression of IFNgR1 protein, and DNA sequencing of the IFNGR1 gene revealed the aected patients to be homozygous for a point mutation resulting in creation of a premature termination codon. The simultaneous report of an infant with disseminated vaccine-associated BCG infection and a dierent chain terminating mutation in IFNGR1 [79] ®rmly established the importance of IFNg responsiveness in control of both vaccine-associated BCG infection and environmentally-acquired NTM infections. Subsequently we ident-i®ed a child with disseminated M. fortuitum and M. avium complex (MAC) infections, in whom genetic analysis showed an IFNGR2 frameshift mutation which created a premature stop codon and was associated with complete absence of IFNg responsiveness [80]. IFNg receptor mutations have since been described in individuals from many parts of the world and many ethnic groups (Table 1). Missense mutations, small inframe deletions or insertions, nonsense or frameshift mutations resulting in creation of a premature stop codon, and aberrant splicing events resulting in larger deletions have been described. Phenotypeto-genotype correlations are being established as more aected individuals are identi®ed. For IFNg receptor de®ciency, the phenotype appears to depend less on which gene (IFNGR1 vs IFNGR2) is mutated, but rather on the extent to which the mutation reduces IFNg responsiveness.

Complete IFNg receptor de®ciency
Complete absence of IFNg responsiveness due to a mutation in either IFNGR1 or IFNGR2 is associated with a severe clinical phenotype. Such aected individuals characteristically have severe disseminated mycobacterial infections that may involve lungs, viscera, lymph nodes, blood, and bone marrow. Onset of ®rst environmentally acquired mycobacterial infection is usually during infancy. Infections are typically caused either by NTM species that are poorly pathogenic in immunocompetent hosts and presumably acquired from environmental exposure, or by BCG acquired by vaccination. In these children, such infections are usually fatal if untreated. Aggressive and prolonged antibiotic therapy can lead to control of infection in some patients. However, the overall prognosis for these patients is poor since antibiotic therapy apparently does not completely eradicate organisms, and there is continued susceptibility to new mycobacterial infection. Based on the current understanding of IFNg signal transduction, IFNg administration would not be expected to be of therapeutic bene®t in patients with complete absence of IFNg responsiveness in vitro. In a small number of patients, bone marrow transplantation has been eective in curing the genetic defect in hematopoeitic cells, and eliminating the phenotype of heightened mycobacterial infection susceptibility ( [85], JL Casanova, personal communication).
Histologic examination of mycobacteria-infected tissues from patients with complete IFNgR1 or complete IFNgR2 de®ciency typically shows granulomas which are poorly circumscribed, poorly dierentiated, and multibacillary (lepromatoid), implying that IFNg is required for mature granuloma formation in the setting of mycobacterial infection [79,80,93]. However, tuberculin-speci®c delayed-type hypersensitivity (DTH) responses are typically normal in M. bovis BCGinfected children with complete IFNgR1 de®ciency, implying that IFNg is not necessary for development of DTH responses in humans. In vitro, PBMC from patients with complete IFNg receptor de®ciency produce low amounts of IFNg and IL-12 in response to phytohemagglutinin (PHA), indicating that IFNg plays a role in regulation of itself and IL-12 [81]. In the two identi®ed patients with complete IFNgR2 de®ciency, the clinical features, histopathology, and results of in vitro functional studies are the same as in patients with complete IFNgR1 de®ciency [80,86].
Heterozygous parents and siblings of children with autosomal recessive complete IFNg receptor de®ciency do not appear to have increased susceptibility to mycobacterial infections, although the number of such individuals studied is small and none have been studied in tuberculosis endemic areas. We have found that PBMC from these heterozygous relatives have normal in vitro IFNg responsiveness, as measured by IFNgstimulated TNFa production [81]. Therefore, haploinsuciency does not appear to be associated with an abnormal clinical or in vitro functional phenotype. However, in vitro challenge of these cells with a biologic stimulus such as M. tuberculosis has not been performed.

Dominant negative partial IFNgR1 de®ciency
Dominant negative eects have been shown conclusively to result from one group of IFNGR1 mutations [89]. These mutations result in a premature stop codon in the proximal intracellular protein domain, and they confer partial, but not complete, loss of IFNg responsiveness. The IFNGR1 mutations with autosomal dominant eects are 818del4 and 818delT [89], and 817insA (data not shown). IFNGR1 818del4 is the most common, occurring in at least 11 unrelated kindreds. Surrounding nucleotide analysis supports a model of slipped mispairing during replication as the mechanism causing 818del4 mutations [89,94]. Mutant proteins are expressed on the cell surface and bind IFNg ligand, but cannot transduce signal due to absence of JAK1 and STAT1 binding sites (Fig. 4A). Moreover, the absence of the IFNgR1 recycling motif results in an increased number of mutant proteins expressed on the cell surface (Fig. 4B). Residual IFNg responsiveness is mediated by the normal IFNgR1 proteins expressed from the normal allele in these heterozygous individuals. PBMC TNFa production in response to IFNg plus lipopolysaccharide is approximately three-fold lower in patients with autosomal dominant IFNGR1 818del4 mutations than in normals (Dorman and Holland, unpublished data).
The clinical phenotype associated with this group of autosomal dominant (AD) mutations is milder than that seen in children with complete absence of IFNg responsiveness. Environmental mycobacterial infec-  [92] a Abbreviations: BCG, bacille Calmette Guerin; c-IFNgR1, complete interferon gamma receptor 1 de®ciency; AD p-IFNgR1, autosomal dominant partial interferon gamma receptor 1 de-®ciency; AR p-IFNgR1, autosomal recessive partial interferon gamma receptor 1 de®ciency; c-IFNgR2, complete interferon gamma receptor 2 de®ciency; p-IFNgR2, partial interferon gamma receptor 2 de®ciency; c-IL-12p40, complete IL-12 p40 de®ciency; c-IL-12Rb1, complete IL-12 receptor b1 de®ciency. b Deceased brother of patient 44; mutation not identi®ed but likely to be Q214R. tions may ®rst occur during childhood rather than infancy, may be localized rather than disseminated, and are usually responsive to appropriate antimicrobial therapy. Granulomas are usually paucibacillary and mature ( [89]). Interestingly, we have observed that the majority of patients with IFNGR1 818del4 mutations develop multifocal NTM osteomyelitis, often without infection at other sites. The pathophysiologic basis for this is unclear. Anecdotal evidence supports the adjunctive use of IFNg therapy in patients with AD partial IFNgR1 de®ciency (Dorman and Holland, unpublished data). However, controlled studies of IFNg therapy during episodes of active infection, or IFNg prophylaxis to prevent infections have not yet been performed.

Autosomal recessive partial IFNgR1 and IFNgR2 de®ciencies
Two siblings with autosomal recessive (AR) partial IFNgR1 de®ciency have been described [87]. At age 1 month, the elder child developed disseminated vaccineassociated BCG infection which was associated with mature granulomas on histopathologic examination of aected tissue, and which responded well to antibiotic therapy. The younger sibling did not receive BCG vaccination, but at age 3 years she developed an illness compatible with primary tuberculosis that responded well to antituberculous therapy. IFNGR1 gene sequencing revealed both children to be homozygous for a single nucleotide substitution leading to replacement of an isoleucine by a threonine at position 87 (I87T) that is part of an N-glycosylation site in the extracellular protein domain. Constructs of this mutation were associated with diminished but not absent responsiveness to IFNg in vitro. The degree of IFNg responsiveness as measured by nuclear translocation of STAT1 in EBV-transformed B lymphocytes from these siblings was greater than that from children with AD partial IFNGR1 de®ciency. This may account for the absence of environmentally acquired NTM infections in the siblings with AR partial IFNgR1 de®ciency. If so, then subtle alterations in IFNg mediated pathways may allow the development of disease due to virulent mycobacteria like M. tuberculosis but protect against disease caused by less virulent environmental NTM.
Recently a patient with autosomal recessive partial IFNgR2 de®ciency was described [88]. As an infant this patient had vaccine-associated disseminated BCG infection which was cured with antibiotics. Over one decade later she developed disseminated M. abscessus infection which could not be controlled with antibiotics but was cured after the addition of adjunctive subcutaneous IFNg. Both infections were associated with mature paucibacillary granulomas. Genetic analysis showed a homozygous nucleotide substitution in IFNGR2 causing an amino acid substitution in the extracellular protein domain (R114C). IFNgR2 protein was present on the surface of patient monocytes, and IFNg responsiveness was diminished but not abolished. This case report established that the genotypephenotype correlations established for IFNGR1 also apply to IFNGR2. This case also raises intriguing questions about the nature of the interactions between IFNgR1 and IFNgR2 in the IFNg receptor complex.

IL12 and IL12 receptor de®ciency
Patients with severe mycobacterial disease and autosomal recessive mutations in the genes encoding IL-12 p40 [90] or IL-12Rb1 [91,92] have recently been ident-i®ed (Table 1). In each case, the mutation precluded protein expression. Each patient suered from severe infection with either NTM or vaccine-associated BCG, and most had severe Salmonella infections. However, in most instances, infection was eectively treated with antibiotics. In several patients, administration of adjunctive IFNg along with antibiotics was associated with substantial clinical improvement. Well-organized, mature, tuberculoid granulomas were observed on histopathologic examination of aected tissues from IL-12Rb1-de®cient patients [91,92], suggesting that IL-12dependent IFNg induction is not required for mature granuloma formation. Tuberculin-speci®c DTH testing was normal in IL-12Rb1-de®cient patients with BCG infection [92], implying that, like IFNg, IL-12 is not required for development of DTH. In vitro, activated T lymphocytes and NK cells from patients had markedly diminished but not absent IFNg production [90± 92]. This residual IL-12-independent IFNg production in IL-12p40 and IL-12Rb1 de®cient patients may account for their milder clinical phenotype compared with patients with complete IFNg receptor de®ciency. Findings in IL-12p40-, and IL-12Rb1-de®cient patients further support that IFNg is critical in control of mycobacteria and Salmonella infections, and that a principal role of IL-12 in control of these infections is to stimulate IFNg production.

Human IFNg de®ciency?
To date human IFNg de®ciency has not been described, despite identi®cation of at least ten dierent human IFNg receptor mutations. The current model of IFNg ligand-receptor interactions does not provide a ready explanation for this discrepancy. The IFNg knockout mouse model indicates that in mice, IFNg is not required for normal growth and development. Moreover, disease due to experimental infection with HSV1 or vaccinia virus is less severe in IFNg knock-out mice than in IFNgR1 knockout mice [64]. Immunologic and genetic evaluation of more patients with heightened susceptibility to infections caused by intracellular pathogens may shed light on this issue. If human IFNg de®ciency does exist and is associated with a clinical phenotype, then it may be correctable with administration of exogenous IFNg. 8. Tuberculosis in IFNg receptor, IL-12Rb1, and IL-12 p40 de®cient patients Among the described patients with known IFNg or IL-12 pathway defects, only one case of probable tuberculosis has been diagnosed [87]. In a 3-year-old girl with AR partial IFNgR1 de®ciency who developed cough, pneumonia, and erythema nodosum, a clinical diagnosis of tuberculosis was made on the basis of development of delayed-type hypersensitivity to tuberculin puri®ed protein derivative and clinical response to administration of anti-tuberculosis antibiotics. Unfortunately, no microbiologic diagnosis was made. A possible second case is the mother of two children with autosomal dominant partial IFNGR1 818del4 mutations. She reportedly died at age 33 of disseminated tuberculosis after three episodes of invasive tuberculosis [89]. However, genetic material was not available and her genotype therefore remains unknown.
The role of IFNg and IL-12 pathway defects in human susceptibility to tuberculosis is clearly an important issue, given that tuberculosis remains a leading cause of infectious disease mortality worldwide. The apparent low incidence of tuberculosis in patients with IFNg or IL-12 pathway defects may be due to a combination of lack of exposure in patients described to date (most of whom live in developed countries where the incidence of tuberculosis is low), and lack of genetic evaluation in patients with tuberculosis who live in developing countries where the incidence of tuberculosis is higher. Alternatively, human host defense against M. tuberculosis may not be dependent on IFNg or IL-12 pathways, although this seems unlikely. As more patients with IFNg or IL-12 pathway defects are ident-i®ed, this issue may be resolved. It will be important to determine if subtle functional changes due to gene polymorphisms are sucient to confer protection against poorly pathogenic mycobacteria but insucient for protection against virulent organisms like M. tuberculosis. 9. Nonmycobacterial infections in IFNg receptor, IL-12Rb1, and IL-12 p40 de®cient patients While mycobacterial infections have been the major recognized cause of morbidity and mortality in IFNg receptor, IL-12Rb1, and IL-12 p40 de®cient patients, infections with other intracellular microorganisms have been described. Severe infections with Salmonella species have been diagnosed in a small number of reported IFNg receptor de®cient patients [78,87], 70% of reported IL-12Rb1 de®cient patients [91,92], and the single reported IL-12 p40 de®cient patient [90]. One patient with L. monocytogenes meningitis [85], one patient with refractory disseminated Histoplasma capsulatum infection [89], and two siblings with pneumonitis thought due to Mycoplasma pneumoniae (one of whom also had serologic evidence for a pneumonia due to a Legionella species) [87] have also been reported. The severity of some viral infections (including herpes viruses, parain¯uenza, and respiratory syncytial virus) is increased in some patients with IFNg receptor de®ciency [95]. The increased severity of herpes virus infections parallels the heightened susceptibility of IFNg and IFNg receptor knockout mice to herpes viruses [55,56]. However, some patients with IFNg receptor de®ciency have had normal recovery from infections caused by RSV and varicella, or immune serologies for HSV, EBV, and CMV without histories of clinical disease [62]. These observations support a role for IFNg in human host defense against some viral infections, but indicate that for viral infections, but not infections due to poorly pathogenic mycobacteria, other immunologic mechanisms may compensate in the absence of IFNg responsiveness. As more children with IFNg pathway defects are ident-i®ed, a broader spectrum of infection susceptibility may become apparent.

Conclusions
Identi®cation of humans with mutations in genes for IFNg receptor proteins, IL-12 p40, and IL-12Rb1 has highlighted the importance of IFNg pathways in human host defense against intracellular pathogens including mycobacteria, Salmonella, and some viruses. Phenotype to genotype correlations are emerging as more patients are identi®ed. In patients with IFNg receptor de®ciency, phenotype, as assessed by infection severity and histopathology, is related to degree of IFNg responsiveness. Children with complete absence of IFNg responsiveness typically have severe disseminated mycobacterial infections, with lepromatoid granulomas in aected tissues. Patients with partial IFNg responsiveness due to either AR or AD IFNg receptor mutations usually have less severe mycobacterial disease associated with tuberculoid granulomas. IL-12 p40 de®ciency and IL-12Rb1 de®ciency are also associated with heightened susceptibility to infections with BCG, NTM, and Salmonella, although the clinical phenotype is typically milder than that of complete IFNg receptor de®ciency.

Future directions
Recognition of IFNg's role in human host defense against intracellular pathogens emphasizes the importance of research to understand the mechanisms by which IFNg activates macrophage killing of intracellular organisms, and the mechanisms by which pathogens such as M. tuberculosis apparently circumvent macrophage killing. Better understanding these mechanisms will lead to the development of rational preventive and therapeutic strategies directed against M. tuberculosis and other intracellular pathogens. It is intriguing to speculate that genetic changes causing subtle functional disturbances in IFNg or IL-12 pathways might contribute to tuberculosis susceptibility at the population level.