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Impairment of Mycobacterial Immunity in Human Interleukin-12 Receptor Deficiency

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Science  29 May 1998:
Vol. 280, Issue 5368, pp. 1432-1435
DOI: 10.1126/science.280.5368.1432

Abstract

In humans, interferon γ (IFN-γ) receptor deficiency leads to a predisposition to mycobacterial infections and impairs the formation of mature granulomas. Interleukin-12 (IL-12) receptor deficiency was found in otherwise healthy individuals with mycobacterial infections. Mature granulomas were seen, surrounded by T cells and centered with epithelioid and multinucleated giant cells, yet reduced IFN-γ concentrations were found to be secreted by activated natural killer and T cells. Thus, IL-12–dependent IFN-γ secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas due to IL-12–independent IFN-γ secretion.

Bacille Calmette-Guérin (BCG) and nontuberculous mycobacteria (NTM) are poorly virulent mycobacteria that may cause disseminated disease in otherwise healthy children (1-3). The identification of inherited IFN-γ receptor ligand-binding chain (IFN-γR1) deficiency provided the first genetic etiology for this syndrome (4) and highlighted the importance of IFN-γ, a pleiotropic cytokine secreted by natural killer (NK) and T cells (5), in the control of mycobacteria in humans. The lack of mature mycobacterial granulomas showed that their formation is strictly IFN-γ–dependent. Partial, as opposed to complete, IFN-γR1 deficiency is associated with mature granulomas and a milder course of mycobacterial infection (6). However, a number of disseminated BCG and NTM infections remain unexplained (7).

Four patients from three unrelated kindreds were investigated in this study (8). All suffered from disseminated mycobacterial infections attributable to BCG orMycobacterium avium, and two of them also had non-typhi salmonella infections. No well-defined immunodeficiency could be detected in these otherwise healthy children, and the diagnosis of IFN-γR1 deficiency was excluded (9). Mutations in IFN-γ and in IFN-γR1–associated molecules within the receptor complex (10), as well as mutations in either of the two IL-12 p70 subunits (p35 and p40), a potent IFN-γ–inducing heterodimeric cytokine secreted by dendritic cells and phagocytes (11), were also excluded.

Mutations in each of the two IL-12 receptor subunits (β1 and β2), expressed on NK and T cells (12), were sought. A nonsense nucleotide substitution (AAG → TAG) at position 913 of the IL-12Rβ1 cDNA coding region was identified in patient 1 (13) (Table1). The patient was found to be homozygous for the corresponding genomic mutation, which was inherited as an autosomal recessive trait in the kindred (14) (Fig.1A). Two specific monoclonal antibodies (mAbs) failed to detect IL-12Rβ1 molecules at the cell surface of phytohemagglutinin (PHA)–activated peripheral blood T cells and PHA-activated Herpesvirus saimiri–transformed T cells (15) (Fig. 1B). Recessive mutations precluding expression of IL-12Rβ1 were also identified in the other two kindreds (Table 1).

Figure 1

A nonsense recessive mutation in the IL-12Rβ1 gene. (A) Segregation of the genomic mutation in kindred 1, as shown by 2% agarose gel electrophoresis of Mbo II digests of the amplified IL-12Rβ1 gene around position 913. The Mbo II site in the wild-type sequence is altered by the mutation (GAAGA → GTAGA); the overrepresentation of undigested products in heterozygous carriers is attributable to the presence of heteroduplex molecules after amplification. (B) Flow cytometry analysis of IL-12Rβ1 on PHA-activated Herpesvirus saimiri–transformed T cells with specific mouse mAb 12Rb.3F12 (dashed lines), compared with an isotypic control (black lines), in a control (C) and in patient 1 (P).

Table 1

Patients with inherited IL-12Rβ1 deficiency.

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There were normal numbers of NK cells in the blood of patient 1, and the efficient destruction of K562 cells by peripheral blood mononuclear cells (PBMCs) attested that NK cells were also functional (Fig. 2A) (16). Addition of recombinant IL-12 did not further up-regulate the cytotoxicity of the patient's NK cells, whereas it did up-regulate the cytotoxicity of control NK cells; this confirmed that IL-12Rβ1 deficiency results in impaired IL-12 receptor function (12). However, efficient NK cytotoxicity was accompanied by secreted IFN-γ concentration only 1% of that of control activated NK cells (Fig. 2B). Impairment of IFN-γ secretion by otherwise functional NK cells also occurs in mice genetically deprived of IL-12p40 or IL-12Rβ1; these mice are termed IL-12 knockout (IL-12KO) mice (17).

Figure 2

Impaired IFN-γ secretion by NK and T lymphocytes. (A) NK cells from a control individual (C) and patient 1 (P) were tested for natural cytotoxicity against K562 cells alone or in the presence of recombinant IL-12. A single representative experiment is shown; the experiment was done twice. E/T, effector/target ratio. (B) Production of IFN-γ from NK cells, tested under the same conditions as in (A), and T cells stimulated by PHA or tuberculin from a control individual and patient 1; means of two experiments are shown. (C) T cells were also tested under the same conditions for proliferation.

When T cells from the patient were incubated with PHA (18), they produced only 5% of the IFN-γ of control cells (Fig. 2B). Normal mitogen-driven T lymphocyte proliferation in vitro showed that impaired IFN-γ secretion did not result from impaired T cell activation (Fig. 2C). Secretion of IFN-γ from tuberculin-activated T lymphocytes was reduced by 99% relative to control T cells. This finding implies that most specific T cells primed in vivo by mycobacterial antigens, in the absence of costimulation by IL-12, could not secrete normal amounts of IFN-γ when restimulated. Impairment of IFN-γ secretion by otherwise functional T cells also occurs in IL-12KO mice (17).

Tuberculin-specific delayed-type hypersensitivity (DTH) (19) was normal in BCG-infected patients with IL-12Rβ1 deficiency, as seen in patients with IFN-γR1 deficiency (4). This further suggests that the cooperation between antigen-presenting cells and tuberculin-specific memory T cells in vivo was not globally affected by the lack of IL-12 stimulation, which in turn implies that neither IL-12 nor IFN-γ are essential for DTH in humans. Antigen-specific DTH in IL-12KO mice seems somewhat impaired, however (17).

Paucibacillary, well-circumscribed granulomas with epithelioid and giant multinucleated cells were identified in the lymph nodes and liver of BCG-infected patient 1 (Fig. 3), unlike in BCG-infected tissue from children with complete IFN-γR1 deficiency (20). Immunostaining revealed CD3-positive lymphocytes, including mostly helper (CD4+ and CD45RO+) and smaller numbers of cytotoxic (GMP-17+, CD8+, and CD45RO+) memory T cells, as in control children with benign local BCG infection (BCG-itis) (20). Thus, even though the kinetics of BCG granuloma formation or the ability to develop mature granulomas in response to other mycobacterial species were not determined, mature BCG granulomas were seen in the absence of IL-12–mediated immunity. Lung granuloma formation is impaired in IL-12KO mice infected withMycobacterium tuberculosis (21).

Figure 3

Mature BCG granulomas. (A andB) Hematoxylin and eosin stainings of a BCG-infected lymph node from patient 1 at magnifications of 100× (A) and 400× (B). (C to G) Immunohistochemical stainings of 4-μm-thick serial sections of a granuloma from the same tissue sample with CD3- (C), CD8- (D), CD4- (E), GMP-17– (F), and CD45RO-specific (G) antibodies (magnifications, 400×).

IL-12KO mice are highly susceptible to M. tuberculosis(21), whereas infections with less virulent mycobacteria, such as BCG and NTM, have not been reported to date. In humans, IL-12 seems important in protective immunity to M. tuberculosis(22), M. avium (23), and M. leprae (24). Evidence from the four patients in the present study (who were genetically deprived of IL-12–mediated immunity) reveals that IL-12 is irreplaceable for protective immunity to even poorly pathogenic mycobacteria. It is likely that IL-12 is also essential in the control of more virulent mycobacterial species, and perhaps other, milder IL-12Rβ1 mutations may lead to a predisposition to clinical tuberculosis in the general population (25).

IL-12KO mice were also found to be highly susceptible toLeishmania major infection (26). Much experimental evidence suggests that IL-12–mediated immunity may be important in the control of a wide range of viral, bacterial, and parasitic microorganisms in mice and humans (27). However, despite probable exposure to most childhood pathogens and environmental microorganisms, patients with IL-12Rβ1 deficiency did not suffer from infections due to microbes other than mycobacteria (and, to a lesser extent, salmonella); this suggests that IL-12 in humans is not necessary to control most other infections. Three other patients sharing this phenotype are described in an accompanying report (28), but the identification of more kindreds is probably needed to better appreciate the range of potential pathogens.

The selective susceptibility to mycobacterial infections is shared by IL-12Rβ1–deficient and IFN-γR1–deficient children (29). Two cytokines, IFN-γ and IL-12, appear to play both a selective and an essential role in human defense against mycobacteria. However, the clinical phenotype shared by children with each of these two genotypes is likely to arise from a single pathogenic mechanism. First, in IL-12Rβ1–deficient patients, IFN-γ production by otherwise functional NK and T lymphocytes is markedly impaired. Second, therapeutic use of IFN-γ cured the mycobacterial infection in IL-12Rβ1–deficient patient 3 (8). Insufficient IFN-γ production thus appears to be the main pathogenic mechanism in IL-12Rβ1–deficient patients.

Mycobacterial infections in IL-12Rβ1–deficient patients tend to have a milder course than in children with complete IFNγR1 deficiency (4), and such patients more closely resemble children with partial IFN-γR1 deficiency (6). Children with IL-12Rβ1 deficiency and partial IFN-γR1 deficiency have mature BCG granulomas, unlike children with complete IFN-γR1 deficiency. The milder phenotype is probably attributable to IL-12–independent pathways of IFN-γ production (30); residual amounts of IFN-γ were produced by IL-12Rβ1–deficient activated lymphocytes. The process of mature granuloma formation in response to mycobacterial infection is strictly IFN-γ–dependent. Although IL-12–dependent IFN-γ induction is not necessary for mature granuloma formation, it is essential for protective mycobacterial immunity in humans.

  • * To whom correspondence should be addressed. E-mail: casanova{at}ceylan.necker.fr

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