Severe Mycobacterial and Salmonella Infections in Interleukin-12 Receptor-Deficient Patients

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Science  29 May 1998:
Vol. 280, Issue 5368, pp. 1435-1438
DOI: 10.1126/science.280.5368.1435


Interleukin-12 (IL-12) is a cytokine that promotes cell-mediated immunity to intracellular pathogens by inducing type 1 helper T cell (TH1) responses and interferon-γ (IFN-γ) production. IL-12 binds to high-affinity β1/β2 heterodimeric IL-12 receptor (IL-12R) complexes on T cell and natural killer cells. Three unrelated individuals with severe, idiopathic mycobacterial and Salmonella infections were found to lack IL-12Rβ1 chain expression. Their cells were deficient in IL-12R signaling and IFN-γ production, and their remaining T cell responses were independent of endogenous IL-12. IL-12Rβ1 sequence analysis revealed genetic mutations that resulted in premature stop codons in the extracellular domain. The lack of IL-12Rβ1 expression results in a human immunodeficiency and shows the essential role of IL-12 in resistance to infections due to intracellular bacteria.

IL-12 is a heterodimeric cytokine that consists of two disulfide-linked subunits, p40 and p35, and is produced by activated antigen presenting cells (dendritic cells, macrophages), particularly upon infection with intracellular microbes (1,2). IL-12 promotes the development of TH1 responses and is a powerful inducer of IFN-γ production by T cells and natural killer (NK) cells (1, 2). The receptor for IL-12 is composed of two distinct subunits, β1 and β2, that assemble to form a high-affinity IL-12R complex expressed on T cells and NK cells (3-6). IL-12 and IFN-γ appear to be both essential for the development of protective cell-mediated immunity to tuberculous mycobacterial pathogens in mice (7) and in humans (8).

Idiopathic disseminating mycobacterial infections due to nontuberculous species (Mycobacterium fortuitum, Mycobacterium avium, Mycobacterium chelonei, Mycobacterium smegmatis) or Mycobacterium bovis bacillus Calmette–Guérin (BCG) have been described in patients without previously recognized immunodeficiencies (9-13). Peripheral blood mononuclear cells (PBMCs) of some patients were deficient in IFN-γ production upon mitogenic polyclonal stimulation (10, 14). In several cases genetic analyses identified inactivating mutations in the IFN-γR1 gene, resulting in complete (10, 12-14) or partial (15) deficiencies in IFN-γR expression or function. In another family with similar disseminating M. avium infections, IL-12 production was reported to be deficient in affected members, but the underlying mechanism was not elucidated (16).

We have examined type-1 cytokine and TH1 responses in three unrelated individuals with recurrent, severe mycobacterial and Salmonella infections. Patient 1 is a 26-year-old female who developed a severe Salmonella paratyphi sepsis at the age of 3 years, which was complicated by abdominal abscesses, and at the age of 22 years presented with a M. avium sepsis with extensive mediastinal lymphadenopathy. Patient 2, a 19-year-old female, presented with recurrent systemic M. avium intracellulare infections at ages 4, 13, and 17 years and with severe systemicSalmonella type B infections at ages 4, 7, and 14 years. Patient 3 is a 3-year-old female who developed progressive M. bovis BCG infection after vaccination at the age of 1 year, followed by severe and nearly fatal S. typhimurium sepsis at the age of 2 years. Upon histological examination, the BCG lesion of patient 3 contained well-organized granulomatous infiltrates (17). None of the three patients had any recognized immunodeficiencies or alterations in expression of T, B, NK, or macrophage cell surface markers (18). All three patients could be treated effectively with antibiotic therapy (18).

To investigate the nature of these immunodeficiencies, we stimulated PBMCs derived from patients and healthy controls (n = 4) with mitogenic combinations of T cell-specific monoclonal antibodies (mAbs) to CD2 and CD28 (19, 20), IL-2, or the lectin phytohemagglutinin (PHA). Although PBMCs from patients as well as controls proliferated to all these stimuli (Figs. 1A and 2B), patients' PBMCs were deficient in IFN-γ production [Figs. 1B and 2, C and E (21); 85 to 99% reduction compared with controls]. Similar deficiencies in IFN-γ production were observed in response to the pathogens M. avium, M. tuberculosis,and S. paratyphi (21). These results were consistently reproducible in multiple experiments.

Figure 1

PBMCs from patients and controls. (A) Proliferative responses. CD28 mAb alone did not induce any proliferation or IFN-γ production. Results represent median counts per minute (cpm) + SEM of triplicate cultures. Contr, healthy control; pat, patient; see (33). (B) IFN-γ production. The second y axis (right) refers to results plotted in the rightmost panel only. All results shown were obtained in the same, representative experiment (34). (C) IFN-γ responsiveness as measured by IL-12 p70 production in whole blood cultures. Similar results were obtained in IL-12p40 ELISAs (35).

Figure 2

(A) Lack of IL-12Rβ1 expression in patients 1 to 3. PBMCs stimulated with PHA for 3 days or short-term IL-2-propagated T cell lines were analyzed by flow cytofluorometry for cell surface expression. Cells were incubated with mAb to IL-12Rβ1 (2-4E6, line 2) (23), CD25 (line 3), or control mouse IgG1 (line 1) and stained with fluorescein isothiocyanate–conjugated goat antibody to mouse (Fab)2. Cells were analyzed with a FACSCAN analyzer (B & D). (B) Lack of IL-12–induced proliferation of PBMCs of patients 1 and 2. The second y axis (right) refers to results plotted in the rightmost panel only. Triplicate cultures of freshly isolated PBMCs were stimulated as described in (34). Human rIL-12 p70 (from S. F. Wolf, Genetics Institute) was added at concentrations of 1 or 5 units/ml. Proliferative responses and cytokine production were measured on day 4. One of three representative experiments is shown. (C) Lack of IL-12–induced IFN-γ production of PBMCs from patients 1 and 2. See (B) and (33). (D) IL-12 independence of residual IFN-γ production in patients 1 and 2. Cultures were set up as described above, in the presence or absence of anti-IL-12 (α-IL-12) mAbs (C8.6, 2 to 3 μg/ml; C8.1, 10 to 15 μg/ml; G. Trinchieri) (36). These were the optimal concentrations (32, 36). Addition of the IL-12 binding, but nonneutralizing, mAb 11.79 did not inhibit these responses (21). (E) Lack of IFN-γ production of PBMCs from patient 3. Proliferative responses in the control and in patient 3 were comparable in the same experiment: 63,120 and 67,764 cpm to CD2 and CD28; 120 and 85 cpm to CD2 alone; 265 and 271 cpm to rIL-2; 32,770 and 12,454 cpm to CD2 and rIL-2, respectively.

To exclude the possibility that defective IFN-γ production in the patients was associated with a lack of IFN-γR expression (10, 13), we determined cell surface expression of IFN-γR (CD119) and responsiveness to exogenous recombinant IFN-γ (rIFN-γ). CD119 expression on freshly isolated CD14+ monocytes from patients was similar to that of controls (21), and incubation of patient and control cells with rIFN-γ enhanced tumor necrosis factor-α (TNF-α) production triggered by lipopolysaccharide (LPS) (22). rIFN-γ also synergized with M. avium in inducing the production of IL-12 p40 and IL-12 p70 (Fig. 1C). LPS alone also induced IL-12 p70 release in patients. Thus, all three patients were deficient in IFN-γ production, yet they displayed normal IL-12 p70 production, IFN-γR expression, and IFN-γR function.

Therefore, we examined IL-12R expression and function. We measured IL-12Rβ1 expression on PHA-activated T cells from patients and controls by using the IL-12Rβ1-specific mAb 2.4E6 (23). Cells from patients did not express detectable IL-12Rβ1 cell surface molecules, whereas expression of the T cell activation marker CD25 was similar on cells from patients and controls (Fig. 2A).

The combination of CD2 mAb with exogenous recombinant IL-12 (rIL-12) synergized in inducing proliferation and IFN-γ production by PBMCs from healthy individuals (Fig. 2, B, C, and E). In contrast, rIL-12 failed to synergize with CD2 mAb in inducing proliferation or IFN-γ production in patients' PBMCs (Fig. 2, B, C, and E). rIL-2, however, synergized with CD2 mAb in inducing proliferation of patients' PBMCs (Fig. 1), showing that patients' PBMCs generally are not defective in their capacity to respond to cytokines. IL-12 also could not induce tyrosine phosphorylation of the IL-12R–associated signal transducer and activator of transcription STAT-4 in patients' cells (24).

Only small amounts of IFN-γ were produced by patients' cells when triggered by the mitogenic combination of CD2+ and CD28 mAbs. However, this could not be inhibited by neutralizing IL-12 mAb; thus, the low release of IFN-γ in these patients is independent of endogenous IL-12 (Fig. 2, D and E) (21). In contrast, IFN-γ release in control cultures was reduced by >70% in the presence of saturating concentrations of mAbs to IL-12 (Fig. 2, D and E) (21). Although IL-12 is a major promoter of IFN-γ production (1, 2), some IFN-γ can be produced in the absence of IL-12 or IL-12Rβ1 in IL-12 p40−/−(25) and IL-12Rβ1−/− mice (26). Although it is unclear how such a pathway is regulated (27,28), the results reported here likely reveal a similar pathway in humans. This IL-12R–independent pathway of (low) IFN-γ production probably accounts for the lack of enhanced IL-4 production in our patients (29).

Collectively, these results show that these three patients do not express functional IL-12R complexes and that their remaining T cell–dependent IFN-γ production is independent of endogenous IL-12.

cDNA and genomic IL-12Rβ1 DNA sequence analysis identified distinct genetic mutations. Patient 1 was homozygous for a nonsense mutation at nucleotide position 94 (C → T) at the cDNA and the genomic level. Both parents were heterozygous, which indicates an autosomal recessive segregation pattern. This mutation introduces a premature stop codon in the extracellular domain (Table1). Patient 2 appeared homozygous for a nonsense mutation at nucleotide position 1126 (C → T) both at the cDNA and the genomic DNA level. This mutation also results in a premature stop codon in the extracellular domain. Again, both parents were heterozygous (30). In patient 3, a deletion was found in the cDNA that extended from nucleotide position 409 to position 549. This deletion led to a frameshift that introduced a premature stop codon at nucleotide positions 570 to 572 (TGA) in the extracellular domain of the IL-12Rβ1 gene. Both parents were heterozygous for this deletion, whereas the patient's healthy sibling was unaffected, which conforms to an autosomal recessive segregation pattern (Table 1) (30). This deletion most probably results from a splice mutation that leads to the skipping of one exon, but one of the flanking introns could not be amplified.

Table 1

Patients with genetic lack of IL-12R expression. Mutations are indicated according to (37). X is a stop codon; Q is glutamine. Mutations shown are homozygous in the patients only; all parents were heterozygous (30). Each of the three mutations leads to a premature stop codon in the extracellular domain-encoding region, resulting in lack of membrane expression. See text for discussion.

View this table:

The six parents as well as the tested healthy siblings of the three patients all expressed functionally competent IL-12R complexes (21).

The genetic lack of IL12-Rβ1 expression represents a human immunodeficiency and is sufficient to explain the observed phenotypes in all three patients. The defect in IFN-γ production and the extreme susceptibility to mycobacterial and Salmonellainfections in these patients most probably result directly from the lack of IL-12R expression and signaling. The selective susceptibility to mycobacterial and Salmonella infections shows that the type-1 cytokine pathway is essential for controlling resistance to these pathogens and that no other redundant protective immune mechanism can compensate for this deficiency in these patients. The patients did not develop any abnormal infections with other viral, bacterial, or fungal pathogens (18), which suggests that IL-12 may be dispensable for protection to pathogens other than intracellular bacteria.

It is not yet known how many nonfunctional alleles have been maintained in the human population (31) and whether partially defective IL-12R alleles also exist, as described for the IFN-γR1 gene (15). Such allelic variants could contribute to genetically controlled disease susceptibility not only in infectious diseases but also in other conditions that rely on IL-12R signaling, such as cancer. The definition of unusual immunodeficiencies reveals fundamental insights into cytokine and cellular effector pathways of protective immunity and may allow the design of appropriate and effective therapeutic regimens.

  • * To whom correspondence should be addressed. E-mail: ihbsecr{at}


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