Human Molecular Genetics Advance Access originally published online on November 17, 2005
Human Molecular Genetics 2005 14(24):3847-3855; doi:10.1093/hmg/ddi409
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Molecular complementation of IL-12Rß1 deficiency reveals functional differences between IL-12Rß1 alleles including partial IL-12Rß1 deficiency
1Department of Infectious Diseases and 2Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, C5-38, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
* To whom correspondence should be addressed. Tel: +31 715261784/2613; Fax: +31 715266758; Email: e.van_de_vosse{at}lumc.nl
Received September 8, 2005; Accepted October 26, 2005
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ABSTRACT |
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Patients with mutations in IL12RB1, the gene encoding IL-12Rß1, suffer from combined IL-12R/IL-23R deficiency and are unusually susceptible to nontuberculous mycobacteria and salmonellae. The functional effects of amino acid changes in IL-12Rß1, however, have not been determined at the molecular level. Molecular complementation studies are essential to demonstrate how structural amino acid changes affect IL-12Rß1 function, and whether functionally different IL-12Rß1 alleles can be distinguished. Thirteen different IL-12Rß1 alleles, including 11 amino acid substitutions and the two major haplotypes (214Q-365M-378G and 214R-365T-378R), were retrovirally transduced in IL-12Rß1 deficient human T cells. We provide functional evidence that L77P, R173P, C186S, R213W and Y367C are deleterious mutations leading to non-functional proteins. Conversely, S74R, R156H, H438Y, A525T and G594E are fully functional IL-12Rß1 variants. The C198R mutation leads to a partially functional IL-12Rß1, representing the first molecularly proven partial IL-12Rß1 deficiency. Interleukin-12 (IL-12) induced not only Interferon-
but also IL-10 in all responder but not in null-mutant alleles, with intermediate levels in C198R. The QMG allele was found to be a higher IL-12 responder allele compared with the RTR allele. These results have implications for understanding IL-12R/IL-23R structure-function and the role of IL-12R/IL-23R in human disease. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Human host immunity against intracellular pathogens such as mycobacteria and salmonellae depends on an effective cell-mediated immune response mediated by type-1 cytokines (1
). Upon bacterial stimulation of pattern recognition receptors, including Toll-like receptors, dendritic cells (DCs) and type-1 macrophages (m
1) produce IL-23, IL-12 and IL-18 which are recognized by complementary receptors (IL-23R, IL-12R and IL-18R) on type-1 helper T (Th1) cells and natural killer (NK) cells (2). These cells subsequently produce Interferon- (IFN-) (3) which binds to the IFN- receptor (IFN-R), present on macrophages and DCs, which are activated to produce increased levels of IL-12 and to enhance both antigen presentation and bactericidal activity. In addition, IL-10 production has been observed in both T cells and NK cells after IL-12 stimulation (4–6), but this has not been studied as extensively.
Around 140 patients have been described with unusually severe infections caused by otherwise poorly pathogenic mycobacteria and salmonellae, a condition also known as Mendelian susceptibility to mycobacterial infection (MSMD [MIM 209950
[OMIM]
]). MSMD is a heterogeneous disorder that can be caused by mutations in various genes involved in IL-12/-23/IFN- cytokine signaling. In these patients, genetic defects have been identified in five genes in this pathway, namely IL12B, IL12RB1, IFNGR1, IFNGR2 and STAT1 (reviewed in 7). More than a third of the patients that have been described so far had defects in IL12RB1, the gene encoding the ß1 chain (IL-12Rß1) of the IL-12R and the IL-23R (7).
The majority of the mutations in IL12RB1 result directly (through nonsense mutations) or indirectly (through splice site mutations or small deletion/insertion events) in premature stop codons, making the effect of these mutations immediately clear. However, about a quarter of the mutations in IL12RB1 described are missense mutations, of which the functional effect is not immediately obvious. We have previously predicted the effect of amino acid changes on the function of the IL-12Rß1 protein based on predicted changes in the three-dimensional structure, the degree of conservation of the amino acids between species, and the function of the domains they were located in reference (8). Observed amino acid changes in IL-12Rß1 in patients with IL-12R/IL-23R signaling defects may also represent rare variants, and the underlying genetic defect might be a mutation in a signal transducing protein (e.g. Tyk2). Therefore, molecular proof using comparative mutant analyses in the same background is essential to be able to conclude whether a variation represents a mutation or a rare variant.
Here, we have cloned IL-12Rß1 variants into a retroviral expression vector and transduced the variants into cells of a patient with a null mutation to study the effect of the variations on IL-12Rß1 expression and function in vitro. The variants analyzed are six published mutations, R213W (9,10), R173P (11) C198R (12), L77P (13), C186S and Y367C (14), several variants that had not been experimentally proved to be mutations or selectively neutral variants yet [S74R, R156H (8), H438Y, A525T and G594E (9)] and the two common haplotypes 214Q-365M-378G (QMG) and 214R-365T-378R (RTR) which are present in the population in about equal proportions (8,15). We have analyzed the production of various cytokines in response to IL-12 and IL-18 stimulation in cells transduced with these variants, and in normal control cells to designate a variant as either a deleterious mutation or a benign variant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Retroviral transduction restores IL-12Rß1 membrane expression and IL-12R function in IL-12Rß1 deficient cells
The use of the retroviral expression vector pLZRS ensures transcription and expression of the IL12RB1 and green fluorescent protein (GFP) genes in tandem and allows for selection of transduced cells by fluorescence-activated cell sorting (FACS) sorting for the GFP signal. Transduction efficiency was typically between 4 and 17%; after FACS sorting 96–99.6% of the cells (98% average) were GFP positive.
Normal control phytohemagglutinin (PHA) blasts (BCA) express IL-12Rß1 on their cell surface and do not express GFP (Fig. 1A). Cells from a patient with a null mutation (P1) do not express IL-12Rß1 or GFP (Fig. 1B). After transduction of either one of the two common IL12RB1 haplotypes (QMG and RTR), into cells from P1, IL-12Rß1 expression on the cell surface was restored, whereas in cells transduced with the empty vector (GFP) it was not (Fig. 1C–E).
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IL-12 and IL-18 synergize in the IFN-
production in normal controls (BCA and BCC in Fig. 2A). IFN- production in response to IL-12 in the absence or presence of IL-18 was restored in IL12RB1 common haplotype transduced cells but not in untransduced cells (P1) or empty vector (GFP) transduced cells (Fig. 2B). Likewise, cell proliferation in response to IL-12 (in the absence or presence of IL-18) was restored in the IL12RB1 common haplotype transduced cells but not in cells with empty vector (data not shown). Cells complemented with the QMG haplotype consistently produced more IFN- (Fig. 2B) and proliferated better (data not shown) than cells that had received the RTR haplotype.
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IL-12Rß1 transduction also restores IL-23R function and IL-23 acts in synergy with IL-18
IL-12Rß1 is an essential subunit of both the IL-12R and the IL-23R. Functional integrity of the IL-12Rß1 subunit can therefore also be assessed by measuring responses to IL-23, e.g. by measuring IFN-
production. Upon stimulation with IL-23 normal controls produced only marginal amounts of IFN-. In accordance with previously published findings (16), IL-23 and IL-18 synergized in the IFN- production in normal controls (Fig. 2C and data not shown). We therefore measured IFN- production upon stimulation with IL-23 and IL-18 to evaluate integrity of the IL-23R in genetically complemented cells. IL-23R function was indeed fully restored in cells transduced with the IL12RB1 haplotypes, QMG and RTR, but not in the untransduced cells (P1) or cells transduced with empty vector (GFP) (Fig. 2C). Although the IL-23R is restored, we observe a difference in IFN- production in response to IL-23 between control cells and the two IL12RB1 haplotypes. IL-12 is known to be essential for sustaining Th1 memory T cells, and a lack of memory T cells has been shown in a IL-12Rß1 patient (17). We therefore suspect that in the peripheral blood monocytes (PBMCs) from patient P1 relatively few memory T cells were present. Oppmann et al. (18) found that IL-23 primarily acts on human memory T cells. Together this may explain why in the PHA blasts of P1, the IL-23 response is limited compared with normal controls.
IL-12Rß1 expression of IL12RB1 variants on the cell surface and intracellularly
All known IL12RB1 amino acid substitutions identified to date were cloned in the appropriate background allele (QMG or RTR) when known. Variants of which the background was unknown (H438Y, L77P, C186S and Y367C) were cloned into the QMG background (Table 1).
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We first determined whether the various IL12RB1 variants were expressed at the cell surface. GFP could be detected in all transduced cells indicating that the constructs were all transcribed and translated (Fig. 3 A–L). The IL-12Rß1 protein of the variants S74R, R156H, H438Y, A525T and G594E could be detected on the cell surface of transduced cells (Fig. 3A, C, I, K, L) using IL-12Rß1 antibody 2.4E6. The IL-12Rß1 protein of the variants L77P, R173P, C186S, C198R, R213W and Y367C could not be detected on the cell surface with the antibody 2.4E6 (Fig. 3B, D–H). A second antibody against IL-12Rß1, 2B10, however, did detect cell surface expression of the L77P protein (Fig. 4C). This antibody also revealed low level of cell surface expression (7.7% of cells) of C198R, particularly in cells with higher levels of GFP expression (Fig. 4F and Table 2). This was not observed in R173P, C186S, R213W or Y367C transduced cells (maximally 0.7% of cells) (Fig. 4D, E, G, H and Table 2). Interestingly, a similar trend for the C198R allele was observed with antibody 2.4E6 that was not observed for any of the other non-expressing variants that were strongly positive for GFP (Fig. 3). These findings suggest that low levels of C198R IL-12Rß1 protein are expressed on the cell surface in transduced cells.
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To further quantify the apparent (low level of) extracellular expression of C198R, we quantified the correlation between the GFP and the IL-12Rß1 expression (as determined with antibody 2B10) in all variants using the program FlowJo (TreeStar) for linear regression of FACS data (Table 2). There was a clear difference between the positive incline of the trendlines calculated for the common haplotypes and functional alleles (all >3.5), and the negative incline found in case of the null-mutant alleles (all <–0.005). The L77P mutation containing cells that were positive for IL-12Rß1 (Fig. 4C) showed an incline of only 0.4, a factor 10 lower than the QMG or RTR transduced cells. C198R transduced cells showed a positive incline of 0.02, confirming that a small but significant amount of the protein is indeed expressed on the cell surface.
We next analyzed intracellular expression levels of all IL-12Rß1 proteins that were undetectable at the cell surface, as we had previously found C198R to be expressed at high levels intracellularly in cells from the patient in which this mutation was identified (12). Using monoclonal antibody (mAb) 2.4E6, IL-12Rß1 mutant protein was indeed detectable intracellularly in R173P, C186S, C198R, R213W and Y367C (Fig. 5D–H) but not in L77P (Fig. 5C). Owing to the small size of the GFP protein, the GFP signal is largely lost when the cells are permeabilized for intracellular labeling.
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Functional integrity of IL12R in transduced IL-12Rß1 variants
To determine whether the constructs produced functional IL-12Rß1 proteins, IL-12R function was tested. IFN-
production of cells transduced with each of the variants or empty pLZRS was determined after stimulation with increasing amounts of IL-12, IL-18 or IL-12 plus IL-18. The cells transduced with empty vector (GFP) did not produce IFN- (Fig. 6). The QMG haplotype consistently produced more IFN- than the RTR haplotype, in line with the results in Fig. 2B.
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The constructs containing the mutations L77P, R173P, C186S, R213W and Y367C showed no production of IFN-
in response to IL-12 and only a marginal response to IL-18 (Fig. 6). No increase in cell proliferation in response to these cytokines tested was observed (data not shown). On the other hand, cells with the C198R construct produced low but significant amounts of IFN- in response to IL-12 or IL-18, and high levels of IFN- in response to the synergizing combination of IL-12 plus IL-18 (Fig. 6). Cell proliferation of C198R in response to increasing amounts of IL-12 and IL-12 plus IL-18 (not to IL-18 alone) was also significantly increased (data not shown). The cells transduced with the variants S74R, R156H, H438Y, A525T and G594E all produced large amounts of IFN- in response to IL-12 and in response to IL-12 plus IL-18 and low amounts in response to IL-18 alone (Fig. 6). In these variants, cell proliferation was strongly increased in response to IL-12 and IL-12 plus IL-18 (data not shown).
Although all IL12RB1 construct-containing cells produce IFN- in response to IL-18, this is, in contrast to normal controls, only a small amount. It is known that in normal controls, IL-18R expression is up-regulated in response to IL-12 thus allowing the cells to respond to IL-18. We have also always observed only a marginal response to IL-18 in cells from IL-12Rß1 deficient patients (data not shown). The low IL-18 responsiveness in the IL12RB1 construct-containing cells is most likely due to a lack of IL-18R expression, this is strengthened by the observation that the response to IL-18 is fully restored in the functional variants once IL-12 is added as well.
Production of IL-10 and TNF-
in response to IL-12 and IL-18 stimulation in functional IL-12Rß1 variants
To determine whether the production of other cytokines was similarly dependent on the presence of the IL-12Rß1, we tested the production of tumor necrosis factor- (TNF-) and IL-10 after stimulation with IL-12. When testing a normal control (BCA), cells from P1 or cells transduced with the QMG or RTR alleles, no correlation could be found between presence of an intact IL-12R and the levels of TNF- produced (data not shown).
However, cells from a normal control (BCX) or cells from P1 transduced with the IL12RB1 QMG allele yielded high IL-10 production in response to IL-12 (Fig. 7A). Because the production of IL-10 was higher in cells from P1 transduced with IL12RB1 than in normal control cells (BCX) (Fig. 7A), we determined whether the presence of pLZRS constructs enhanced IL-10 production, even though GFP only transduced cells were negative (Fig. 7A). To this end, we transduced cells of a normal control (BCC) with empty vector (GFP) and with the two common IL12RB1 haplotypes (QMG and RTR) and compared their IL-10 production to the cells without construct. The presence of a construct clearly did not influence IL-10 production (Fig. 7B). A clear correlation between IL-12 dose and IL-10 response could be observed (Fig. 7B).
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IL-10 production in response to IL-12 in all of the IL12RB1 variants (Fig. 7C) was in accordance with the IFN-
production results in that cells from P1 transduced with vector only (GFP) or the known mutants L77P, R173P, C186S, R213W, Y367C failed to produce any IL-10. In contrast, the QMG and RTR alleles, the variants S74R, R156H, H438Y, A525T, G594E, the partial mutation C198R and the normal control (BCA) all produced significant amounts of IL-10. The IL-10 production was, again similar to the results for IFN- production, higher for the QMG allele than for the RTR allele. While the IL12RB1 variants were all tested in the same background (PHA blasts from P1) thus allowing for a comparison of IL-10 production between the variants, the normal controls we tested (all QMG/RTR heterozygotes) show a wide range of IL-12 induced IL-10 production likely due to differences in background. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this study, we provide evidence, using a molecular complementation approach, that several known amino acid changes in IL-12Rß1, L77P, R173P, C186S, C198R, R213W and Y367C are indeed deleterious mutations. All other missense variations resulting in amino acid changes did not affect cell surface expression and function and should be considered selectively neutral variants. The proteins R173P, C186S, R213W and Y367C were not expressed on the cell surface and did not respond to IL-12 by IFN-
production, cell proliferation or up-regulated IL-18R expression. The only mutant protein that had been transduced into cells before was R213W, which was found not to be expressed on the cell surface of HEKC293 cells and was not detectable by western blotting (9). The human embryonic kidney (HEK) cells used in that study are, however, not physiologically IL-12Rß1 expressing cells, which might impede testing of IL-12R/IL-23R function, e.g. due to the absence of essential downstream signaling components. We, instead, used human T cells from a patient with a null mutation (Q32X) as recipient cells, as the truncated protein that is produced because of this null mutation is highly unlikely to interfere with experiments. The first 24 amino acids contain a signal peptide that is normally cleaved off to produce the mature protein, resulting in a 7 amino acid peptide that contains only a fraction of the 638 amino acid mature protein. We here show that R213W, as well as R173P, C186S, C198R and Y367C, are abundantly expressed intracellularly, indicating these proteins are produced but retained within human T cells. This is most likely due to the protein quality control system in the endoplasmic reticulum that prevents transport of mutant, misfolded or incorrectly complexed proteins (8). Together, findings are summarized in Figure 8.
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Importantly, we confirm that the C198R mutation generates a partially functional protein as reported previously (12
). While in the patient's peripheral T cells, IL-12Rß1 protein was undetectable at the cell surface (12), the C198R IL-12Rß1 protein can be detected at the cell surface when it is (over)expressed in the transduced cells. This is reflected in a significant IFN- and IL-10 production in response to IL-12, and a strongly increased IFN- production in response to IL-12 in synergy with IL-18. These data for the first time prove at the molecular level that C198R is a partially functional mutation. This is consistent with the intermediate clinical phenotype with milder, localized infection that was seen in the patient in whom this mutation was originally identified (12).
The L77P mutation has been found in a patient with disseminated Mycobacterium bovis BCG, Salmonella typhimurium and Paracoccidioides brasiliensis infections (13) and was expected to represent a complete null mutation. Unlike the other amino acid substitutions that result in phenotypical null mutations, however, the L77P protein can be detected at high levels at the cell surface by antibody 2B10 although at a 10-fold lower level than the common IL12RB1 haplotypes. The protein could not be detected by another antibody (2.4E6) directed at IL-12Rß1, suggesting that this amino acid substitution directly or indirectly affects the 2.4E6 binding site. IFN- and IL-10 productions in response to IL-12 (in synergy with IL-18) were negligible. Together these data suggest that, although the L77P protein is expressed at the cell surface, probably at lower levels compared with normal IL-12Rß1, its function is negligible. The L77P substitution therefore most likely directly affects the IL-12 binding site (Fig. 8).
We had previously predicted the functional effect of a number of variations in the IL-12Rß1: S74R, R156H, H438Y, A525T and G594E (8). On the basis of the IFN- and IL-10 production results presented here, we conclude that all five encode fully functional proteins and are expressed abundantly at the cell surface.
A significant difference in IFN- production, cell proliferation and cytotoxicity in response to IL-12 between individuals homozygous for the QMG (allele 1 in 15) or for the RTR (allele 2 in 15) haplotypes was reported by Akahoshi et al. (15). These responses were tested in peripheral blood CD2+ cells (T cells and NK cells) from eight individuals each. They found a higher IFN- response to IL-12 stimulation in individuals homozygous for the QMG allele than in individuals homozygous for the RTR allele. Using cells from individuals, one cannot exclude the possibility that the difference observed between the QMG and RTR homozygotes is caused by confounding variations. By expressing these alleles in identical backgrounds, we now provide the first molecular proof that the QMG allele is indeed a high IL-12 responder allele compared with the RTR allele; this was further substantiated by the observed higher IL-10 production by cells transduced with QMG. Synergy of IL-12 and IL-2 in production of IL-10 has been observed before in human PHA-stimulated T cells, T cell clones and NK cells (4–6,19). We show here that an intact IL-12Rß1 is essential for the production of IL-10 in response to IL-12 and that measuring IL-10 production provides a clear, unambiguous read-out of IL-12R function.
We conclude that a spectrum of functionally different IL-12Rß1 alleles exists, ranging from non-functional alleles without cell surface expression (most mutant alleles), non-functional alleles with cell surface expression (L77P) and partially functional alleles (C198R) to the common haplotypes with either low (RTR) or high responder function (QMG). The different classes of mutant alleles, the two major haplotypes, and likely also as yet unknown more subtle variations, may differentially affect the outcome of inflammatory and infectious disease. Indeed, the RTR allele was already found in a case–control study to be associated with tuberculosis (15). Future studies will shed light on the contribution of these and other subtle IL-12Rß1 variations to the development of inflammatory and infectious disease.
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MATERIALS AND METHODS |
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Patients and controls
Patient 1 has been described before (20
) as having a Q32X null mutation. Cells from three anonymous blood bank donors, BCA, BCC and BCX, who were heterozygous for the common QMG/RTR haplotypes were used as positive controls.
Cloning IL12RB1 variants into a retroviral expression vector
Full-length IL12RB1 coding sequences were PCR amplified using cDNA from healthy controls. The PCR products were first cloned into pGEM-T-Easy (Promega) before transfer into pLZRS–IRES–GFP (21). Variations in IL12RB1 were introduced in the constructs either by digestion/ligation of PCR-amplified material from patients into the pLZRS–IL12RB1 construct or by site-directed mutagenesis (22). As a negative control, pLZRS–IRES–GFP without IL12RB1 insertion is used. All constructs were verified by sequencing. Helper-free recombinant retrovirus was produced after introducing the constructs into a 293T-based amphotropic retroviral packaging cell line, Phoenix (23) using a calcium-phosphate transfection kit (Invitrogen). The virus producing cells were subsequently cultured for 2–3 weeks under 2 µg/ml puromycin (Clontech) selection after which a 20 h supernatant was harvested.
Cells, culture conditions and retroviral transduction
PBMCs were isolated from heparinized blood by Ficoll-Amidotrizoate density gradient centrifugation. Cells were cultured in IMDM (Bio-Whittaker) supplemented with 20 mM GlutaMAX (Gibco-BRL), 10% FCS, 100 U/ml Penicillin, 100 µg/ml Streptomycin (Gibco-BRL) and 30 U IL-2 (Chiron). To generate PHA blasts, PBMCs were stimulated in the previous culture medium supplemented with 800 ng/ml PHA-16 (Murex). On day 2, after PHA-stimulation, 0.5x106 PHA blasts were transduced with virus particles in CH-296 coated (RetroNectinTM, Takara Shuzo) 48-wells plate according to the protocol by Heemskerk et al. (24) with minor modifications. On day 14, cells were FACS sorted on GFP signal after which 0.3–0.5x106 cells were restimulated at least twice with PHA in the presence of 1x106 irradiated allogeneic T cells (pool from five donors) and 0.1x106 irradiated B-LCL. After each restimulation, cells were allowed to proliferate for 14 days.
FACS and functional analyses
Cells were labeled directly with PE-conjugated mouse anti-human IL-12Rß1 mAb 2.4E6 or indirectly with rat anti-human IL-12Rß1 mAb 2B10 (BD Biosciences) in combination with the biotin-conjugated mouse anti-rat mAb G28-5 (BD Biosciences) and streptavidin-PE (BD Biosciences) to determine extracellular IL-12Rß1 cytokine receptor expression by FACS analysis. For intracellular IL-12Rß1 expression, cells were fixated using 4% formaldehyde and permeabilized with 0.1% saponin before labeling with IL-12Rß1 antibodies. Cells were tested for their ability to respond to increasing concentrations of exogenous IL-12 (R&D Systems) and/or 75 ng/ml recombinant human IL-18 (MBL) and to PMA/ionomycin (50 and 100 ng/ml, respectively) (Sigma). The concentration of IFN-, TNF- and IL-10 in the supernatant was determined by cytokine-specific ELISAs (PeliKine/Sanquin). Proliferation was determined using a 3H incorporation assay. Mitogenic stimuli were anti-CD28 (CLB-CD28/1, Sanquin) and anti-CD3 (LUMC pharmacy) or anti-CD2 (CLB-T11.1/1 and CLB-T11.2/1 at 1:1, Sanquin).
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ACKNOWLEDGEMENTS |
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We would like to thank Dr Ozden Sanal (Children's Hospital, Hacettepe University, Ankara, Turkey), Dr Taco Kuijpers (Amsterdam Medical Center, Amsterdam, the Netherlands) and Dr Frank Kroon (Leiden University Medical Centre, Leiden, the Netherlands) for providing patient materials.
Conflicts of Interest statement. The authors have no conflicting financial interests.
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FOOTNOTES |
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Shared senior authorship. 
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