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The Journal of Immunology, 2003, 170: 597-603.
Copyright © 2003 by The American Association of Immunologists

Impaired Accumulation and Function of Memory CD4 T Cells in Human IL-12 Receptor {beta}1 Deficiency1

Aileen M. Cleary*, Wenwei Tu*, Andrea Enright*, Thierry Giffon*, Rene Dewaal-Malefyt{dagger}, Kathleen Gutierrez* and David B. Lewis2,*

* Department of Pediatrics and the Immunology Program, Stanford University School of Medicine, Stanford, CA 94305; and {dagger} DNAX Research Institute, Palo Alto, CA 94304


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Defects in IL-12 production or IL-12 responsiveness result in a vulnerability to infection with non-viral intracellular organisms, but the immunological mechanisms responsible for this susceptibility remain poorly understood. We present an immunological analysis of a patient with disseminated Salmonella enteritidis and a homozygous splice acceptor mutation in the IL-12R{beta}1-chain gene. This mutation resulted in the absence of IL-12R{beta}1 protein on PBMC and an inability of T cells to specifically bind IL-12 or produce IFN-{gamma} in response to either IL-12 or IL-23. The accumulation of memory (CD45R0high) CD4 T cells that were CCR7high (putative central memory cells) was normal or increased for age. Central memory CD4 T cells of the patient and age-matched controls were similar in having a low to undetectable capacity to produce IFN-{gamma} after polyclonal stimulation. In contrast, the patient had a substantial decrease in the number of CCR7neg/dull CD45R0high memory CD4 T cells (putative effector memory cells), and these differed from control cells in having a minimal ability to produce IFN-{gamma} after polyclonal stimulation. Importantly, tetanus toxoid-specific IFN-{gamma} production by PBMC from the patient was also significantly reduced compared with that in age-matched controls, indicating that signaling via the IL-12R{beta}1-chain is generally necessary for the in vivo accumulation of human memory CD4 T cells with Th1 function. These results are also consistent with a model in which the IL-12R{beta}1 subunit is necessary for the conversion of central memory CD4 T cells into effector memory cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The generation of protective Ag-specific CD4 T cells from naive precursors requires engagement of the TCR by specific peptide/MHC complexes as well as an array of other specific interactions between costimulatory molecules and their ligands and between cytokines and their receptors (1, 2). These interactions result in the immediate generation of short-lived, Ag-specific effector CD4 T cells, which participate in the acute immune response, and memory CD4 T cells, which persist as a functional cell population for decades and act promptly and vigorously upon Ag rechallenge (3, 4, 5).

Human memory CD4 T cells can be distinguished from antigenically naive CD4 T cells based on their CD45RAneg/lowCD45R0high and CD45RAhighCD45R0neg/low surface phenotypes, respectively (5). Memory CD4 T cells from adult peripheral blood can be further divided based on surface expression of the CCR7 chemokine receptor (6). Memory CD4 T cells that are CCR7high have been reported to lack expression of the CCR3 and CCR5 chemokine receptors and to produce little or no IFN-{gamma} and IL-4 after stimulation via the {alpha}{beta}-TCR/CD3 complex (6). These have been called central memory cells, based on the prediction that these cells will be similar to naive T cells, which are uniformly CCR7high, in mainly trafficking between the circulation, and lymph nodes and spleen. The importance of CCR7 expression in conferring this trafficking pattern has been directly shown in mice using selective gene disruption (7). In contrast, human CCR7neg/dull memory CD4 T cells were enriched for CCR3 and CCR5 expression, which would be expected to facilitate their entry into infected tissues expressing cognate chemokines for these receptors (8). These CCR7neg/dull CD4 T cells accounted for virtually all IFN-{gamma} and IL-4 production by human CD4 T cells after {alpha}{beta}-TCR/CD3 stimulation (6) and for this reason were termed effector memory cells. These findings as well as the observation of shorter telomere lengths for memory CD4 T cells that are CCR7neg/dull compared with those that CCR7high suggest that the central memory CD4 T cell population may be an intermediate between naive CD4 T cells and effector memory cells (8). However, this model remains controversial (9).

IL-12 is a 70-kDa heterodimeric cytokine composed of a 35-kDa (p35) and a 40-kDa (p40) subunit (10). IL-12 binds to a specific high affinity cell surface receptor composed of a {beta}1 (IL-12R{beta}1) and a {beta}2 (IL-12R{beta}2) subunit (10, 11). IL-12 treatment of T cells results in the expression of multiple proteins that promote Th1 cell differentiation, proliferation, and survival, including IFN-{gamma} and the IL-12R{beta}2 subunit (10). The engagement of the IL-12R on T cells triggers activation of the Janus kinases, Tyk2 and JAK2, which are associated with the IL-12R{beta}1 and IL-12R{beta}2 cytoplasmic domains, respectively (12, 13, 14). This results in phosphorylation and activation of STAT4, which is associated with the IL-12R{beta}2 cytoplasmic domain (15) and which is critical for the generation of Th1 responses in vivo (16).

Recently, a new heterodimeric cytokine, termed IL-23, has been identified that consists of the IL-12 p40 subunit and a novel 19-kDa protein (p19), which is homologous to IL-12 p35 (17). Similar to IL-12, IL-23 stimulates T-lineage cells to produce IFN-{gamma}, appears to bind to the IL-12R{beta}1 subunit with high affinity, and activates STAT4 (17). The IL-23R consists of a newly identified chain, IL-23R, that appears to function in conjunction with the IL-12R{beta}1 subunit (18). This is based on the ability of Abs against either IL-23R or the IL-12R{beta}1-chain to block the IL-23 responsiveness of a NK cell line that naturally expresses both chains or of Ba/F3 cells that express these receptor molecules following transfection (18). However, the role of IL-12R{beta}1 chain signaling in IL-23 responsiveness has not been defined for human T cells.

Targeted gene disruption in mice of IL-12p35, IL-12p40, IL-12R{beta}1, IL-12R{beta}2, or STAT4 has established that all these components are essential for Th1-type adaptive immunity to non-viral intracellular pathogens (19, 20, 21, 22) In humans, deficient production of IL-12 p40 and gene defects in IL-12R{beta}1 have been associated with recurrent or severe bacterial infections, particularly Salmonella and Mycobacteria (19, 23, 24, 25, 26, 27, 28). Although these studies have documented an essential role for IL-12 and the signaling pathway used by IL-12 and IL-23 in the control of intracellular pathogens in mammals, the role of this pathway in memory CD4 T cell generation, particularly in humans, remains unclear.

We have identified a patient with disseminated Salmonella infection and a single-point mutation in the exon 15 splice acceptor site of the IL-12R{beta}1 subunit gene, and present an immunological analysis demonstrating that this mutation results in a quantitative and qualitative deficiency of effector memory (CCR7neg/dull) CD4 T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Human PBMC were isolated by Ficoll-Hypaque density gradient centrifugation and were used directly or primed into T cell blasts by incubation with PHA and recombinant human (rh) IL-2 as previously described (17, 29) Cells were cultured in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (Life Technologies) at 37°C in a 5% CO2 humidified atmosphere.

Cytokine production assay

IL-12 production by PBMC was determined after incubation with 30,000 IU/ml of rhIFN-{gamma} (Intermune, Burlingame, CA) for 18 h, which primes for IL-12 expression, followed by the addition of LPS (Pseudomonas aeruginosa, Calbiochem, La Jolla, CA; 1 µg/ml) for 8 h. To assay for cellular responsiveness to IL-12, the production of IFN-{gamma} by PBMC was assessed after 18 h of treatment with 15 ng/ml rhIL-12 (Genetics Institute, Boston, MA), alone or in combination with bead-associated CD3 and CD28 mAbs. CD3/CD28 mAb beads were produced by covalently conjugating (30) purified mouse anti-human CD3 and CD28 mAbs (BD PharMingen, San Diego, CA) to tosylated paramagnetic beads (Dynal Biotech, Lake Success, NY). To assay for cellular responsiveness to IL-23, the production of IFN-{gamma} by T cell blasts was assessed after 72 h of incubation with 50 ng/ml of rhIL-23 (17) or, for comparison, with 15 ng/ml of rhIL-12. IFN-{gamma} production in response to tetanus toxoid was assessed from cultures of patient and age-matched (aged 4–12 years) control PBMC after 6 days of incubation with 0.2 mM tetanus toxoid (Calbiochem) in RPMI medium with 10% human AB serum. To assay for cellular responsiveness to IFN-{gamma}, the production of TNF-{alpha} by PBMC was determined after their incubation for 2 h with rhIFN-{gamma} (30,000 IU/ml) to prime for TNF-{alpha} production, followed by addition of 1 µg/ml of LPS for an additional 4 h. Cell culture supernatants were assayed by ELISA for IL-12p70 (R&D Systems, Minneapolis, MN), IFN-{gamma} (BD PharMingen), and TNF-{alpha} (BD PharMingen) in triplicate.

Staining for IL-12 binding and IL-12R components

Staining was performed in PBS with 10% heat-inactivated human AB serum and 0.1% sodium azide (staining buffer) at 4°C, with extensive washing with this buffer between each incubation step. To assay IL-12 binding, T cell blasts were sequentially incubated with 30 nM rhIL-12 or, as a negative control, vehicle alone (RPMI medium) for 1 h at 4°C, mouse anti-hIL-12 mAb (IgG1 isotype; Caltag, Burlingame, CA), and PE-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates, Birmingham, AL). To stain for IL-12R{beta}1 surface expression, PBMC were sequentially incubated with goat anti-hIL-12R{beta}1 antiserum or control goat IgG (R&D Systems) and PE-conjugated (Fab')2 donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). To stain for IL-12R{beta}2, T cell blasts were sequentially incubated with mouse anti-hIL12R{beta}2 mAb, 5A7 (IgM isotype) (31), goat-anti-mouse IgM biotin (Caltag), and PE-conjugated streptavidin (Caltag). T cell blasts were used for analysis of IL-12R{beta}2 expression, since this protein is present at low to undetectable levels on freshly isolated PBMC.

cDNA and genomic cloning and sequencing

Random hexamer-primed, reverse-transcribed total RNA was subjected to 35 cycles of PCR (denaturing for 30 s at 95°C, annealing/extension for 3 min at 68°C) using the Advantage 2 system kit (Clontech, Palo Alto, CA) and IL-12R{beta}1 subunit cDNA oligonucleotide primers (nt 7F, 5'-TGAACCTCGCAGGTGGCAGA-3'; nt 2089R, 5'-TCGGGCGAGTCACTCACCCT-3') (32). PCR products were subcloned into a blunt cloning vector (Invitrogen, Carlsbad, CA) and sequenced using the T7 and M13 reverse primers. Genomic DNA was isolated from PBMC using the Puregene DNA Isolation Kit (Gentra, Minneapolis, MN). PCR was performed for 35 cycles (denaturing for 30 s at 95°C, annealing for 45 s at 53°C, extension for 1 min at 72°C) using 100 ng of genomic DNA as template, Taq polymerase (Life Technologies), and genomic sequence primers (forward, 5'-GCACTCCAGCCTGGGCAACAG-3'; reverse, 5'-GCATGTGCACCCAATAAAAAG-3') that flank exon 15 of the IL-12R{beta}1 subunit gene. Primers were designed based on IL-12R{beta}1 gene exon and intron boundaries, as determined by comparison of the sequences of mRNA (U03187) with the genomic chromosome 19 working draft (NT_011288). PCR products were subcloned into a TA cloning vector (Invitrogen) and sequenced.

RT-PCR assay for aberrantly spliced IL-12R{beta}1 transcripts

Random hexamer-primed reverse transcribed total RNA from a healthy control, the patient, and the patient’s mother was subjected to PCR (a touchdown program of 20 cycles of denaturation (15 s at 95°C), annealing (30 s at starting at 60°C, with a decrease of 0.5°C/cycle), and extension (30 s at 72°C)). IL-12R{beta}1 cDNA oligonucleotide primers flanking the exon 15 segment (nt 1753U, 5'-CGTCCTTGGCTACCTT-3'; nt 2068L, 5'-CTGAGCCTCAACGATCACATC-3') (32) were used. Aliquots of the PCR product were electrophoresed using a 2% agarose gel. These primers amplify a PCR product of 315 bp for a full-length IL-12R{beta}1 transcript and a 240-bp product for transcripts with a deletion of the exon 15 segment.

Staining for CXCR3, CCR7, and intracellular IFN-{gamma}

PBMC were stimulated with CD3/CD28 mAb microbeads for 6 h, with 10 µg/ml of brefeldin A (Sigma-Aldrich, St. Louis, MO) present for the last 5 h of the incubation. Stimulated cells were sequentially incubated with mouse anti-human CCR7 mAb (IgM isotype; BD PharMingen), biotinylated rat anti-mouse IgM (BD PharMingen), and a combination of Tricolor-conjugated CD4 mAb (Caltag), PE-conjugated streptavidin and either allophycocyanin-conjugated CD45RA or CD45R0 mAbs (BD PharMingen). Cells were washed, treated with FACS lysis buffer and permeabilizing solution (BD PharMingen), and incubated with FITC-conjugated hIFN-{gamma} mAb (BD PharMingen). Alternatively unstimulated PBMC were stained with a combination of Tricolor-conjugated CD4 mAb (Caltag), PE-conjugated CXCR3 (R&D Systems), allophycocyanin-conjugated CD45RA mAb (BD PharMingen), and FITC-conjugated CD45R0 mAb (BD PharMingen).

Flow cytometric analysis

Stained cells were fixed with 2% (w/v) paraformaldehyde (Electron Microscopy Science, Fort Washington, PA) in PBS and analyzed using a FACScan flow cytometer (BD PharMingen). Lymphocytes or lymphoblasts were included by gating, based on forward and side scatter properties. Circulating memory CD4 T cells were identified by gating on CD4-positive events that were either CD45RAneg/dull or that were CD45R0high. Gating was performed on CD45RAneg/dull events when it allowed a sharper demarcation between naive and memory CD4 T cell populations. Determination of positive cells and mean fluorescence intensity was performed using CellQuest (BD PharMingen) software based on background staining obtained using appropriate fluorochrome-conjugated and isotype-matched control murine mAbs or goat antisera.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A patient with disseminated Salmonella infection

A 6-year-old boy of Iranian descent was evaluated immunologically. He was born in the United States to parents who were first cousins. He had been fully immunized based on current recommendations for the United States without complications, and had not received Mycobacterium bovis bacille Calmette-Guérin (BCG)3 vaccine. He had no significant medical problems until 5 years of age, when he presented with Salmonella group D gastroenteritis, which persisted despite antimicrobial therapy. Eight months later he was brought to surgery for presumed appendicitis, and a large, necrotic mesenteric mass was removed that contained viable Salmonella enteritidis, group D. There was no family history of recurrent infections among his parents, 12-year-old brother, or other relatives. An initial immunological workup revealed that the circulating numbers of total lymphocytes, CD4 and CD8 T cells, and B cells were normal for age. The serum IgG level was elevated (1970 mg/dl) above normal (608–1229 mg/dl), while serum levels of IgM and IgA were within normal limits, as was total hemolytic complement activity.

Absent IL-12- and IL-23-induced IFN-{gamma} production by patient PBMC and T cell blasts

Further immunological evaluation focused on excluding genetic defects in IL-12 production or in cellular responsiveness to IL-12 or IFN-{gamma}, as these disorders have been associated with recurrent and disseminated Salmonella infection (19, 23, 26, 33, 34). PBMC from the patient, after priming by incubation with IFN-{gamma}, secreted equivalent amounts of IL-12 in response to LPS compared with cells from a healthy adult control (Fig. 1GoA), indicating an intact capacity for IL-12 production. The ability of the patient’s PBMC to secrete TNF-{alpha} in response to IFN-{gamma} and LPS was also unaffected (Fig. 1GoB) excluding defects in the IFN-{gamma}R or associated intracellular signaling molecules. However, there were striking differences in the cellular responsiveness of the patient vs controls to IL-12 (Fig. 1GoC). Control PBMC treated with IL-12 secreted IFN-{gamma}, and this was augmented by CD3/CD28 mAb-containing microbeads, which served as a polyclonal T cell stimulus. In contrast, patient PBMC produced almost undetectable amounts of IFN-{gamma} in response to either IL-12 alone or the combination of IL-12 and CD3/CD28 mAb microbeads. This suggested a defect in either components of the IL-12R or its downstream signaling pathway. Neither control nor patient PBMC had detectable levels of IL-4 by ELISA with any of these stimuli, indicating that a lack of response to IL-12 did not result in the accumulation of memory T cells with a Th2 phenotype (data not shown).



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  		FIGURE 1. Patient PBMC and T cell blasts show decreased responses to IL-12 or IL-23. IL-12 (A) and TNF-{alpha} (B) levels in supernatants of PBMC from the patient or a healthy adult control after culture with medium (no additives), IFN-{gamma}, LPS, or both LPS and IFN-{gamma}. C, IFN-{gamma} levels in supernatants from control or patient PBMC cultured with medium, CD3/CD28 beads, IL-12, or both beads and IL-12. D, IFN-{gamma} levels in supernatants from patient or control T cell blasts after culture with medium, IL-12, or IL-23. The mean ± SD are shown ({blacksquare}, control cells; {square}, patient cells). All data are representative of two to six independent experiments. *, p < 0.02 vs the age-matched control based on a two-tailed unpaired Student’s t test.

 
It has previously been shown that IL-23 is similar to IL-12 in inducing IFN-{gamma} production by T cells in a STAT4-dependent manner, and that the human IL-12R{beta}1 subunit confers on transfected heterologous cells the ability to bind IL-23 (17, 18). Treatment of a human NK cell line with IL-12R{beta}1 Ab also inhibits the response to IL-23, suggesting that IL-12R{beta}1 is a component of functional IL-23R (18). To determine whether the defect in the patient also affected IL-23-induced IFN-{gamma} production by T cells, IFN-{gamma} production by T cell blasts derived from adult controls or the patient was compared following IL-23 treatment. Control T cell blasts secreted IFN-{gamma} in response to IL-23, although this was lower in comparison with treatment of an equimolar concentration of IL-12, in agreement with previous results (17) In contrast, T cell blasts from the patient did not secrete IFN-{gamma} in response to either IL-23 or IL-12, indicating a receptor or postreceptor defect that affected responsiveness to both cytokines (Fig. 1GoD).

Decreased IL-12R{beta}1 and IL-12R{beta}2 cell surface expression despite normal levels of mRNA

The inability of patient PBMC or T cell blasts to respond to IL-12 or IL-23 could reflect either receptor defects or post-cytokine binding intracellular defects. We screened for these possibilities by using real-time PCR to determine mRNA expression by PBMC for IL-12R{beta}1, IL-12R{beta}2, and STAT4. The levels of these mRNAs or a positive control transcript ({beta}-actin) did not differ significantly for total RNA from PBMC of the patient vs those of a healthy adult (data not shown). This excluded a defect in the IL-12R{beta}1, IL-12R{beta}2, or STAT4 genes that resulted in either decreased transcription and/or decreased stability of their transcripts.

The patient’s cells were next evaluated for their ability to bind IL-12. T cell blasts derived from the patient had undetectable surface binding of IL-12, while such binding was readily demonstrated on control cells (Fig. 2GoA), strongly suggesting a defect that either compromised IL-12R cell surface expression and/or altered IL-12 binding. PMBC from the patient were completely deficient in surface expression of the IL-12R{beta}1 subunit component, based on the results of staining with a polyclonal antiserum (Fig. 2GoB), suggesting a mutation of the gene encoding this protein. Interestingly, while T cell blasts from both the patient and controls expressed substantial amounts of IL-12R{beta}2 cell surface protein based on staining with mAb, there was clearly less on T cells from the patient (Fig. 2GoB).



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  		FIGURE 2. IL-12 binding and IL-12R expression is reduced on T cell blasts and PBMC from the patient. A, Flow cytometric analysis of T cell blasts that were incubated in the presence ({square}) or the absence ({blacksquare}) of IL-12 and stained for IL-12 surface binding. B, Flow cytometric analysis of IL-12R{beta}1 and IL-12R{beta}2 surface expression ({square}) vs isotype control ({blacksquare}) on control and patient PBMC and T cell blasts, respectively, after immunofluorescent staining. For all histograms, the percentage of cells that stained positive is indicated in the upper right corner. Data are representative of at least three independent experiments.

 
Identification of a splice acceptor point mutation for exon 15 of the IL-12R{beta}1 gene

An examination of IL-12R{beta}1 cDNA sequence derived from patient and control PBMC revealed a complete deletion of exon 15, which encodes the membrane-proximal portion of the cytoplasmic tail and contains the conserved box 1 sequence characteristic of hemopoietin family receptors (35). Sequencing of genomic DNA surrounding exon 15 identified a point nucleotide substitution splice acceptor site (GT to GG) mutation in the patient (Fig. 3GoA). This mutation resulted in the deletion of the segment encoded by exon 15 of the IL-12R{beta}1 subunit gene, accounting for this aberrant exon-splicing event. This aberrant splice also caused a downstream frameshift and premature stop codon in the sequence encoded by exon 16.



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  		FIGURE 3. Splice acceptor mutation of exon 15 of the IL-12R{beta}1 subunit gene. A, Shown is a schematic of the IL-12R{beta}1 subunit detailing the amino acid sequence predicted to be deleted in the patient’s protein. The transmembrane (TM) region, the amino acid sequence motifs (C/CXW and WSXWS), and boxes 1 and 2, all of which are characteristic of hemopoietin cytokine receptor family members are shown, with the conserved box 1 enclosed in a rectangle. Control and patient genomic nucleotide sequences that surround exon 15 are shown, with splice donor and acceptor sites indicated in bold and underlined. Chromatograms demonstrating the acceptor site GT to GG mutation, in the genomic DNA of the patient, are also depicted. B, Agarose gel electrophoresis of PCR products obtained using cDNA from PBMCs as template and oligonucleotide primers for IL-12R{beta}1 cDNA sequence that flank the exon 15 segment. The PCR products shown are: lane 1, healthy control; lane 2, patient; and lane 3, the patient’s mother. The locations of the predicted PCR product of 315 bp, in which the exon 15 sequence is present, and of 240 bp, in which exon 15 is aberrantly spliced, are indicated by arrows.

 
We used a RT-PCR-based assay to confirm that the IL-12R{beta}1 exon 15 segment was skipped for IL-12R{beta}1 transcripts of the patient’s PBMC and to analyze other family members for this mutant allele. Oligonucleotide primers for IL-12R{beta}1 cDNA that flanked the exon 15 segment were designed, and PCR was performed on cDNA obtained from PBMC of a healthy control, the patient, and the patient’s mother. As predicted, using control cDNA as template yielded only the expected 315-bp product, while the patient’s cDNA template resulted in only a 240-bp product that had deleted the exon 15 segment (Fig. 3GoB). The patient’s mother, father, and brother were heterozygous for this mutation, as the 315- and 240-bp products were both present (Fig. 3GoB and data not shown).

IL-12R{beta}1 is necessary for the accumulation of memory CD4 T cells with Th1 effector function

We next determined whether the lack of IL-12R signaling perturbed the accumulation of memory (CD45ROhighCD45RAneg/dull) CD4 T cell subsets by comparing these populations in the patient and age-matched controls. Memory CD4 T cells, which include virtually all CD4 T cells capable of producing IFN-{gamma}, are essentially absent from healthy newborns and gradually increase with age into adulthood (36). Therefore, cells from age-matched donors (age range, 4–15 years) were used as positive controls for these studies. A substantially and consistently lower proportion of memory CCR7neg/dull CD4 T cells (putative effector memory cell subset) (6, 8) was found in the patient compared with all age-matched controls (Fig. 4Go, A and B). Interestingly, based on mean fluorescence intensity measurements, the amount of CCR7 protein on this memory CCR7neg/dull CD4 T cell fraction was higher in the patient than in controls (Fig. 4Go, A and B). In addition, the percentage of memory CD4 T cells that expressed the CXCR3 chemokine receptor, a cell fraction that is enriched in the capacity for IFN-{gamma} production (9), was lower in PBMC from the patient compared with cells from age-matched controls (data not shown).



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  		FIGURE 4. CD4 effector memory cell accumulation and function are impaired in IL-12R{beta}1 subunit deficiency. A, Shown are dot plots from representative age-matched controls (left panels) or the patient (right panels). The upper panels compare CD45RA and CCR7 expression, gating on total CD4 T cells. The lower panels compare intracellular IFN-{gamma} accumulation after 6 h of stimulation with CD3/CD28 mAb beads vs CCR7 expression, gating on memory (CD45RAneg/dull) CD4 T cells. The position of the bars indicates the level of fluorescence that exceeds 99% of cells stained with appropriate isotype-matched controls of irrelevant specificity. Quadrant statistics are indicated. Data are representative of at least four independent experiments. Of note, the lower panels show that the percentage of putative effector memory (CCR7neg/dull) CD4 T cells that secreted IFN-{gamma} was 7.3% for the control vs 0.0% for the patient. B, Overlays of CCR7 expression on patient (solid) and age-matched control (dashed) memory (CD45RAneg/dull) CD4 T cells. C, IFN-{gamma} levels in supernatants from age-matched control or patient PBMC cultured with tetanus toxoid for 6 days. The mean ± SD are shown ({blacksquare}, age-matched control cells; {square}, patient cells). Data are representative of three independent experiments. *, p < 0.05 vs the age-matched control based on a two-tailed unpaired Student’s t test.

 
The capacity of these memory CD4 T cell populations for IFN-{gamma} production was next evaluated. We confirmed a previous report by others (6) that virtually all IFN-{gamma} production by adult human peripheral blood CD4 T cells after engagement of the {alpha}{beta}-TCR/CD3 complex and CD28 is mediated by the memory CCR7neg/dull cell subset rather than by memory cells that were CCR7high (putative central memory cell subset; Fig. 4GoA) (6, 8). However, the reduced, but detectable, CCR7neg/dull subset of memory CD4 T cells of the patient almost completely lacked IFN-{gamma} secretion, while these cells in age-matched control produced robust amounts of this cytokine (Fig. 4GoA).

This generalized reduction in IFN-{gamma} production by memory CD4 T cells from the patient suggested that a functional IL-12R{beta}1 subunit might be of generic importance in the postnatal accumulation of memory CD4 T cells with Th1-type effector function rather than only being required for Th1 responses to non-viral intracellular pathogens. To test this, we examined PBMCs from the patient and age-matched controls, all of whom had received a complete childhood series for tetanus toxoid vaccine, for their CD4 T cell-mediated recall responses to tetanus toxoid. Control cultures of PBMC incubated with tetanus toxoid secreted substantially more IFN-{gamma} than did cultures of PBMC from the patient (Fig. 4GoC). In contrast, PBMC from the patient and controls had similar levels of tetanus-specific cell proliferation, based on cellular incorporation of [3H]thymidine (data not shown). This suggested that clonal expansion of CD4 T cells capable of producing IL-2, but not IFN-{gamma}, occurred normally in the patient. Interestingly, although the patient had a protective (>0.01 IU/ml) Ab level for diphtheria (0.18 IU/ml), he had indeterminate levels for both tetanus Ab (0.33 IU/ml; >0.5 IU protective) and Haemophilus influenzae type b Ab (0.85 µg/ml; >1.0 µg/ml protective). Taken together, these data indicate that lack of a functional IL-12R{beta}1 subunit results in a global defect in the accumulation and function of Th1 memory CD4 T cells, including in the setting of vaccination, and that this is associated with decreased humoral immune responses to T cell-dependent Ags.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified a patient with a splice acceptor mutation affecting exon 15 of the IL-12R{beta}1 subunit gene, which encodes the cell membrane-proximal portion of the cytoplasmic tail of this protein, including the box 1 motif. Box 1 is a conserved, proline-rich, eight-amino acid sequence, which in other hemopoietin receptor family members is important for mitogenesis and inhibition of apoptosis (35) and is likely to mediate similar effects by the IL-12R. This splice acceptor mutation resulted in the complete loss of surface expression of this IL-12R subunit as well as decreased surface levels of the IL-12R{beta}2 subunit. T cells from this patient were unable to bind IL-12 and did not secrete IFN-{gamma} in response to either IL-12 or IL-23, demonstrating that the IL-12R{beta}1 subunit is absolutely required for T cell responsiveness to IL-23 and excluding the possibility of compensation by IL-23R using subunits other than IL-12R{beta}1. Strikingly, this patient also lacked most memory CD4 T cells of the CCR7neg/dull subset of CD4 T cells, and those present had a uniformly and markedly reduced capacity to produce IFN-{gamma}, the signature Th1 cytokine, in response to polyclonal stimulation. In addition, the CD4 T cell recall response to soluble tetanus toxoid was impaired as far as IFN-{gamma} production, consistent with a genetic deficiency of IL-12R{beta}1 resulting in a generalized defect in Th1 cell function, including responses to Ags other than those contained in intracellular pathogens.

Based on in vitro studies, Sallusto and colleagues (6, 8) have proposed a two-step progression from human naive CD4 T cells to central memory cells to memory cells with effector cytokine (Th1 or Th2) function. Our results, in which T cell activation was achieved by engagement of the {alpha}{beta}-TCR/CD3 complex and CD28 molecules, agree with this earlier study, in that IFN-{gamma} production by CD4 T cells from normal donors was completely accounted for by the CCR7neg/dull subset of memory cells. However, a recent study in which adult peripheral blood CD4 T cells were stained for CCR7 and intracellular cytokines after pharmacological stimulation using ionomycin and PMA found that a substantial number of CCR7high memory CD4 T cells expressed detectable IFN-{gamma} protein (9). These discrepant results may reflect the different stimuli used for T cell activation, and there is precedence for alterations in the cytokine profile of human CD4 T cells depending on the mode of T cell activation employed (37).

Our data suggest a general and essential role for cytokine effects dependent on the IL-12R{beta}1 chain in the generation and/or survival of memory CD4 T cells with Th1 effector function. IL-12 or IL-23 signaling, via the IL-12R{beta}1 subunit, is probably responsible for the expansion and postconversion survival of CD4 Th1 cells that have already been committed to the Th1 lineage as a result of effects of master regulatory transcription factors, such as T-bet (1, 38, 39). Consistent with such a role, IL-12 has previously been shown to act as an anti-apoptotic and mitogenic stimulus (1, 16, 39, 40). Further, IL-12 p40 has been shown to be essential in several infection models with intracellular pathogens for sustaining memory and effector CD4 T cells with Th1 function (41, 42, 43). Together, these observations argue that IL-12 may have a role in maintaining a memory CD4 T cell response that is independent of its promotion of IFN-{gamma} expression and Th1 differentiation. Thus, even if alternative IL-12-independent pathways for Th1 differentiation may exist that are mediated by other cytokines, such as IFN-{gamma} or IFN-{alpha}/{beta}, these pathways may not result in normal homeostasis of functional CD4 Th1 memory cells. Studies of the effect of IFN-{gamma} therapy on this patient’s immunological responses and pathogen clearance are in progress.

The markedly decreased capacity of the memory CD4 T cells to produce IFN-{gamma} production compared with that of age-matched controls suggested that a functional IL-12R{beta}1 subunit might be of generic importance in generating and/or maintaining a Th1 response rather than limited to Th1 responses to intracellular pathogens. Such a generic role is supported by the observation that PBMC from the patient also had significantly reduced tetanus toxoid-specific IFN-{gamma} production compared with age-matched controls. Since this recall response is mainly due to memory CD4 T cells, these results indicate that a functional IL-12R{beta}1 subunit is also required for an optimal Th1 response to a purified soluble protein vaccine. Moreover, the observation that Ab titers to both tetanus and H. influenzae type b conjugate vaccine were decreased in this patient suggest that IL-12R{beta}1 signaling may also play a role in the generation of humoral immunity to T cell-dependent Ags. Whether this decrease in Ab titers reflects compromised CD4 T cell help for B cell activation and differentiation or T cell-independent mechanisms, such as decreased IL-12-dependent activation of naive B cells by dendritic cells (44), remains to be determined.

Interestingly, the CCR7neg/dull subset of memory CD4 T cells had increased expression of CCR7 per cell compared with this cell subset in age-matched controls. In addition, the percentage of memory CD4 T cells that expressed CXCR3, a chemokine receptor preferentially expressed by Th1 cells (8, 9), was higher for age-matched controls than for the patient’s PBMC. Together, these data raise the possibility that signaling dependent on the IL-12R{beta}1 subunit may be involved in both down-regulation and up-regulation of particular chemokine receptors in vivo and, thus, has an important influence on the trafficking patterns of CD4 T cells. Others have shown that CCR7high memory CD4 T cells can be induced to proliferate and differentiate into a CCR7neg/dull population using a cocktail of cytokines (IL-6, IL-7, IL-10, IL-15, and TNF-{alpha}) (45). Whether IL-12 also acts directly to decrease CCR7 expression in vivo or, rather, helps maintain memory cells that have previously down-regulated CCR7 remains to be determined.

We found that IL-12R{beta}2 surface expression was reduced on T cell blasts that were genetically deficient in the IL-12R{beta}1 subunit. It is possible that this reduced IL-12R{beta}2 expression may reflect an inability of these cells to produce sufficient IFN-{gamma} to optimally up-regulate IL-12R{beta}2 (46), in accordance with the capacitation/development hypothesis (47). In support of this theory, recent data show that IFN-{gamma} induces both T-bet and IFN-{gamma} production in a positive feedback loop and may act early in T cell differentiation to increase IL-12R{beta}2 (48). Another possibility that is not mutually exclusive with the first is that the presence of IL-12R{beta}1 subunit may be necessary for optimal IL-12R{beta}2 expression secondary to stabilization of the receptor at the cell surface.

Our results clearly demonstrate in humans the apparently absolute requirement of the IL-12R{beta}1 subunit for signaling in response to IL-23, at least in T cells. However, the relative importance of IL-12 vs IL-23 in the generation and/or maintenance of effector memory CD4 T cells remains unclear. An independent role for IL-23 in CD4 Th1 development and effector memory cell generation and function may be elucidated by further experimentation with either IL-12p35 knockout mice or, more directly, p19 knockout mice if these become available. To date, studies of IL-12p35 knockout mice have found a milder course for experimental infections than for IL-12p40 knockout animals (49), suggesting a role for IL-23 in host defense. Since IL-23 shares the IL-12p40 subunit with IL-12, it is likely that humans with IL-12R{beta}1 or IL-12p40 deficiency would have a more severe immunological phenotype than patients with a selective deficiency of the IL-12p35 or the IL-23p19 subunits.

This is, to the best of our knowledge, the first reported case of a patient with a mutation in the cytoplasmic tail of the IL-12R{beta}1 subunit gene. The patient’s mutation carries a 75-bp deletion of DNA in the proximal region of the cytoplasmic tail, which encodes the Box 1 motif, followed by a frameshift mutation that results in a premature stop codon. Despite normal transcript levels, there was no detectable surface expression of the protein encoded by this mutant IL-12R{beta}1 chain allele. Recent studies of the erythropoietin receptor suggest that the docking of JAK kinases with the receptor occur intracellularly before surface expression, and that this interaction may be required for the normal intracellular trafficking of the receptor to the cell membrane (50). It will therefore be of interest to determine whether the mutant IL-12R{beta}1 allele of this patient is expressed at the protein level intracellularly and is compromised in its cellular transport by an analogous mechanism.

In summary, we have found that the absence of the IL-12R{beta}1 subunit in humans results in an inability to generate IFN-{gamma} by T cells in response to either IL-12 or IL-23 and a striking quantitative and qualitative defect in Th1 immunity mediated by memory CD4 T cells.


   Acknowledgments
 
We thank Dr. Jerome Ritz (Dana-Farber Cancer Institute, Boston, MA) for providing the 5A7 mAb, and Dr. Christopher Wilson (University of Washington, Seattle, WA) for providing rhIL-12. We thank Dr. Martin Dahl for critical reading of this manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R01HD36291 and a Jeffrey Model Foundation Center Grant for Research and Clinical Care of Primary Immunodeficiencies (to D.B.L.), a Walter V. and Idun Y. Berry Fellowship in Children’s Health (to A.M.C.), a Stanford Medical School Dean’s Fellowship (to W.T.), and a Siegelman Fellowship for Pediatric Research (to T.G.). DNAX Research Institute is supported by Schering-Plough Corp. Back

2 Address correspondence and reprint requests to Dr. David B. Lewis, Division of Immunology and Transplantation Biology, Department of Pediatrics, CCSR Building, Room 2115b, 269 Campus Drive, Stanford, CA 94305-5164. E-mail address: dblewis{at}leland.stanford.edu Back

3 Abbreviations used in this paper: BCG, bacille Calmette-Guérin; rh, recombinant human. Back

Received for publication August 7, 2002. Accepted for publication November 1, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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