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Isoforms into Multiple DistinctIL12RB1Human

Inflammatory Signals Direct Expression of

T. RobinsonWaukau, Christine Bengtson, John M. Routes and Richard Nicole R. Ford, Halli E. Miller, Allison E. Reeme, Jill

http://www.jimmunol.org/content/189/9/4684doi: 10.4049/jimmunol.1200606September 2012;

2012; 189:4684-4694; Prepublished online 28J Immunol 

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The Journal of Immunology

Inflammatory Signals Direct Expression of Human IL12RB1into Multiple Distinct Isoforms

Nicole R. Ford,* Halli E. Miller,* Allison E. Reeme,* Jill Waukau,† Christine Bengtson,†

John M. Routes,† and Richard T. Robinson*

IL12RB1 is essential for human resistance to multiple intracellular pathogens, including Mycobacterium tuberculosis. In its

absence, the proinflammatory effects of the extracellular cytokines IL-12 and IL-23 fail to occur, and intracellular bacterial

growth goes unchecked. Given the recent observation that mouse leukocytes express more than one isoform from il12rb1, we

examined whether primary human leukocytes similarly express more than one isoform from IL12RB1. We observed that human

leukocytes express as many as 13 distinct isoforms, the relative levels of each being driven by inflammatory stimuli both in vitro

and in vivo. Surprisingly, the most abundant isoform present before stimulation is a heretofore uncharacterized intracellular form

of the IL-12R (termed “isoform 2”) that presumably has limited contact with extracellular cytokine. After stimulation, primary

PBMCs, including the CD4+, CD8+, and CD56+ lineages contained therein, alter the splicing of IL12RB1 RNA to increase the

relative abundance of isoform 1, which confers IL-12/IL-23 responsiveness. These data demonstrate both a posttranscriptional

mechanism by which cells regulate their IL-12/IL-23 responsiveness, and that leukocytes primarily express IL12RB1 in an

intracellular form located away from extracellular cytokine. The Journal of Immunology, 2012, 189: 4684–4694.

The cytokines IL-12 and IL-23 influence numerous aspectsof adaptive and innate human immunity, including thedifferentiation of helper T and NK cell subsets (1–6),

enhancing cytolytic T cell responses (7), promoting B cell pro-duction of select Ig isotypes (8), and regulating dendritic cellactivity (9). The biological activities of IL-12 and IL-23 are de-pendent on IL-12Rb1, a type I integral membrane protein thatserves as a low-affinity receptor for the p40 subunit of bothcytokines (10–12). Reflecting the multifaceted influence of IL-12Rb1 on human immunity, deleterious mutations in the geneIL12RB1 associate with susceptibility to numerous maladies (e.g.,atopic dermatitis, Crohn’s disease, neurofibromatosis type I, andsarcoidosis) (13–17) and intracellular bacterial infections (e.g.,salmonellosis, leishmaniasis, coccidioidomycosis, and mycobac-terial disease) (18–23). In the case of Mycobacterium tuberculosisinfection, mouse models demonstrate that IL-12 is important forT and NK cell secretion of IFN-g; in its absence, activation ofinfected macrophages is reduced and intracellular M. tuberculosisgrowth goes unchecked (24–26). As further evidence of its im-

munological significance, interruption of IL-12/IL-23’s interactionwith IL-12Rb1 is the target of psoriasis treatments ustekinumaband ABT-874 (27–29). Targeting IL-12Rb1 activity via IL-12 isalso being investigated as a cancer therapeutic (30).To relay the immunological effects of IL-12 or IL-23, IL-12Rb1

must both physically associate with each cytokine and signal incomplex with either IL-12Rb2 or IL-23R, respectively (10–12).Although the extracellular portion of IL-12Rb1 contains thecytokine-binding region essential for physical association withIL-12 or IL-23, the cytoplasmic portion acts in concert with IL-12Rb2/IL-23R to transmit intracellular signals via preassociatedJAKs TYK2 and JAK2 (31). TYK2 associates with the cytoplas-mic tail of IL-12Rb1, whereas JAK2 associates with either IL-12Rb2 or IL-23R (31, 32). Reflecting the sensitivity of IL-12Rb1activity to changes in either its extracellular or intracellular re-gions, amino acid substitutions in either of these regions can en-hance or interrupt IL-12Rb1 function (33). Posttranscriptionalmodifications to these same amino acids (e.g., spliceosome-mediateddeletions) are also likely to influence IL-12Rb1 function and,possibly, the efficacy of any current or experimental therapy tar-geting them.Alternative RNA splicing has been demonstrated to produce

cytokine receptors with distinct, isoform-specific functions (34).For example, the IL-1 type II receptor and soluble IL-6Ra aregenerated through alternative RNA splicing and serve, respec-tively, as an antagonist and agonist of their cognate cytokines (35,36). Given the recent observation that mouse leukocytes expresstwo distinct mRNA isoforms from il12rb1 (37) (the mouse ho-molog of human IL12RB1), we sought to determine whether hu-man leukocytes and tissues similarly express more than oneIL12RB1 mRNA isoform. We observed that under both homeo-static and inflammatory conditions, leukocytes and lung tissuestranscribe IL12RB1 into as many as 13 unique mRNA isoforms:2 major isoforms (1 located on the cell surface and another inan intracellular compartment), as well as 11 minor isoforms. Thepredicted amino acid sequences of 12 of these isoforms deviatefrom the IL-12Rb1 amino acid sequence originally reported (10).These isoforms are generated through a combination of alternate

*Department of Microbiology and Molecular Genetics, Medical College of Wiscon-sin, Milwaukee, WI 53226; and †Department of Pediatrics, Medical College of Wis-consin, Milwaukee, WI 53226

Received for publication February 23, 2012. Accepted for publication August 30,2012.

This work was supported by the Medical College of Wisconsin, the Medical Collegeof Wisconsin Center for Infectious Disease Research, Advancing Healthier Wiscon-sin Grant 5520189, and BD Biosciences Immunology Grant 2000212978.

The sequences presented in this article have been submitted to National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/) under accession numberBC029121.

Address correspondence and reprint requests to Dr. Richard T. Robinson, MedicalCollege of Wisconsin, BSB 2nd Floor, 8701 Watertown Plank Road, Milwaukee, WI53226. E-mail address: rrobinson@mcw.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: Ct, threshold cycle; hnRNP, heterogeneous nuclearribonucleoprotein; MCW, Medical College of Wisconsin; PDI, protein disulfideisomerase; STX, syntaxin; UTR, untranslated region.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

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39 splice- and polyadenylation-site activation within the IL12RB1genomic sequence, alternate 39 exon inclusion, exon skipping, par-tial exon inclusion, as well as alternate 59 exon site usage. Fur-thermore, the relative representation of these isoforms varies withactivation or disease status. The variable expression of multipleIL12RB1 isoforms in different tissues and lymphocyte lineagesmay contribute to tissue- and lineage-specific responses to IL-12and IL-23.

Materials and MethodsIsolation of PBMCs

Units of blood from healthy adult donors were collected into sodium citrate-treated bags at the Blood Center of Wisconsin (Milwaukee, WI); donorswere excluded if they were taking (or had taken within 2 wk before col-lection) any of the following categories of immunosuppressant medications:antineoplastic agent, antiviral agent, corticosteroid (either dermatologicalor nondermatological), disease-modifying antirheumatic drug, or immuno-suppressive mAb drugs. After deidentification, blood units were delivered toour laboratory via courier the same day for PBMC isolation. For this, bloodwas diluted 1:2 in sterile PBS (Ca2+/Mg2+ free) and immediately spun overa room-temperature Ficoll-Histopaque 1107 gradient (GE Healthcare,Piscataway, NJ) according to established protocols (38). Buffy coats werewashed twice in PBS, counted, and then used for either total RNA puri-fication, positive selection of leukocyte subsets, or in vitro PHA-P stim-ulation. All studies using human cells and tissues were approved by theMedical College of Wisconsin (MCW) Institutional Review Board. Writ-ten informed consent was received from participants before their inclusionin our study.

Purification of leukocyte subsets

For purifying leukocyte subsets of interest, PBMCs were suspended ina slurry of magnetic bead-Ab conjugates recognizing either human CD4(clone L200), CD8 (clone SK1), or CD56 (clone NCAM16.2) accordingto the manufacturer’s protocols (BD Biosciences, San Diego, CA). Afterpositive selection and two rounds of washing in PBS, subsets were countedand immediately lysed for RNA extraction. Leukocyte subsets were pu-rified away from PBMCs both before and after PBMC culture in PHA tocompare IL12RB1 isoform 1 and 2 mRNA expression levels in lineages ofthe same donor.

In vitro PHA stimulation

After PBMCs were purified, they were cultured in complete RPMI 1640containing CD2-activator (39) PHA-P (1 mg/ml final concentration;Sigma-Aldrich, St. Louis, MO). After 3 d, cells were washed and eitherlysed for RNA or used for leukocyte subset purification as detailed earlier.

Lung tissue specimens

All tissues were supplied by the National Resource Center of the NationalDisease Research Interchange. Relevant donor information is presented inSupplemental Table II. Between 1 and 5 h postmortem, affected areas ofsarcoid lungs (or from control lungs) were resected and snap frozen for theNational Disease Research Interchange repository. Upon transfer to ourlaboratory, these tissues were divided into smaller pieces for RNA ex-traction, ELISA analysis of select cytokine levels, or histological analysis.

RNA extraction

Total RNAwas extracted using the Qiagen RNeasy method (Valencia, CA).For total mononuclear cells and select lymphocyte populations, cells werecounted and pelleted for RNA extraction. For frozen lung tissue, portions ofsarcoid or control lung were placed in a mass-adjusted volume of QiagenBuffer RLT (with 2-ME) and immediately homogenized at high speed usinga Miltenyi gentleMACS dissociator (specifically, using the “RNA 02.01”program).

cDNA synthesis

First-strand cDNA synthesis was performed using Roche Transcriptor HighFidelity cDNA Synthesis Kit (Indianapolis, IN). Anchored poly-dT primers(supplied with the kit) were used to initiate reverse transcription.

IL12RB1 isoform sequencing and amplification

cDNA samples were amplified using the Roche FastStart High Fidelity PCRSystem (Indianapolis, IN) with any of the following primer pairs: 59-IL-

12RB1 and 39-isoform 1 (for amplification of IL-12RB1 isoform 1), 59-IL-12RB1 and 39-isoform 2 (for amplification of IL-12RB1 isoform 2), orrGAPDH_F and rGAPDH_R (for amplification of GAPDH). Because wewere able to amplify the entire 59-untranslated region (59-UTR) with thesame primer for both isoforms, the transcription start site is presumably thesame for both isoforms. The sequences of all primers used can be found inSupplemental Table I. All PCR amplicons were either cloned into bacterialvector pUC19 using standard PCR cloning methods or TA-cloned usingthe Promega pGEM-T system (Madison, WI). Individual clones (∼30 perdonor) were sequenced by Sanger sequencing using an ABI 3730xl DNASequencer (Functional Biosciences, Madison, WI). Chromatograms wereindividually analyzed to ensure the integrity of each sequence (Sequencher5.0; Gene Codes Corporation, Ann Arbor, MI).

Formultiplex analysis of isoform1 and isoform 2 expression levels, 2ml ofeach cDNA sample was placed in a 50-ml reaction comprising the following:10 mMTris-HCl (pH 7.4), 2 mMMgCl2, 50 mMKCl, 200mMdNTPs, 2.5 UTaq polymerase, 0.4mMprimer Ex9_F, 0.1mMprimer Ex9b_R, and 0.4mMprimer Ex11_R. The parameters for amplification were as follows: incuba-tion at 95˚C for 2 min (1 cycle); denaturation at 95˚C for 30 s, annealing at60˚C for 1 min, and extension at 72˚C for 1 min (35 cycles) on a MyCyclerPCR machine (Bio-Rad, Hercules, CA). Products were visualized on a 1%agarose gel using standard electrophoresis methods.

IL12RB1 isoform copy number assay

The mRNA copy number of both IL12RB1 isoforms 1 and 2 was deter-mined by real-time quantitative PCR. A 25-ml reaction was used with2x iQ SYBR Green (Bio-Rad), 5% DMSO, cDNA derived from indicatedcells/tissue, and the following 0.4 mM primers: 12F and 12/13_R foramplification of isoform 1, 9/9bF and 9b_R for amplification of isoform 2(sequences are listed in Supplemental Table I). The parameters for thesereactions were the following: incubation at 95˚C for 3 min (1 cycle); de-naturation at 95˚C for 10 s, annealing at 60˚C for 30 s, and extension at72˚C for 30 s (40 cycles) on iQ5 RT-PCR machine (Bio-Rad). The thresholdcycles (Ct values) for individual amplifications were determined using iQ5software. IL12RB1 isoform 1 and 2 cDNAs cloned into pUC19 served asstandards to calculate mRNA copy number from the Ct values given.Melting curve analysis was used to determine amplification specificity andwas performed immediately after the amplification reaction. The relativeabundance of isoform 1 to isoform 2 was determined via dividing thenumber of isoform 1 mRNA copies in a cDNA sample by the number ofisoform 2 mRNA copies in the same sample.

Determining IL12RB1 mRNA isoform half-life

To determine the relative half-lives of IL12RB1 isoforms in the presence orabsence of PHA, PBMCs were purified as detailed earlier and placed incomplete media in the presence or absence of PHA. Four hours later, eitheractinomycin D (1 mg/ml final concentration) or vehicle control (DMSO)was added to each culture. For a given PBMC preparation, multiple wellswere set up in this manner (5 3 105/well in six-well plates) to collect datafrom three experimental replicates per condition per time point. Cells werecollected at designated times after actinomycin D or DMSO treatment;total RNA was isolated using the Qiagen RNeasy method and frozen forfuture analysis (allowing us to perform, in one setting, the IL12RB1 iso-form copy number assay described earlier with samples from all timepoints). For cDNA synthesis, the concentration of total RNA of eachsample was normalized (as determined via NanoDrop) and cDNA preparedfor all samples using the same lot of reverse transcription reagents. ThemRNA copy number of both IL12RB1 isoforms 1 and 2 was determined asdetailed earlier and normalized to the total concentration of RNA col-lected. Data were analyzed using GraphPad Prism and its bundled softwarevia nonlinear regression analysis; both goodness of fit (R2) and calculatedhalf-life values are indicated.

Cell lines and transfection

HEK293T cells were cultured in DMEM supplemented with 5% newborncalf serum and 5% FBS in the presence of puromycin (Sigma-Aldrich) asneeded. HEK293T cells stably expressing IL12RB1 isoform 1 or 2 cDNAswere generated by Lipofectamine-mediated transfection (Invitrogen,Carlsbad, CA) with the vector pPyCAGIP (40) containing either cDNA(together with their 39-UTRs) under the CAG promoter. Isoforms weresubcloned into a multiple cloning site upstream of an internal ribosomalentry site and puromycin N-acetyl transferase gene (pac) to allow for puro-mycin selection. HEK293T cells stably transfected with empty pPyCAGIPserved as negative controls for some experiments. BSC40 cells (usedfor localization studies) were cultured in DMEM supplemented with10% FBS and transfected with pPyCAGIP-isoform 1, pPyCAGIP-isoform

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2, pPyCAGIP-empty, or pAcGFP-N1 (Clontech, Mountain View, CA)using the Lipofectamine method (Invitrogen). Transient transfectants wereused for immunofluorescent analyses. The leukemic Jurkat T cell line wasused for our initial assessment of IL12RB1 isoform expression (see Fig.1A) and was cultured with or without indicated concentrations of Con A(Sigma-Aldrich) in complete RPMI 1640.

Western analysis

Protein lysates were prepared from indicated cell populations in SDS re-ducing sample buffer, run on 7% SDS-PAGE gels, and transferred ontonitrocellulose membrane via a Semi-Dry Transfer system (Bio-Rad).Membranes were blocked in a milk and TBS solution, and probed witha rabbit polyclonal anti-human IL-12Rb1 Ab (ProteinTech Group, catalogno. 13287-1-AP) generated against an amino acid sequence shared be-tween both IL12RB1 isoforms 1 and 2 (aa 44–381; for the relative locationof these amino acids in isoforms 1 and 2, see Fig. 2B, 2C). After incu-bation with an HRP-conjugated secondary Ab and multiple washes in TBS,blots were exposed to film for indicated lengths of time.

Immunoprecipitation

IL12RB1 isoforms 1 and 2 were immunoprecipitated using the Protein ADynabeads System (Invitrogen). Specifically, cells transfected with bothisoform 1 and 2 cDNAs were puromycin selected (as detailed earlier) andlysed in NP-40 lysis buffer for immunoprecipitation with polyclonal anti–IL-12Rb1/Protein A beads (per the manufacturer’s instructions). Fornegative controls, immunoprecipitates were similarly prepared from bothempty-vector transfectants (i.p. using anti–IL-12Rb1/Protein A) and fromisoform-transfected cells (i.p. using nonspecific IgG/Protein A). Afterimmunoprecipitation, samples were linearized and treated with recombi-nant PNGase F (New England Biolabs), per the manufacturer’s instruc-tions. These treated samples, as well as untreated controls, then weresubjected to Western analysis as described earlier.

Immunofluorescence

BSC40 transfectants grown on glass coverslips werewashed with PBS, fixedwith 4% paraformaldehyde in PBS, permeabilized with 0.5% saponin inPBS and 3% BSA, incubated with primary Abs (polyclonal anti–IL-12Rb1[mentioned earlier], anti-MHC class I [clone W6/32]), or anti-protein di-sulfide isomerase [anti-PDI; clone 34/PDI]), washed, and incubated withAlexa 488- or 594-conjugated secondary Abs (Invitrogen). Cells wereimaged using a Nikon Eclipse TE2000-U inverted microscope, with a 603oil immersion objective, and analyzed using Nikon Elements software(Nikon Instruments, Melville, NY).

Histological analysis

Frozen tissues were sectioned and stained with H&E by the MCWChildren’s Research Institute Histology Core. Visual analysis of serialsections (100 mm apart) was used to confirm the relative abundance ofinflammatory cells in the sarcoid lung compared with control specimens.Images were taken with a Nikon Upright Microscope with PhotometricsCamera and, when necessary, were digitally stitched together with Fiji(ImageJ) to provide high-resolution images of a large area.

ELISA analysis

Lung specimens of similar size were homogenized at high speed in ice-coldPBS using a Miltenyi gentleMACS dissociator (specifically, using the“Protein” program). Homogenized lungs were then centrifuged to separateany debris; the supernatants were then aliquoted for ELISA analysis ofhuman IFN-g, TNF-a, and IL-12p40 levels (BD Biosciences).

Graphing and statistics

Graphs were prepared using Graph Pad Prism version 5.0. Statisticalanalyses used the bundled software of Prism; comparisons involving mRNAlevels of IL12RB1 isoform 1, IL12RB1 isoform 2, or the ratio of these twoisoforms to one another used a two-tailed paired t test. Differences be-tween groups were considered significant if p , 0.05 and are graphicallyindicated by an asterisk.

ResultsPrimary human leukocytes express two major IL12RB1isoforms

Because mouse leukocytes express two distinct il12rb1 isoforms(37), we predicted that human leukocytes would also express morethan one IL12RB1 isoform. Consistent with this, amplification of

cDNA from PBMCs of healthy donors, syngeneic PBMCs stim-ulated with PHA-P, or the Jurkat T cell leukemia line with primersspanning IL12RB1 produced two major isoforms (Fig. 1A): apreviously characterized larger isoform (10) (hereafter referredto as “isoform 1”) and an uncharacterized shorter isoform (“iso-form 2”; Figs. 1B, 2C, 2D) originally cloned from human testes(National Center for Biotechnology Information accession no.BC029121; http://www.ncbi.nlm.nih.gov/nuccore/bc029121).Whereas isoform 1 results from transcriptional inclusion of all 17exons of the IL12RB1 gene (Fig. 2A, 2B), isoform 2 results fromthe inclusion of exons 1–9, an alternate 39-exon and alternate 39-UTR that are otherwise excluded from the sequence of isoform 1(Figs. 1B, 2). This alternate 39-exon (hereafter referred to as “exon9b”) is 125 nt in length, and contains a coding sequence termi-nated by a stop codon and an alternate polyadenylation site (Fig.2D). Although the 39-UTR of isoform 1 is 47 nt long, the 39-UTRadjacent of isoform 2 is 647 nt long and contains severalpyrimidine-rich elements (Fig. 2D). The translated product of theisoform 2 mRNA, as deduced from its sequence, retains the cy-tokine binding region but loses the transmembrane and intracel-lular sequences of isoform 1 (Fig. 2A–C); these lost sequences arereplaced with a distinct, shorter C-terminal sequence (Fig. 2C). Inthis manner, human IL12RB1 isoform 2 differs from its homologin mice (il12rb1Dtm), which is created by the skipping of mouseil12rb1 exon 14 (37). Thus, human leukocytes express two majorisoforms from the IL12RB1 gene, the splicing of which is differentfrom mice because it entails transcription of an alternative 39exon, alternative polyadenylation site, and a unique 39-UTR.

Leukocyte activation alters the relative representation ofIL12RB1 isoforms 1 and 2

Visual analysis of IL12RB1 isoform 1 and 2 expression by syn-geneic PBMCs before and after PHA stimulation suggested thatthe ratio of these two isoforms changes with stimulation status(Fig. 1A, compare lanes 3 and 4). This result would indicate thatthe pathways activated by PHA initiate the alternative RNAsplicing of IL12RB1 isoforms 1 and 2. To quantify these changesand determine whether they could be generalized to PBMCs ofmultiple individuals, we determined via real-time PCR analysisthe mRNA copy numbers of both isoforms before and after PHAstimulation. Real-time PCR allowed us to reliably detect a mini-mum of 102 to 103 mRNA copies for each isoform (SupplementalFig. 1A–C). For isoform 1, PHA stimulation decreased the Ct

value at which this isoform was detected (Fig. 1C), indicating thatPHA increased production of this mRNA. Although occasionallyPHA stimulation decreased the Ct at which isoform 2 was de-tected, the difference was greater for isoform 1 than for isoform2 (compare Fig. 1C with 1D). When expressed as mRNA copynumber, the data demonstrate that although PBMCs from sevenindividuals increased expression of isoform 1 after PHA exposure(Fig. 1E), the relative abundance of isoform 2 remained un-changed (Fig. 1F). As a consequence, the ratio of each isoform tothe other changes in the presence of PHA (Fig. 1G). This changein the relative representation of IL12RB1 isoforms 1 and 2 withPHA exposure was observed across multiple unrelated donors(Fig. 1G) and was not associated with changes in the mRNA half-life of either isoform (Supplemental Fig. 1D–L). We concludefrom these data that PHA-dependent signaling pathways direct thealternative RNA splicing of IL12RB1 isoforms 1 and 2 in PBMCs.

Leukocyte activation alters the appearance of multiple minorIL12RB1 isoforms

In addition to expressing two major isoforms, sequencing ofmultiple mRNA clones before and after PHA exposure revealed

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that PHA also influenced the extent to which IL12RB1 was furtherprocessed into additional minor isoforms. These minor isoformsare listed in Table I together with their associated changes to theamino acid sequence. The frequency with which each minor iso-form appears in the presence or absence of PHA (as a percentageof the total number of clones analyzed) is shown in Fig. 3A and3B. With the exception of minor variant dex16, PHA exposurechanged the relative representation of all minor splice variantsfor isoform 1 (Fig. 3A). PHA exposure also changed the rela-tive representation of half of all minor splice variants observedfor isoform 2 (Fig. 3B). In several instances, more than one mi-nor splicing event was observed in a single clone (Fig. 3C). PHA-driven alternative splicing was gene specific, as multiple GAPDHmRNA clones showed no evidence of alternative splicing ofits nine exons (Fig. 3C). Collectively, these data demonstratethat immunogen exposure induces alternative RNA splicing ofboth IL12RB1 isoforms 1 and 2, with the relative abundance ofboth major and minor isoforms being dependent on activationstatus.

The extent of IL12RB1 RNA splicing is lineage dependent andcan vary between individuals

PBMCs represent an accumulation of multiple distinct leukocytelineages. As with any population-based analysis, changes in geneexpression observed at a total population level may not reflect whatoccurs in select subpopulations. For this reason, we determinedwhether changes in IL12RB1 splicing were observed in CD4+,

CD8+, and CD56+ lineages of the same individual before and afterPBMC culture with PHA. These lineages were chosen because ofthe well-described effects of IL-12 on each (30). The results ofthese experiments are shown in Fig. 4. For two out of three donors,CD4+, CD8+, and CD56+ lineages increased their expression ofisoform 1 relative to isoform 2 after their culture in total PBMC/PHA (Fig. 4A, 4B). For donor A, CD8+ cells expressed the highestratio of isoform 1 to isoform 2 after PHA exposure (Fig. 4A). Fordonor B, CD56+ cells expressed the highest ratio of isoform 1 toisoform 2 after PHA exposure (Fig. 4B). For a third donor, therelative ratio of isoform 1 to isoform 2 in CD4+ and CD8+ cellsactually decreased after PHA exposure, whereas CD56+ cellsexpressed a small increase (Fig. 4C). From these data, we con-clude that although leukocyte activation alters the relative repre-sentation of both major IL12RB1 isoforms, the extent and di-rection of alternative IL-12RB1 splicing (i.e., whether isoform 1or 2 is preferentially expressed after activation) is lineage specific.

IL12RB1 isoforms 1 and 2 have distinct protein characteristics

Because the 39-UTR sequence of IL12RB1 isoform 2 mRNAcontains several pyrimidine-rich elements associated with mRNAdegradation (41) (see highlighted sequences in Fig. 2D), wewished to determine whether this mRNA was translated into aprotein product. For this, the entire IL12RB1 isoform 2 mRNA(with the 39-UTR intact) was cloned into mammalian expressionvector pPyCAGIP (40) upstream of an internal ribosomal entrysite and puromycin-resistance (pac) cassette. Puromycin-resistant

FIGURE 1. Human leukocytes express two major IL12RB1 isoforms. (A) mRNA samples from either PBMCs, PBMCs stimulated with PHA, or Con A-

stimulated Jurkat cells were converted to cDNA and amplified with primers spanning intron and exon sequences. This multiplex amplification produced two

major IL12RB1 isoforms as visualized by agarose electrophoresis (lanes 3–9). No template and no reverse transcriptase controls were also amplified to

confirm cDNA specificity (lanes 1, 2). The amplification products of PBMC cDNA were cloned into pUC19 for downstream sequencing of individual

mRNA clones. (B) Isoform 1 mRNA results from transcriptional inclusion of all 17 exons of the IL-12RB1 gene. Isoform 2 mRNA results from inclusion of

exons 1–8, skipping of exon 9, and inclusion of an intron sequence excluded from isoform 1. This excluded sequence is hereafter referred to as exon 9b. (C

and D) Real-time PCR was used to determine the absolute copy number of both isoforms before and after PHA stimulation. Shown are representative

amplification curves of (C) isoform 1 or (D) isoform 2 before and after PHA exposure. (E–G) Ct values were converted to actual mRNA copy numbers as

described in Materials and Methods; shown are number of (E) isoform 1 and (F) isoform 2 mRNA copies as expressed by the PBMCs of seven separate

individual donors before and after PHA stimulation. Samples are normalized for amount of input RNA. (G) The ratio of isoform 1 to isoform 2 levels both

before and after PHA stimulation as expressed by PBMCs of the same donor; significance was determined using a paired t test (*p # 0.05).

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(pac+) HEK293T cells were subsequently selected and their ex-pression of isoform 2 protein assessed via Western analysis. Blotswere probed with a polyclonal Ab generated against a regionshared between isoforms 1 and 2 (see Materials and Methods).pac+ HEK293T cells expressing isoform 1 or empty vector alonewere also generated and used as positive and negative controls,respectively. As shown in Fig. 5A, both isoform 1 and 2 cDNAsexpressed protein products recognized by polyclonal anti–IL-12Rb1, whereas empty vector containing HEK293T cells did not.

Rather than form discrete bands, all products were characterizedby smears; this was especially true of the 120-kDa product ofisoform 1 and is likely the result of glycosylation. Consistent withthis, six asparagine residues of isoform 1 are predicted glycosyl-ation sites (42) (Fig. 2B); two of these residues are shared withisoform 2 (Fig. 2C). Also consistent with glycosylation is thatPNGase F treatment of isoforms 1 and 2 (immunoprecipitatedfrom cells transfected with both cDNAs) resulted in a more dis-crete electrophoretic banding pattern than that of non–PNGase-treated controls (Supplemental Fig. 2A, 2B). Whereas isoform 1-expressing cells produced two products (∼250 and ∼120 kDa),isoform 2-expressing cells produced three products (∼250, ∼90,and ∼40 kDa; Fig. 5A). Despite loading equal amounts of lysate,isoform 1 consistently reacted more strongly with polyclonal anti–IL-12Rb1 than did isoform 2; this is demonstrated by differentexposure times shown in Fig. 5A (5 min versus 30 s).To determine whether changes in PBMC IL12RB1 isoform

mRNA abundance (Fig. 1E–G) were reflected at the protein level,denatured lysates from untreated and PHA-treated PBMCs weresubjected to Western analysis and stained with polyclonal anti–IL-12Rb1. Lysates prepared from stable HEK293T cells expressingisoform 1 and 2 cDNAs were used as controls. As shown in Fig.5B, PBMC lysates probed with anti–IL-12Rb1 had a differentelectrophoretic pattern than that of either isoform 1- or isoform2-expressing HEK293T cells. In the absence of PHA, PBMCsexpressed a minimum of five different proteins that reacted withpolyclonal anti–IL-12Rb1 (Fig. 5B, bands 1, 2, 3, 5, and 6). In thepresence of PHA, a minimum of six different proteins reacted withpolyclonal anti–IL-12Rb1 (Fig. 5B, bands 1–6). We consideredthe possibility that isoform 2 may be a secreted form of IL-12R.However, attempts to immunoprecipitate isoform 1 or 2 from themedia of either transfected HEK293T cells or activated PBMCswere unsuccessful (Fig. 5C). Similar results were obtained usingnickel-mediated pull-down of a tagged version of isoform 2expressing 6x-His downstream of the N-terminal signal peptide(data not shown). We conclude from these analyses that IL12RB1isoform 1 and 2 cDNAs both express proteins with distinct mi-gration patterns, and that PBMCs express multiple proteins rec-ognized by anti–IL-12Rb1 whose electrophoretic migration pat-tern changes after PHA stimulation.

IL12RB1 isoform 2 localizes to an intracellular compartment

A BLAST search of the C-terminal amino acid sequence of iso-form 2 indicated that this portion of isoform 2 bears homology toproteins known to localize to either the endoplasmic, trans-Golginetwork or intracellular endosomes (Supplemental Fig. 2C), in-cluding VKORC1 (43), SLC48A1 (44), ADRA1A (45), syntaxin 6(STX6) (46), and STX8 (47). These similarities, along with thedistinct electrophoretic patterns of isoforms 1 and 2, led us toquestion whether the cellular localizations of isoforms 1 and 2were also different. BSC40 cells transiently expressing isoforms 1or 2 were stained with anti–IL-12Rb1 and used for direct visu-alization by fluorescent microscopy to test this hypothesis. Todiscriminate between expression on the cell surface and expres-sion inside the cell, cells were also stained with anti-MHC class I.As we anticipated, cells containing only empty vector displayedminimal reactivity with anti–IL-12Rb1 (data not shown), whereasGFP-transfected cells displayed a predominantly nuclear fluores-cence signal (48) (Fig. 6A). In agreement with the ability of iso-form 1 to bind extracellular IL-12 (10), IL12RB1 isoform 1 dem-onstrated a pattern of fluorescence consistent with cell surfacelocalization (compare Fig. 6B with that of MHC class I stainingshown in Fig. 6E). In contrast with isoform 1, isoform 2 localizedto an intracellular compartment within transfected cells (Fig. 6C).

FIGURE 2. IL12RB1 isoform 2 is distinct from isoform 1 and is derived

from alternate 39 exon inclusion. (A) The genomic organization of human

IL12RB1. IL12RB1 is located on autosomal chromosome 19 at location

19p13.1 (1817037-18197697) and comprises exons 1–9, 9b, and 10–17.

The locations of each exon along the IL12RB1 sequence are shown; exons

1–9 and 10–17 are shown as blue boxes, and exon 9b and its 39-UTR are

shown as red boxes. (B) A schematic of the protein domains of IL12RB1

isoform 1 protein as reviewed and modeled in van de Vosse et al. (42). On

the left of the schematic are indicators of which IL12RB1 exons encode

which portions of the isoform protein; to the right of the schematic are the

individual domain names alongside the corresponding amino acid number.

Also indicated by green triangles are predicted glycosylation sites (spe-

cifically, aa 121, 329, 346, 352, 442, and 456 of isoform 1), the relative

locations of the two cysteine pairs (purple) in the first fibronectin type III

(FB1) domain, and the WSxWS motif in the FB2 domain (pink). (C) A

schematic of the protein domains of IL12RB1 major isoform 2 as predicted

from its mRNA sequence. This protein retains the cytokine binding region

(CBR), two potential glycosylation sites, and the WSxWS motif, but

contains a unique C-terminal sequence because of the inclusion of exon 9b

sequence. (D) The mRNA sequence of IL12RB1 exon 9b comprises 125 bp

of protein coding sequence (highlighted yellow with the translated amino

acid sequence shown beneath) that is terminated by a stop codon (under-

lined); this is followed by a 647-bp 39-UTR that contains several pyrimi-

dine-rich regions (highlighted purple) and a polyadenylation site (high-

lighted red). Note that the first nucleotide of the sequence shown (a

guanine; circled) is derived from exon 9 and not exon 9b.

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Isoform 2 staining exhibited a reticular pattern; nevertheless, thispattern was distinct from that observed for ER-specific PDI(Supplemental Fig. 2D). The absence of isoform 2 on the cellsurface was confirmed by examining isoform 2-transfected cellsstained with both anti–IL-12Rb1 and anti-MHC class I (compareFig. 6D with 6E). Therefore, we conclude from our microscopicanalysis that IL12RB1 isoforms 1 and 2 have distinct localization

patterns, with isoform 1 being expressed on the cell surface andisoform 2 being expressed in an intracellular compartment.

Alternative IL12RB1 splicing is characteristic of inflammatoryresponses in vivo

Having observed PHA-responsive IL12RB1 splicing in vitro (Figs.1–4), we wished to determine whether alternative splicing of

Table I. Eleven minor IL12RB1 isoforms are expressed by human leukocytes

Minor IsoformNo. of NTDeleted

No. of NTAdded AA Sequence

DomainAffected

dex3 12 …G----S……GSLPGS…

Pre-CBR

Dex5 140 …SVKYEPPLG……SGRLRTSG*…

CBR (FB1)

dex5 54 …QVGAEVGFRHRTPSSPWKLG……Q------------------G…

CBR (FB1)

dex8 21 …P-------E……PVGLVLIAE…

CBR (FB2)

Dex10 168 …TEPVALNISVGTNGTTMYWPARAQSTTYCIEWQPVGQDRGLATCSLTAPQDPDPAGMA……T--------------------------------------------------------A…

FB4

Dex11 138 …MATYSWSRESGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNA……M----------------------------------------------A…

FB4

Dex13 135 …SEHPVQPTETQVTLSGLRAGVAYTVQVRADTAWLRGVWSQPQRFSIE……S---------------------------------------------E…

FB5

dex13 72 …S------------------------E……SGCGSPAWPLRSKEVPGLRVASPTAE…

FB5

dex16 13 …DRCKAKM*…DRCDR*

INTRA

dex17 143 …KM*…KTRRATISITTAGY*

INTRA

dex16+dex17 13 143 …DRCKAKM*…DRHEERLSQSQRLVIKHLWHTQPIPSTHMIPYQIPTTTP*

INTRA

Listed are each of the minor IL12RB1 isoforms expressed by either unstimulated or PHA-stimulated PBMCs. Five distinguishing features regarding each isoform arecollimated from left to right: in the first column, “Minor isoform,” a name for each minor splice isoform is given referring to the exon affected and whether the splice results ina d (addition to and/or truncation of that particular exon) or D (complete skip of the exon). The second and third columns, “No. of NT deleted” and “No. of NT added,” refer tothe absolute number of nucleotides (NT) deleted or added as a consequence of alternative splicing, respectively. The fourth column, “AA sequence change,” details the specificamino acid (AA) residues subtracted or added to the major isoform AA sequences; the major isoform sequence is listed on top as a reference, whereas the differing minor isoformsequence is listed on the bottom. Asterisks indicate a stop codon. The last column, “Domain affected,” lists the particular domains of isoforms 1 and 2 affected by the AAsequence change (using the domain assignments of van de Vosse et al. [42] as our reference).

CBR, Cytokine binding region; FB, fibronectin domain; INTRA, intracellular, cytoplasmic tail domain; Pre-CBR, preceding the CBR.

FIGURE 3. Activation influences the extent to which both major IL12RB1 isoforms are further processed into additional minor isoforms. Multiple

IL12RB1 cDNA clones were sequenced from the PBMCs (both unstimulated and PHA stimulated) of eight healthy individuals; shown is a dot plot of the

relative representation of each of the minor splice variants shown in Table I among both (A) isoform 1 and (B) isoform 2 clones in the presence or absence

of PHA. Each minor splice variant has a corresponding color that is indicated in the legend; also indicated above each dot plot is the relative placement of

each splicing event along the isoform 1 or 2 mRNA sequence. (C) Several clones of isoforms 1 and 2 were observed to contain more than one splicing event.

As a control, multiple GAPDH cDNA clones from the same cells were subjected to an identical analysis. Shown are the number of splicing events observed

in all IL12RB1 isoform 1, isoform 2, and GAPDH clones analyzed.

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IL12RB1 mRNA characterized inflammatory responses in vivo.For this, we compared the extent of IL12RB1 mRNA splicing inlungs from individuals with sarcoidosis with that observed inhealthy controls. Sarcoidosis is a chronic inflammatory disease ofundefined cause characterized by the infiltration of activated leu-kocytes and granulomatous formations in the lung (49). cDNAfrom affected lung portions was generated and amplified ina manner identical to that done with PBMC cDNA; relevantclinical information on each tissue donor is listed in SupplementalTable II. Consistent with previous reports, sarcoid lung specimenscontained granulomas comprising both lymphocytes and epithe-lioid histiocytes surrounded by extensive fibrosis (Fig. 7B). Thesefeatures were not observed in control lung specimens (Fig. 7A).Sarcoid lungs were characterized by higher levels of TNF-acompared with control lungs (Fig. 7C); IL-12p40 and IFN-g levelsvaried between the tissues we examined (Fig. 7D, 7E). Associatedwith this inflammatory response in lung sections from two dif-ferent sarcoidosis patients (from tissue immediately adjacent tothat histologically analyzed in Fig. 7B) was an increased ratio ofIL12RB1 isoform 1 to isoform 2 (Fig. 7F, 7G) compared withcontrol lungs. Although isoform 1 comprised 15–26% of allIL12RB1 expression in control sections (Fig. 7F), it comprisedapproximately twice as much (34–60%) in sarcoid sections. Weconclude from these data that, similar to in vitro responses toPHA, alternative IL12RB1 splicing is characteristic of inflamma-tory responses in vivo.

DiscussionIL12RB1 is critical for IL-12– and IL-23–dependent leukocyteresponses: CD4+ Th cells of individuals homozygous for non-functioning IL12RB1 (i.e., IL12RB1mut) alleles exhibit defec-tive Th1 and Th17 polarization (19, 20, 50), NK cells fromIL12RB1mut individuals are hyporeactive (6), and the frequency ofCD56+ T cells in the circulation of IL12RB1mut individuals is low(6). These combined immunological defects leave IL12RB1mut

individuals more susceptible to infection by mycobacteria, Sal-monella, and Candida species, as reported in a recent extensiveinternational study (20). Smaller and more regional studies havealso shown positive associations between IL12RB1 deficiencies(13, 14, 21, 23, 51–53) or IL12RB1 polymorphisms (16, 22, 54–57) and susceptibility to a number of other maladies and infectiousdiseases. However, notably, beneficial IL12RB1 polymorphismsalso have been discovered (58, 59). It is essential that we under-stand IL12RB1’s basic functions and regulation to fully under-stand how IL12RB1 modulates immunity and how we can use the

expression of this gene or its ligands IL-12 and IL-23 for thera-peutic purposes.In this study, we have demonstrated that 13 distinct IL12RB1

mRNA isoforms are expressed by PBMCs of healthy individuals:

2 major isoforms (isoforms 1 and 2) and 11 minor isoforms (dex3,

Dex5, dex5, dex8, Dex10, Dex11, Dex13, dex13, dex16, dex17,

and dex16+dex17). The relative abundance of each isoform changes

with leukocytes’ activation state (both in vitro and in vivo), as well

as the specific leukocyte lineage or tissue examined. Based on

these data, as well as the observation that isoform 2 localizes

intracellularly, we propose the following model of IL12RB1 post-

transcriptional regulation in leukocytes. Under homeostatic con-

ditions, IL12RB1 is expressed at basal levels predominantly in the

form of isoform 2. This isoform lacks any signaling domains and

is sequestered intracellularly with limited access to extracellular

cytokine (i.e., IL-12 or IL-23). Upon cellular activation, a modi-

fication of RNA splicing occurs, resulting in the preferential ex-

pression of isoform 1 over that of isoform 2. Isoform 1 localizes to

the outer membrane, where it comes into contact with cytokine

and confers cytokine responsiveness. Any changes in the relative

abundance of each isoform by an individual cell likely “tunes” its

response to IL-12/IL-23 in a manner that promotes immunity as a

whole.Because IL12RB1 is a well-known regulator of both adaptive

and innate human immune responses, it is surprising that the

majority of isoforms observed have not been reported previously.

For .20 y, phenotypes associated with IL12RB1mut and poly-

morphic IL12RB1 alleles have been interpreted on the assumption

that only one protein product is expressed from a normal IL12RB1

allele; this protein, IL-12Rb1, corresponds to isoform 1 of our

study and was originally cloned by Chua et al. (10). One possible

reason isoform 1 has been the only IL12RB1 isoform reported to

date was the method of its discovery. This first IL-12R component

was cloned by Chua et al. (10) from COS cells transfected with

a cDNA library from PHA-activated PBMCs; a COS clone ex-

hibiting both surface reactivity with mAb 2.4E6 and an affinity

for extracellular IL-12 was chosen for sequencing (10). Rigorous

studies since that time have shown that isoform 1 is integral to the

plasma membrane (60, 61), binds the p40 domain of IL-12/IL-23

(11, 62), associates with IL-12Rb2/IL-23R to form high-affinity

complexes for IL-12/IL-23 (12, 32), and, because it lacks intrinsic

kinase activity, relies on its association with cytoplasmic Tyk2 for

relaying IL-12 signals (31, 63). However, the original cloning

scheme used by Chua et al. (10) excluded any clones not having

reactivity with the Ab 2.4E6 or extracellular IL-12; consequently,

FIGURE 4. The extent of IL12RB1 RNA splic-

ing is lineage dependent and can vary between

individuals. PBMCs of healthy donors were stim-

ulated with PHA; before and after PHA stimula-

tion, CD4+, CD8+, and CD56+ lineages from the

same PBMC pool were magnetically sorted, and

the levels of IL12RB1 isoform 1 and 2 expression

were determined using real-time PCR. (A–C)

Shown for three individual donors (donors A, B,

and C, respectively) are pie graphs demonstrating

the relative expression levels of isoform 1 and 2

levels in each lineage, both before and after PHA

exposure. The number at the upper right of each

pie graph is the percentage of isoform 1 as a func-

tion of total IL12RB1 transcript levels (i.e., [iso-

form 1 copy number]/[isoform 1 + isoform 2 copy

number]).

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any clones without these reactivities because of either an absenceor shielding of the epitope recognized by 2.4E6, or intracellularlocalization, went unsequenced and uncharacterized. Chua et al.(10) did make mention of a second transcript expressed byPBMCs that can be discriminated by Northern blot via its bindinga cDNA probe specific to the exons encoding the extracellularportion of IL-12Rb1; however, this transcript did not bind a cDNAprobe specific to region encoding the cytoplasmic domain of IL-12Rb1 (10). This transcript was left uncloned and, in hindsight,likely corresponds to either isoform 2 or one of the other isoformsdescribed in this article.Given both the significance of IL12RB1 expression to human

health and our observation that this gene is primarily expressed asheretofore uncharacterized isoform, determining the function ofisoform 2 as it pertains to IL-12– and IL-23–dependent immunityis the obvious next step for future investigations. Specifically, itshould be determined whether isoform 2 competes with isoform 1for access to IL-12Rb2/IL-23R, impacts IL-12/IL-23 binding, orregulates Th1/Th17 lineage commitment. Because isoform 2localizes to an intracellular compartment, it presumably has lim-ited contact with cytokine at the cell surface. We considered thepossibility that isoform 2 was secreted, which would allow it tocome into contact with extracellular IL-12/IL-23; however, im-munoprecipitation attempts from the supernatants of either acti-vated PBMCs or transfected HEK293T cells failed. The homologyof isoform 2’s C terminus to the N terminus of STX6 and STX8(Supplemental Fig. 2C) may prove informative to future func-tional studies of isoform 2. Namely, the N terminus of STX tethersSNARE proteins (expressed on vesicles) to the protein MUNC18(64), which is expressed on target membranes; after SNARE/MUNC18 interaction, vesicle/membrane fusion occurs and vesi-cle contents are released across the target membrane (65, 66). Thisinformation, when combined with the observation of Durali et al.(67) that primary lymphocytes can internalize IL-12, suggests thatisoform 2 may play a role in either recycling internalized IL-12back into the extracellular compartment or promoting a yet to bedefined intracellular IL-12R system. Testing this hypothesis willbe largely dependent on our generating an Ab specific to the Cterminus of isoform 2 and examining the consequences of isoform2 silencing in primary lymphocytes. Also still to be determined iswhat posttranslational modifications (in addition to glycosylation)

FIGURE 5. IL12RB1 isoforms 1 and 2 have distinct protein character-

istics. (A) Denatured lysates of HEK293T cells stably expressing either

IL12RB1 isoform 1, isoform 2, or empty vector were subjected to SDS-

PAGE (7%); subsequently generated blots were probed with polyclonal Ab

generated against an amino acid sequence shared between both isoforms.

Shown is a blot representative of three separate experiments; two exposure

times (5 min and 30 s) are shown to demonstrate the differential reactivity

of anti–IL-12Rb1 with isoforms 1 and 2. (B) An identical analysis is

performed with denature lysates of PBMCs (of the same donor) collected

before (2PHA) and after PHA exposure (+PHA). (C) The supernatants of

HEK293T cells stably expressing empty vector, isoform 1, or isoform 2

were subjected to immunoprecipitation with anti–IL-12RB1/Protein A

beads. Supernatants from the PHA-stimulated PBMCs of three unrelated

donors (as well as FBS containing media alone) were similarly immuno-

precipitated. The resulting pulled-down complexes were denatured and

analyzed as done for (A) and (B). Cellular lysates from HEK293T cells

stably expressing isoform 2 served as a positive control (left lane, indicated

by an arrow left of blot). All blots are representative of either three (A, B)

or five (C) separate experiments.

FIGURE 6. IL12RB1 isoforms 1 and 2 have distinct localization pat-

terns. BSC40 cells expressing (B) IL12RB1 isoform 1 or (C–E) isoform 2

were dual labeled with anti–IL-12RB1 and anti-MHC class 1. As a positive

control for fluorescence, a separate group of BSC40 cells (A) expressing

eGFP was also visualized. Shown are representative images taken at 360

magnification (under oil immersion) demonstrating the localization pat-

terns of (A) eGFP, (B) IL12RB1 isoform 1, and (C) IL12RB1 isoform 2.

Shown in (D) and (E) is the dual-labeling of isoform 2-expressing cells

stained for (D) IL12RB1 isoform 2 and (E) MHC class I. Results are

representative of ∼20 cells examined per transfection condition as done for

three separate experiments.

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account for the discrepancy between the isoform 1’s predictedmolecular mass (∼73 kDa based on amino acid compositionalone) and its actual mass (∼100–120 kDa) as estimated by bothus (Fig. 5) and Chua et al. (10).Regarding changes in relative isoform abundance, immunogen

exposure altered the relative abundance of both major and minorIL12RB1 isoforms in leukocytes. In vitro, this was accomplishedby culturing PBMCs in the presence of PHA. In vivo, an alteredIL12RB1 isoform 1/2 ratio was characteristic of sarcoid lunggranulomas. Although the inflammatory stimulus/stimuli respon-sible for pulmonary sarcoidosis has yet to be determined defini-tively, candidates include both viral and bacterial organisms(including mycobacteria) (49). We conclude from both our in vitroand in vivo data that extracellular signals induce alternativesplicing of IL12RB1. Although alternative splicing of genes iscommon in human leukocytes, incidents of signal-induced alter-native splicing in this population are relatively rare (68). CD44and PTPRC (i.e., CD45) are two other human immune-associatedgenes that undergo signal-induced alternative splicing (69, 70).Regarding the mechanism by which mitogen signals the alterna-tive splicing of CD45, splicing of CD45 is dependent on thespliceosome component PSF (71). In the absence of PMA, thekinase GSK3 phosphorylates PSF, hindering the association ofPSF with the CD45 pre-mRNA (71). Upon stimulation with PMA,however, GSK3 activity is reduced, allowing PSF to associate withthe CD45 pre-mRNA and promote exon exclusion (71). Whethera similar GSK3/PSF-dependent mechanism governs signal-in-duced IL12RB1 splicing will be a focus of future experiments.Another possibility is the involvement of heterogeneous nuclearribonucleoproteins (hnRNPs) (68, 72–74). hnRNPs are known tobe phosphorylated, sumoylated, and arginine methylated (75–78);PHA possibly signals a modification of hnRNP H that altersits poly(A)-promoting activity through associated poly(A) factorsCstF or poly(A) polymerase (72, 73, 79) and consequently de-creases the relative success of poly(A) addition to isoform 2.Finally, we cannot rule out the possibility that IL12RB1 promoterusage varies after cell activation. In mice, the cis elements IRE/ISRE and ETS/PU.1 are located 2508-bp upstream of the il12rb1transcriptional start site (80); these elements promote il12rb1isoform 1 transcription via their recruitment of IRF3 and PU.1,respectively. Because isoform 2 shares the first nine exons ofisoform 1, its transcription is also likely governed by the IRE/ISRE and ETS/PU.1 elements. However, it is possible that addi-tional, as yet unidentified promoters and trans factors promote theactivation of the 39 splice site and polyadenylation site withinintron 9, which terminates the transcript with what we refer to asexon 9b (Fig. 2). Most examples of this form of alternative pro-cessing involve cis elements within the affected exon and theflanking introns (79, 81, 82).Because polymorphisms in IL12RB1 associate with a range of

human maladies and infectious diseases (16, 22, 54–57), it willbe important to determine, in a manner analogous to studies donewith CD45 (83, 84), how these same IL12RB1 polymorphismsinfluence exon inclusion. Furthermore, because modulators of IL-12/IL-23 signaling are being used as approved and experimentaltherapies for a variety of clinical applications (27–30, 85, 86),including vaccine adjuvants (87), it will also be important to de-termine whether differences in IL12RB1 isoform prevalence un-derlie person-to-person variation in IL-12 and IL-23 responses.

AcknowledgmentsWe thank departmental colleagues Dr. Mark McNally and Stephen Hudson

for helpful discussions regarding alternative splicing, Christine Naughton

of the MCW Children’s Research Institute Histology Core, and Matthew

FIGURE 7. Alternative IL12RB1 splicing is characteristic of inflammatory

responses in vivo. (A and B) Frozen sections from either (A) control or (B)

sarcoid lungs were H&E stained to assess the degree of pulmonary inflam-

mation present. Shown are representative micrographs of lungs at34 magni-

fication; regions selected for closer visualization are boxed and shown as310

magnification insets. Areas of lung adjacent to those shown in (A) and (B)

were homogenized in either PBS for (C–E) ELISA determination or (F–G)

RNA extraction buffers for high-fidelity cDNA synthesis and subsequent real-

time PCR analysis. (C–E) For ELISA analysis, lysates were assayed for their

expression of (C) IFN-g, (D) TNF-a, and (E) IL-12p40; data are expressed as

the total amount of cytokine present and are shown for each individual donor.

(F–G) For real-time analysis, the absolute copy number of both IL12RB1 iso-

forms 1 and 2 was determined for (F) control and (G) sarcoid lungs (two sec-

tions of lung per donor were assayed). Resultant data are represented as the

ratio of each isoform to the other; the percentage of isoform 1 as a function of

total IL-12RB1 transcript levels is also shown for each lung. Al, Alveoli; Br,

bronchiole; EH, epithelioid histiocytes; F, fibrosis; L, lymphocytes; Vs, vessel.

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Forsberg of the Interdisciplinary Program in Biomedical Sciences. We

thank Drs. Amy Hudson and Joseph Barbieri for sharing invaluable exper-

tise in immunofluorescent staining and analysis, as well as insightful dis-

cussions of our data. We also thank Dr. Diane Rodi and Rebecca Seevers

for the excellent biosafety training they have provided to our laboratory.

DisclosuresThe authors have no financial conflicts of interest.

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