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The Journal of Immunology, 2008, 180, 1060 -1070
Copyright © 2008 by The American Association of Immunologists, Inc.
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Polymorphisms in the CD3Z Gene Influence TCR{zeta} Expression in Systemic Lupus Erythematosus Patients and Healthy Controls1

Claire L. Gorman2,3,*, Andrew I. Russell2,{dagger}, Zhuoli Zhang*, Deborah Cunninghame Graham{dagger},*, Andrew P. Cope4,* and Timothy J. Vyse4,{dagger}

* Kennedy Institute of Rheumatology and {dagger} Molecular Genetics and Rheumatology Section, Faculty of Medicine, Imperial College London, Hammersmith Hospital, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TCR{zeta} (CD247) functions as an amplification module in the TCR signaling cascade and is essential for assembly and surface expression of the TCR/CD3 complex. The TCR{zeta}-chain is down-regulated in many chronic infectious and inflammatory diseases, including systemic lupus erythematosus (SLE). It is unclear whether reduced TCR{zeta} expression is a cause or a consequence of chronic inflammatory responses. We have addressed this question by adopting a combined genetic and functional approach. We analyzed TCR{zeta} protein expression using a FACS-based expression index and documented considerable, but longitudinally stable, variation in TCR{zeta} expression in healthy individuals. The variation in TCR{zeta} expression was associated with polymorphisms in the CD3Z 3'-untranslated region (UTR) in SLE patients and healthy controls. Detailed mapping of the 3'-UTR revealed that the minor alleles of two single nucleotide polymorphisms (SNPs) in strong disequilibrium (rs1052230 and rs1052231) were the causal variants associated with low TCR{zeta} expression (p = 0.015). Using allelic imbalance analysis, the minor alleles of these 3'-UTR SNPs were associated with one-third of the level of mRNA compared with the major allele. A family-based association analysis showed that the haplotype carrying the low-expression variants predisposes to SLE (p = 0.033). This suggests that a genetically determined reduction in TCR{zeta} expression has functional consequences manifested by systemic autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The TCR is a multisubunit complex, comprising at least eight transmembrane units. The clonotypic TCR {alpha}- and β-chains are responsible for Ag recognition (1), while the invariant chains of the CD3 complex ({delta}, {epsilon}, and {gamma}) and two {zeta} polypeptides couple Ag recognition to downstream signal transduction pathways. The invariant chains possess ITAMs; the CD3 {gamma}, {delta}, and {epsilon} subunits each have one ITAM, while each {zeta}-chain possesses three. Thus, TCR{zeta} (CD247) functions as an amplification module in the TCR signaling cascade (2). The {zeta}-chain is also essential for the assembly and surface expression of the TCR/CD3 complex, as demonstrated in studies of T cells from TCR{zeta}-deficient mice (3). Indeed, biochemical studies have indicated that the synthesis of TCR{zeta} proceeds at a rate ~10% that of other TCR subunits; thus, surface expression of functional TCR/CD3 complexes depends to a large extent on de novo synthesis of TCR{zeta} (4). Because TCR{zeta} is also expressed by NK cells as a component of the activating receptors NK cell protein 46, NK protein 30, and CD16 (Fc{gamma}RIII, low-affinity Fc receptor for IgG,), it follows that changes in TCR{zeta} expression could have profound effects on innate as well as adaptive immune responses (5, 6).

The human TCR {zeta}-chain gene (CD3Z) spans ~88 kb of DNA and has been mapped to chromosome 1q24.2 (www.ensembl.org). The size of the gene is accounted for by a large first intron of ~77.6 kb. CD3Z is composed of eight exons separated by distances of between 0.7 and 8 kb (7, 8). The spliced mRNA product is 1.492-kb long, comprising a 492-bp coding region and a 3'-untranslated region (3'-UTR)5spanning 906 bp. Expression of the TCR{zeta} gene is regulated at transcriptional, posttranscriptional, and posttranslational levels. For example, the proximal promoter region includes two binding sites for the transcription factor E-74-like factor (Elf)-1, shown to be essential for transcription (9). Defects in Elf-1 expression have been associated with down-regulation of constitutive TCR{zeta} mRNA production (10). In contrast, TCR engagement induces rapid internalization of TCR subunits (including the {zeta}-chain) and targeting for degradation in the lysosome (11). Loss of TCR{zeta} expression has also been linked to ubiquitination (12, 13) and both granzyme B- (14, 15) and caspase-mediated degradation (16, 17). However, a growing body of evidence now suggests that posttranscriptional regulation is an important component of TCR{zeta} gene regulation. The 3'-UTR of mRNA plays a key role in posttranscriptional gene regulation by affecting mRNA stability, localization, and transport. Several recent studies highlighting the effects of alternatively spliced 3'-UTR variants on TCR{zeta} mRNA expression have strengthened this concept (18, 19, 20, 21).

The TCR{zeta}-chain is down-regulated in a diverse range of disease states; these include autoimmune diseases such as systemic lupus erythematosus (SLE) (22, 23, 24) and rheumatoid arthritis (RA) (25, 26), neoplastic conditions such as gastric and colonic carcinoma (27), and chronic infections such as leprosy, tuberculosis (28), and HIV (29). T cells found in such conditions are hyporesponsive to stimulation in vitro with Ag or mitogens, implying that TCR{zeta} down-regulation is functionally significant (30, 31). Indeed, synovial fluid T cells in RA, which are also hyporesponsive to TCR ligation, express a lower level of TCR{zeta} than PBLs from the same patient (25, 32). However, whether TCR{zeta} down-regulation is the result of or a perpetuating factor in the cycle of chronic inflammation remains to be determined. We have recently explored the relationship between loss of TCR{zeta} expression and T cell function; we have found that the T cell population expressing low levels of TCR{zeta} (TCR{zeta}dim cells) is enriched for a subset of Ag-experienced memory effector T cells (based on tetramer and cell surface markers) that, while paradoxically hyporesponsive to TCR signaling, express inflammatory cytokines and have an enhanced migratory capacity in vitro and in vivo (33).

SLE is an autoimmune rheumatic disease characterized by both autoantibody production and T cell dysfunction. Multiple genes contribute to the development of SLE, and chromosome 1q22–24 has been linked to disease in genome-wide scans of multicase families (34). Because CD3Z is located within this linkage region, it has been postulated that polymorphism at the CD3Z locus may be implicated in the pathogenesis of SLE. Indeed, many studies have shown that T cells from patients with SLE exhibit both quantitative and qualitative differences in TCR{zeta} expression and function when compared with healthy controls. For example, TCR{zeta} cDNA from SLE patients contains more frequent polymorphisms and aberrantly spliced forms (35, 36). To date, however, the coding region of the CD3Z gene in SLE has been found to be nonpolymorphic (37). Much more revealing, nevertheless, is analysis of the 3'-UTR; in addition to single nucleotide polymorphisms (SNPs), an alternatively spliced variant of the 3'-UTR has been identified in patients with SLE. This 344-bp product (compared with the wild-type 906-bp fragment) results from the deletion of nt 672–1233 in exon VIII and leads to reduced mRNA stability (18, 19, 20, 21). A further TCR{zeta} mRNA splice variant characterized by an exon VII deletion has also been identified in SLE patients; this transcript is also more unstable than wild-type mRNA (38, 39). Although none of these abnormalities have been clearly associated with SLE susceptibility, they do implicate a role for mRNA instability imposed by 3'-UTR variants in the mechanism of TCR{zeta} down-regulation.

Because depressed levels of TCR{zeta} are not specific to one particular disease, it remains unclear whether TCR{zeta} dysfunction is a primary inciting event or arises as a consequence of chronic inflammatory responses. We have addressed this question by taking a combined genetic and functional approach. We have conducted a mutation screen of the CD3Z genomic coding sequence followed by a family-based association study in SLE using polymorphisms within coding and noncoding sequence, substantially expanding upon previous attempts to investigate this gene in SLE susceptibility (37). We have subsequently analyzed TCR{zeta} protein expression using a flow cytometry-based expression index and attempted to associate this with CD3Z polymorphism in patients with SLE and also in healthy controls. After a primary SNP screen suggested a link between CD3Z 3'-UTR polymorphism and low TCR{zeta} expression, we went on to perform a more detailed analysis of allelic variation of the 3'-UTR and its possible functional association with TCR{zeta} mRNA and protein expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
SLE patients, families, and healthy controls

We have established a large collection of genomic DNA and sera from U.K. simplex SLE families. The study cohort includes a random sample of 452 European-Caucasian SLE families that have been described in detail elsewhere (40). All patients satisfied the American College of Rheumatology classification criteria for SLE (41). The study protocol was approved by the London Multicentre Research Ethics Committee.

Peripheral venous blood was drawn from 30 consecutive SLE patients attending the Hammersmith Hospital SLE Clinic (aged between 18 and 56 years, mean 33.7 years) and 60 healthy donors (aged between 23 and 45 years, mean 30.3 years) using an identical protocol. Ninety-three percent of the SLE patients and 62.5% of the healthy controls were female. Ten of the SLE patients had severe lupus complicated by renal and/or neurological involvement, whereas the remaining 20 had a milder form of SLE with cutaneous disease and joint symptoms.

Human lymphocytes

PBLs were obtained by Ficoll-Hypaque density gradient centrifugation from heparinized peripheral venous blood immediately following venesection. Aliquots were prepared and frozen in liquid nitrogen until required for FACS analysis (multiple samples were analyzed simultaneously to reduce batch effects). Genomic DNA was extracted from an aliquot of ~5 x 106 cells by isopropanol/chloroform separation. DNA purity was checked and quantified by an ND-1000 spectrophotometer (NanoDrop Technologies).

Antibodies and chemokines

The following Abs were purchased from BD Biosciences Pharmingen: mouse IgG1-PerCP, anti-CD3-PerCP, anti-CD4-PerCP, and anti-CD8-PerCP. Mouse IgG1-PE and anti-TCR{zeta}-PE (TIA-2, which recognizes a cytoplasmic domain epitope) were purchased from Immunotech (Beckman Coulter).

TCR{zeta} expression analysis

Because the TCR {zeta}-chain has a short nine-amino acid extracellular domain, mAbs that detect the intracellular cytoplasmic domain epitopes after fixing and permeabilization were used to analyze TCR{zeta} expression (TIA-2; Beckman Coulter), as described previously (33). Surface staining of T cell subsets was performed by standard methods. Isotype-matched control Abs were used to confirm expression specificity.

Analysis was performed with an LSR FACScan flow cytometer (BD Biosciences) and CellQuest software (BD Biosciences) using the TCR{zeta} expression index that we have developed. This is based upon two independent sets of variables, as previously described (33) (Fig. 1A): 1) constitutive expression of TCR{zeta} is determined by calculating the ratio of the mean fluorescence intensity (MFI) of the TCR{zeta}+ population to the MFI of the TCR{zeta}– population, comprising B cells and monocytes; and 2) the ratio of the number of circulating TCR{zeta}bright to TCR{zeta}dim cells is calculated. These indices can be determined separately for CD3+, CD4+ and CD8+ lymphocyte subsets. Differences in TCR{zeta} expression between different CD3Z genotypes were determined using the Mann-Whitney U test.


Figure 1
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  		FIGURE 1. Heterogeneity of TCR{zeta} expression in PBL from healthy donors. A, The TCR{zeta} expression index is determined by flow cytometry and has two components, the MFI index and the TCR{zeta}bright/dim ratio. B, Plots of TCR{zeta}bright/dim ratio distribution vs TCR{zeta} MFI index in CD3+ T cells from 60 healthy donors. Each point represents the TCR{zeta} expression index for one individual.

 
In 22 of the healthy controls, TCR{zeta} expression in CD3+, CD4+, and CD8+ T cell subsets was measured serially 10–12 mo apart to assess the longitudinal stability of expression; analysis was performed using the Bland-Altman method of comparison (42) followed by linear regression of these results using Prism software (GraphPad Software). In addition, multiple linear regression was performed in a stepwise fashion with batch and TCR{zeta} genotype (at rs1052230/rs1052231, where the "rs" number is a reference SNP cluster identifer) as independent variables using SPSS software.

TCR{zeta} expression data in the healthy individuals genotyped for the SNPs resulting from the 3'-UTR fine mapping was analyzed using a multiple linear stepwise regression model (SPSS software) to determine which SNP(s) exhibit(s) the strongest association with TCR{zeta} expression. The SNP(s) with the strongest association were selected for TCR{zeta} mRNA expression analysis.

SNP identification and verification

At the outset of this project, only a minimal number of SNPs were available in public databases. Thus, SNPs were identified in the coding and regulatory regions of CD3Z by direct sequencing of each exon together with an additional 1 kb of 5'-flanking sequence in 45 SLE patients representative of the study population (40). The existing cDNA sequence (7) and the full genomic sequence of CD3Z (GenBank accession no. NT_004668; 1854894) were used as references. Direct sequencing was performed in both orientations in order exclude sequencing artifacts. The primers used are listed in Table I.


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  		Table I. Sequences of primers used in SNP identification

 
SNPs were then checked for uniqueness by the Basic Local Alignment Search Tool (BLAST) computer program (www.ncbi.nlm.nih.gov/BLAST/) and cross-referenced against the publicly available SNP databases SNP Consortium (http://snp.cshl.org) and dbSNP (www.ncbi.nlm.nih.gov/projects/SNP/). Approximately 700 bp of the large first intron containing four putative SNPs (rs858555, rs858556, rs858557, and rs858558) were also investigated (this segment was chosen for the purposes of linkage disequilibrium (LD) analysis because it was positioned midway between exons 1 and 2).

Subsequently, the 3'-UTR of the CD3Z locus was fine mapped to identify potential causal variants in the modulation of TCR{zeta} expression. Candidate SNPs in the 3'-UTR and 12kb intergenic region (between CD3Z and the adjacent POU2F1 locus) were selected from the public SNP database, dbSNP and the Utah CEPH families analyzed by the International HapMap Consortium (www.hapmap.org) (43).

Genotyping Methods

PCR-RFLP The SLE nuclear families were genotyped for six CD3Z SNPs (rs16859125, rs858557, rs840016, rs33937946, rs1052230, and rs1052231) by novel PCR-RFLP assays developed using restriction maps constructed by the EMBOSS sequencing software package (www.hgmp.mrc.ac.uk). The assays are summarized in Table II. All restriction enzymes were purchased from New England Biolabs with the exception of AccB7I (Promega). PCR product was digested to completion in appropriate buffer according to the manufacturer’s specifications. The resulting fragments were size separated by electrophoresis through 3% agarose or 10% polyacrylamide gels and visualized by ethidium bromide staining.


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  		Table II. PCR-RFLP genotyping assaysa

 
TaqMan 5' nuclease assay Two SNPs, rs16859030 and a novel SNP (G>A –359 ex 4, or "CD3Z4"), were typed by TaqMan (Applied Biosystems) because neither SNP was amenable to PCR-RFLP. TaqMan assays were performed under standardized conditions with primers and probes designed using File Builder 3.0.1 software (Applied Biosystems).

The position of the allele-specific nucleotide in each probe is underlined; G>A –359 ex4: 5'-TET-TGACATTAGCACCATTTTTCCTCTGGCT-TAMRA-3' (C probe), 5'-FAM-TGACATTAGCACCATTTCTCCTCTGGCT-TAMRA-3' (T probe), 5'-CCTCCTCTGGGAAGATTCTAATACAT-3' (forward primer), and 5'-TGCATGGAACGAGCTTAGTCTAGT-3' (reverse primer); rs16859030: 5'-TET-CAGCTCCTCAAATGTTGTGCCTTACAGT-TAMRA-3' (A probe), 5'-FAM-CAGCTCCTCAAATGCTGTGCCTTACAGT-TAMRA-3' (G probe), 5'-TCTGCTGCTGGACTCTCCCT-3' (forward primer), and 5'-CCTTTCCGTAAGCATTTCCG-3' (reverse primer).

The relative fluorescence of each reporter dye was measured using an ABI PRISM 7700 sequence detection system and allelic discrimination was performed using the SDS software package (Applied Biosystems).

KASPar competitive allele-specific PCR The seven SNPs resulting from the fine mapping of the 3'-UTR were put forward for genotyping in the SLE families and in the 60 healthy donors using KASPar competitive allele-specific PCR (KBioscience). For the samples in which the genotyping result was inconclusive, confirmation was performed by direct sequencing in both orientations.

mRNA expression mRNA expression was quantified using allele-specific real-time PCR. mRNA was obtained from 11 healthy donors heterozygous for rs1052230/rs1052231; mRNA was extracted from an aliquot of 5 x 106 lymphocytes using RNeasy mini kits (Qiagen). Purity was assessed using an Agilent 2100 Bioanalyzer platform (Agilent Technologies). cDNA was synthesized using SuperScript II reverse transcriptase according to the manufacturer’s instructions (Invitrogen Life Technologies). Quality and quantity was assessed using a NanoDrop spectrophotometer.

Allele-specific real-time PCR primers and probes were designed using File Builder 3.0.1 software: 5'-VIC-TTATAGGTCCCAAGTGTTG-TAMRA-3' (T probe), 5'-FAM-TTATAGGTCCCATGTGTTG-TAMRA-3' (A probe), 5'-AAGGCCTCGCAGGAAGAC-3' (forward primer), and 5'-TTTTTCCTGTCCTGCCACTGT-3' (reverse primer).

Real-time PCR were performed under standardized conditions using an ABI 7500 Fast real-time PCR System (Applied Biosystems). The relative fluorescence of each reporter dye (one reporter for each allele) was measured using 7500 Fast System SDS software (Applied Biosystems). Relative expression of each allele was calculated using the Relative Expression Software Tool (REST 2005 version 1.9.12; Corbett Life Science).

Allele-specific mRNA expression was also measured using a complementary technique; following direct sequencing of paired cDNA and genomic DNA samples for each donor, the resulting electropherograms were analyzed quantitatively using PeakPicker software (version 0.5) (44).

Statistical genetics Genotype data from the SLE families were analyzed by the transmission disequilibrium test (TDT) using GeneHunter (version 2.1_r3 beta) software (45) and Haploview (46). Pedcheck (version 1.00) (47) was used to exclude ex-paternity pedigrees. Haplotypes and D' plots were constructed using Haploview. D' plots are shown in preference to r2 plots as we were interested in the overall LD block structure rather than correlation between individual SNPs. Correction for multiple analyses was performed in Haploview by permutation testing (500 permutations). Transmission of single markers was examined in addition to haplotype analysis and ratios of alleles transmitted to individuals with SLE compared with untransmitted alleles (transmitted to untransmitted ratios) were calculated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TCR{zeta} protein expression analysis

TCR{zeta} expression in healthy controls. We analyzed TCR{zeta} expression in 60 healthy individuals using the FACS-based expression index. Intracellular staining of PBLs with PE-conjugated anti-TCR{zeta} Abs revealed distinct lymphocyte subsets, including populations of CD3+TCR{zeta}+ cells, CD3–TCR{zeta}+ cells, and a double negative population of monocytes and B cells that do not express the TCR {zeta}-chain. Furthermore, we found that there was considerable heterogeneity of TCR{zeta} expression in the 60 healthy donors for both components of the TCR{zeta} expression index (Fig. 1B).

In 22 of the 60 healthy individuals analyzed, TCR{zeta} expression in CD3+, CD4+, and CD8+ T cell subsets was measured longitudinally using both components of the TCR{zeta} expression index at two different time points 10–12 mo apart. Linear regression analysis comparing the average expression with the absolute difference between expression at the two time points (Bland-Altman technique) did not differ significantly from zero, with the exception of the TCR{zeta} MFI expression index for the CD3+ cell subset (p < 0.0001) for which there were two outlying values (Fig. 2). These outlying values were not observed in the CD4+ or CD8+ cell subsets, suggesting that they were anomalous. Thus, when the two outlying values were excluded, the p-value was 0.90.


Figure 2
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  		FIGURE 2. Bland-Altman and linear regression analysis of healthy control longitudinal TCR{zeta} expression data. A, CD3+ TCR{zeta} MFI index. B, CD4+ TCR{zeta} MFI index. C, CD8+ TCR{zeta} MFI index. D, CD3+ TCR{zeta}bright/dim ratio. E, CD4+ TCR{zeta}bright/dim ratio. F, CD8+ TCR{zeta}bright/dim ratio. The p value of the data in A when the two outliers are excluded is 0.90. "Difference" refers to the absolute difference between the two time points.

 
TCR{zeta} expression in SLE. We went on to analyze TCR{zeta} expression in 30 individuals with SLE. TCR{zeta} was detectable in PBLs from all 30 patients, none being completely deficient in the protein. When compared with controls, both components of the TCR{zeta} expression index were significantly lower in SLE patients (TCR{zeta}bright/dim ratio (p < 0.0001) and TCR{zeta} MFI index (p < 0.0001) for the CD3+ subset; Fig. 3). A similar pattern of significantly reduced TCR{zeta} expression was observed in CD4+ and CD8+ T cell subsets (CD4+, p = 0.002; CD8+, p = 0.004 for TCR{zeta}bright/dim ratio).


Figure 3
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  		FIGURE 3. Plots of TCR{zeta} expression in CD3+ cells from 60 healthy donors and 30 SLE patients. A, TCR{zeta}bright/dim ratio. B, TCR{zeta} MFI index. In SLE patients, both components of the TCR{zeta} expression index were significantly lower in patients with SLE than in healthy controls (p < 0.0001, Mann-Whitney U test). Each point represents the TCR{zeta} expression index for one individual.

 
LD and haplotype analysis of the CD3Z locus

Due to the inherent variability in TCR{zeta} expression observed in healthy individuals, we hypothesized that genetic variation in the coding or regulatory parts of the CD3Z gene may be contributory (the CD3Z locus is depicted in Fig. 4). Furthermore, such genetic factors may also contribute to susceptibility or severity in SLE. We screened for mutations by direct sequencing of 90 chromosomes from SLE cases. Screening of exons 1 through 8 confirmed one synonymous polymorphism in CD3Z coding sequence, rs33937946, located in exon 4 of the gene adjacent to the sequence encoding ITAM I (36). The rest of the coding sequence and core promoter (~1.2 kb) were nonpolymorphic. The search for SNPs was extended to the limit of 5'-flanking sequence; rs16859125 was identified 1033 nucleotides upstream of the most proximal transcription start site (48), and neither allele altered known transcription factor consensus binding sites. The 3'-UTR of CD3Z contained two polymorphisms 7 bp apart, rs1052330 and rs1052331. An additional five SNPs were identified in intronic regions to increase the density of markers for haplotype analysis, and polymorphisms close to intronic splice sites were prioritized because they were potentially functional. A single SNP was chosen within the large first intron of CD3Z to test for linkage disequilibrium across this region. We also confirmed the presence of a variable number tandem repeat located in intron 5; the results of LD analysis suggested that this variant did not provide additional information. These eight SNPs used in the primary screen are depicted in Fig. 4B.


Figure 4
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  		FIGURE 4. The CD3Z locus. A, The organization of the CD3Z locus on 1q24.2 flanked by the genes POU2F1 (octamer binding transcription factor 1) and CREG (cellular repressor of E1A genes), which maps to ~1 kb upstream of CD3Z. The scale specifies the physical distance in base pairs (from National Center for Biotechnology Information Build 36). CD3Z and CREG1 are reverse transcribed while POU2F1 is forward transcribed (depicted by arrows). B, The genomic structure of the CD3Z gene and the positions of SNPs used in both the primary SNP screen and the fine mapping of the 3' end of CD3Z. Exons are represented by boxes, noncoding sequence by lines. The filled boxes denote translated regions and the open boxes represent UTRs. Exons and introns are drawn to scale except for intron 1, which extends over ~78 kb of DNA.

 
The 452 European-Caucasian SLE families were genotyped for the initially selected eight SNPs in CD3Z. All SNPs were in Hardy-Weinberg equilibrium. Following analysis with Haploview (46), the six 3'-SNPs (numbered 1 to 6 in Fig. 5A) were found to be located in the same haplotype block. Four haplotypes were generated that had a frequency >5% (haplotypes A–D in Fig. 5A). Notably, the 3'-UTR SNPs rs1052230 and rs1052231 were in very strong LD (r2 = 0.96) in this population (haplotype C in Fig. 5A).


Figure 5
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  		FIGURE 5. The D' charts and haplotype blocks for CD3Z SNPs. The resulting haplotypes are shown on the right of each respective chart, labeled A–G. A, CD3Z SNPs used in the preliminary analysis in the SLE families. B, CD3Z SNPs used in the 3'-UTR fine mapping in the SLE families. C, CD3Z SNPs used in the 3'-UTR fine mapping in the HapMap CEPH families. In this panel, SNPs 05 = rs 1214615; 11 = rs1052231; 12 = rs1052230; 14 = rs16859030; 21 = rs952963; 25 = rs1723023; and 27 = rs2995082.

 
The effect of CD3Z genotype on TCR{zeta} expression

The effect of the CD3Z genotype on protein expression was first tested in the 60 healthy controls to exclude a confounding effect from disease. There was no detectable effect from the first six 5'-SNPs (numbered 3 to 8 in Fig. 5A) in CD3+, CD4+, and CD8+ T cell subsets. However, there was a clear effect from the rs1052230 and rs1052231 SNPs: the TCR{zeta}bright/dim ratio in CD3+ cells from heterozygote donors was significantly lower (12.33 ± 2.7) when compared with those homozygous for the common alleles (54.2 ± 8.4; p = 0.0004) using the Mann-Whitney U test (Fig. 6A). Analysis of CD4+ and CD8+ T cell subsets was comparable, although the levels of significance were lower than for the CD3+ population as a whole (CD4+, p = 0.0051; CD8+, p = 0.0082), suggesting that this association was a generic effect independent of cell type. The association with TCR{zeta} expression was less robust using the TCR{zeta} MFI index (CD3+, p = 0.012; CD4+, p = 0.11; CD8+, p = 0.013). In this healthy control cohort, rs1052230 and rs1052231 were in complete LD. In the 30 SLE patients, the 3'-UTR SNPs rs1052230 and rs1052231 were similarly associated with reduced TCR{zeta} expression in CD3+ cells (p = 0.026 for TCR{zeta}bright/dim ratio; not significant for TCR{zeta} MFI index, p = 0.077), with a similar trend in T cell subset analysis (see Fig. 6B).


Figure 6
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  		FIGURE 6. CD3+ TCR{zeta} expression in genotyped healthy controls (A) and patients with SLE (B). TCR{zeta} expression is shown in terms of the MFI index and the TCR{zeta}bright/dim ratio. Mann-Whitney U tests were used to perform statistical calculations.

 
Fine mapping of the CD3Z 3'-UTR

Following the identification of an association between the minor alleles of the 3'-UTR SNPs, rs1052230 and rs1052231, and low TCR{zeta} expression, the 3'-UTR of the CD3Z locus was fine mapped to identify potential causal variants in the modulation of TCR{zeta} expression. Thirty-three candidate SNPs in the 3'-UTR, intron VII, and the 12-kb intergenic region (between CD3Z and the adjacent POU2F1; see Fig. 4A) were selected from the public SNP databases dbSNP (www.ncbi.nlm.nih.gov/projects/SNP/) and HapMap (www.hapmap.org; Ref. 43).

In the HapMap CEPH families, these 33 SNPs lie within the same haplotype block following analysis with Haploview using the solid spine algorithm. From these, five tagging SNPs with minor allele frequencies >5% spanning 7 kb (plus the original two 3'-UTR SNPs; Fig. 4B) were selected for genotyping the healthy donor population. One of these tagging SNPs was further downstream in the intergenic region (rs1214615) and four were further upstream (rs16859030, rs952963, rs1723023 and rs2995082). These four upstream SNPs were all intronic, as no suitable exonic SNPs were identified. Preliminary analysis using the Mann-Whitney U test showed that two SNPs (rs1214615 and rs952963) had a similar pattern of association of low TCR{zeta} expression with the minor allele compared with the two previously selected SNPs (TCR{zeta}bright/dim ratio in CD3+ cells: p = 0.001, p = 0.0002, and p = 0.0005 for rs1214615, rs952963, and rs1052230/rs1052231, respectively). These two SNPs had the highest r2 values with rs1052230/rs1052231 (rs1214615 = 0.82; rs952963 = 0.76), and both were found in the same haplotype as rs1052230/rs1052231 (haplotype C in Fig. 5B). The SNPs rs952963 and rs1214615 plus the original two SNPs were thus used in more detailed multiple linear stepwise regression analyses to select the SNP having the closest relationship with TCR{zeta} expression. These data are shown in Table III. No association of any of the TCR{zeta} indices with age or sex was noted. rs1052230 and rs1052231 were confirmed as the markers with the strongest association between low TCR{zeta} expression and possession of the minor allele (for the CD3+ and CD4+ T cell subsets). These original two SNPs were therefore used in the subsequent mRNA expression analysis.


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  		Table III. Multiple stepwise regression analysis of CD3Z SNPs and TCR{zeta} expressiona

 
To further exclude confounding effects from different batches, multiple linear stepwise regression was used using batch and TCR{zeta} genotype (as defined by SNPs rs1052230/1052231; see below) as independent variables. Batch did not have any significant effect on any of the indices. These results are summarized in Table IV.


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  		Table IV. Multiple stepwise regression analysis of healthy control longitudinal TCR{zeta} expression dataa

 
mRNA expression analysis

cDNA from 11 healthy donors heterozygous for rs1052230/rs1052231 was synthesized and analyzed by allele-specific real-time PCR. Differential expression of mRNA between the minor and major alleles of these tightly linked SNPs was evident after correction for the difference in allelic expression in genomic DNA (likely to be due to inherent differences in dye fluorescence between the allelic probes). Carriage of the major allele was found to be associated with >3-fold more mRNA upon analysis with REST after correction for differences between genomic DNA.

To refine the analysis, differential allelic mRNA expression was confirmed using PeakPicker software (44) (Fig. 7). The mean ratio of the peaks of the major allele to the minor allele for rs1052231 in cDNA from 11 heterozygous individuals was 1.79 (±0.13) compared with a mean ratio of 1.05 (±0.11) in genomic DNA (p < 0.0001 using the Mann-Whitney U test). For rs1052230, the major to the minor allele expression ratio was 2.10 (±0.15) compared with a mean ratio of 1.02 (±0.09) in genomic DNA (p < 0.0001). As this analytic method is controlled internally, it provides compelling evidence that the minor allele of these 3'-UTR SNPs, rs1052230/rs1052231, expresses mRNA at a level ~2-fold lower than the major allele.


Figure 7
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  		FIGURE 7. mRNA expression from the alleles of SNPz rs1052330/rs1052231. A summary of the results obtained using PeakPicker software to analyze the ratio of the major to minor allele mRNA (cDNA) expression of rs1052230 and rs1052231 SNPs from 11 healthy heterozygotes. The corresponding expression ratio in genomic DNA (gDNA) was close to 1 for both SNPs. Mann-Whitney U tests were used to perform statistical calculations.

 
Family-based tests for association with SLE

Because we had confirmed that TCR{zeta} expression is reduced in lupus T cells and had shown an association between these 3'-UTR SNPs and low TCR{zeta} expression in SLE, we tested for an etiological genetic association with SLE in our European-Caucasian lupus family cohort.

The CD3Z genotypes from the primary SNP screen were subsequently examined for association with SLE by TDT analysis using Genehunter (45). The minor allele of rs1052230 was overtransmitted to affected individuals (p = 0.035; transmitted to untransmitted allele ratio, 1.53:1). In addition, the major allele of rs16859030 was overtransmitted to affected individuals (p = 0.024; transmitted to untransmitted ratio, 2.3:1). However, there was no difference in the transmission of rs1052231 or in any of the other SNPs. Although rs1052230 and rs1052231 were in complete linkage in the healthy control cohort, there was a recombination rate of 4.47% in the SLE cohort that may have caused the discrepancy between these two SNPs. This difference in recombination is presumably due to the smaller size of the control cohort. Nevertheless, these two 3'-UTR SNPs were still in very tight linkage in the SLE families (r2 = 0.99). These results were confirmed using Haploview.

We went on to test for an association between the SNPs from the fine mapping of the 3'-UTR and SLE susceptibility. Using Haploview, all seven markers were located in the same linkage block defined using the solid spine algorithm (see Fig. 5B), implying that all SNPs are in LD with each other. Individuals were excluded that had >50% missing genotypes. Seven haplotypes that had a frequency of >1% were generated (haplotypes A–G in Fig. 5B). Upon TDT analysis with Genehunter and Haploview, no individual allele was overtransmitted or undertransmitted to affected individuals (see Table V). However, the haplotype containing both the minor alleles of rs1052230 and rs1052231 (haplotype C) was weakly associated with SLE susceptibility (p = 0.045 using Haploview; p = 0.033 using Genehunter; see Table VI). Upon correction for multiple testing, however, these results were not significant, as determined by 500 permutations.


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  		Table V. Single SNP TDT (3'-UTR SNPs)a

 

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  		Table VI. Haplotype TDT (3'-UTR SNPs)a

 
A comparable analysis using the same SNPs was performed in the HapMap CEPH families; the haplotypes obtained were similar to those in the SLE families with the exception that there was no haplotype F. Furthermore, the frequency of haplotype C was lower in the CEPH than in the SLE families, and no recombination between rs1052230 and rs1052231 was observed.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
TCR{zeta} is a pivotal component of T cell signaling machinery and is thus vital in maintaining the inflammatory response. In this article we report a wide variation in the expression of TCR{zeta} in healthy individuals that has not been previously reported in large cohorts. Furthermore, our data provide evidence that this variation in the expression of TCR{zeta} is linked to polymorphisms in the 3'-UTR of the human CD3Z gene; the minor alleles of two 3'-UTR SNPs (rs1052230 and rs1052231) are shown to be variants associated with low TCR{zeta} expression. This association is evident in both healthy controls and in patients with SLE.

Using allele-specific real-time PCR, we found that the minor allele of these 3'-UTR SNPs expresses less mRNA than the major allele in healthy individuals; this suggests that posttranscriptional gene regulation may be responsible for the reduced TCR{zeta} protein expression observed in individuals who possess the minor allele of these SNPs. It is conceivable that variants of the CD3Z 3'-UTR might reduce TCR{zeta} mRNA stability, for example, resulting in TCR{zeta} down-regulation. Several recent studies highlighting the mRNA destabilizing effects of alternatively spliced 3'-UTR variants on TCR{zeta} mRNA expression have strengthened this concept (18, 19, 20, 21). This may occur through the association of RNA-binding proteins with cis-elements, analogous to the adenine-uridine-rich elements (AREs) found in the 3'-UTR of inflammatory genes such as cyclooxygenase-2 and TNF-{alpha} that have important roles in regulating mRNA stability (49, 50, 51). Three AREs have been identified in the 3'-UTR of CD3Z (52); although the SNPs that we have studied are not in the vicinity of the AREs, this does not preclude their indirect involvement through effects on RNA secondary structure. Although verification of the nature of such mechanisms will require further investigation in, for example, mRNA stability studies, these results do imply important functional consequences of this CD3Z 3'-UTR allelic variation.

To make sense of this genotype-phenotype association, we considered the immunobiology of T cells expressing low levels of the {zeta}-chain (i.e., TCR{zeta}dim T cells). Because TCR{zeta} is a vital part of the T cell signaling machinery, it is likely that loss of TCR{zeta} will impair T cell signaling. T cells expressing low levels of TCR{zeta} are found in chronic infectious and inflammatory diseases along with neoplastic conditions; these T cells are hyporesponsive to stimulation in vitro with Ag or mitogens. It has been demonstrated that, in some T cells from patients with certain tumors (53) or with SLE (54), Fc{epsilon}R{gamma}, a member of the {zeta} family, can replace TCR{zeta}, resulting in near-normal TCR cell surface expression. However, because Fc{epsilon}R{gamma} possesses only one ITAM compared with the three on the {zeta}-chain, the T cell signal may be quantitatively and/or qualitatively different. Moreover, such substitutions of Fc{epsilon}R{gamma} for TCR{zeta} have not been observed in studies describing TCR{zeta} down-regulation (reviewed in Ref. 30). Rarely, cases of absolute TCR{zeta} deficiency in humans have been described; these have resulted in severe immunodeficiency with autoimmune features (55, 56, 57).

Recently, we have reported a detailed phenotypic and functional analysis of TCR{zeta}dim T cells, demonstrating that this circulating population is enriched for cells that have previously engaged Ag and are primed for cytokine production (33). For instance, the TCR{zeta}dim subset is enriched for CD45RO+ T cells while the TCR{zeta}bright subset is populated by many CD45RA+ T cells. Furthermore, the TCR{zeta}dim subset is enriched for TNF-{alpha}- and IFN-expressing cells when compared with the TCR{zeta}bright population. TCR{zeta}dim cells are also potent activators of monocytes through cell contact-dependent pathways. These data have demonstrated that although TCR{zeta}dim cells are relatively refractory to TCR-induced proliferation, they are not anergic or senescent, being capable of inflammatory cytokine expression in response to cytokine receptor or costimulatory signals. These characteristics give TCR{zeta}dim T cells a unique effector status. Thus, low TCR{zeta} expression (or an increased circulating effector TCR{zeta}dim T cell population) is likely to have important functional consequences, including the generation of a proinflammatory environment. Our data suggest that CD3Z 3'-UTR allelic variants may be associated with increased numbers of these effector T cells during immune and inflammatory responses.

A genetic determinant of TCR{zeta} expression may, therefore, contribute to the pathogenesis of chronic inflammatory disease. Having defined genetic variants associated with reduced TCR{zeta} expression, a feature of lupus T cells, we went on to test for association between polymorphisms in the CD3Z gene and SLE by conducting a family-based study. There was the suggestion of a trend between SLE susceptibility and the 3'-UTR polymorphisms that have been found to be associated with low TCR{zeta} expression. Results from the fine mapping of the 3'-UTR of CD3Z also suggested a trend between these 3'-UTR SNPs and SLE disease susceptibility. If this trend was to be substantiated as a true association, however, analysis of an additional 500 families would be required in further replication studies. Upon comparison with the genotype data from the International HapMap Consortium, the haplotype containing the minor alleles of these 3'-UTR SNPs was observed at a slightly lower frequency in the HapMap CEPH families and at a slightly higher frequency in the Yoruban (Nigerian) families (data not shown).

It is possible that the CD3Z 3'-UTR allelic variation may represent a genetic predisposition to chronic inflammatory diseases such as SLE by virtue of TCR{zeta} down-regulation (i.e., increasing the circulating TCR{zeta}dim effector T cell population). Alternatively, reduced levels of TCR{zeta}, predisposing directly to attenuation of TCR signaling, may promote autoimmunity through a number of different mechanisms that could include positive selection of autoreactive T effector cells in the thymus and negating effects on the Treg cell population. New insights come from animal models such as the SKG mouse in which a mutation in the TCR proximal protein tyrosine kinase, ZAP-70, leads to a systemic autoimmune inflammatory polyarthritis resembling RA. According to this model, refractory TCR signals lead to a genetically determined selection shift during thymic development and the development of a highly autoreactive repertoire of peripheral T cells (58). Interestingly, numbers of naturally occurring CD4+CD25+Foxp3+ regulatory T cells are increased in SKG mice compared with their wild-type littermates, but their function is impaired. The link between defects in TCR signaling and autoimmunity and lymphoproliferative syndromes is further supported in mice bearing germline mutations/deletions of TCR signaling molecules, including LAT (linker for activation of T cell) (59), and by the development of autoimmunity in rare cases of primary human immunodeficiency, such as those due to TCR{zeta} deficiency (55, 56, 57). It is conceivable that in patients with autoimmune disease who carry gain-of-function PTPN22 mutants that also impair TCR responsiveness, pathways of central and peripheral immune tolerance are perturbed through similar mechanisms. This genetic predisposition could be considered as the "first hit" in disease pathogenesis, with the effects of inflammatory mediators and oxidative stress on TCR{zeta} expression as the "second hit." In contrast, these CD3Z 3'-UTR SNPs may be disease severity rather than disease risk markers; in the setting of immune activation, these 3'-UTR variants might bias immune responses toward a TCR{zeta}dim-associated "inflammation mode" characterized by disordered and sustained Ag-independent effector responses in inflamed tissues, as our recent data might imply (41). Indeed, the relationship between low TCR{zeta} expression and the minor alleles of the 3'-UTR SNPs was stronger with one of the two components of the TCR{zeta} expression index, the TCR{zeta}bright/dim ratio. Because this component reflects the actual number of TCR{zeta}dim cells rather than constitutive TCR{zeta} expression, it is possible that these polymorphisms influence re-expression of TCR{zeta} (after activation-induced down-regulation) rather than constitutive TCR{zeta} expression. As shown in the regression analysis, the effect of the minor allele of rs1052231 alters the TCR{zeta}bright/dim ratio by a factor of –0.40 in the CD3+ population (Table III). This is insufficient, however, to account for the observed difference in TCR{zeta} expression between SLE cases and controls in this study and in previous studies by other groups (20, 21, 22, 23, 24, 39). Thus, additional genetic and/or disease-related factors must contribute to the reduction of TCR{zeta} expression in SLE.

In conclusion, we have identified genetic factors that may underlie the wide variation in TCR{zeta} expression in healthy people as well as in patients with SLE. The resultant skewing toward low TCR{zeta}-expressing TCR{zeta}dim cells may predispose to chronic Ag-independent effector responses and hence chronic tissue inflammation. The identification of a genetic factor with functional consequences that may shape the immune architecture in health and autoimmune disease has wide-reaching implications beyond that of disease-based research.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funding from an European Union Framework 6 Integrated Project (AutoCure), the Wellcome Trust United Kingdom, the Arthritis Research Campaign, and a Clinical Research Fellowship awarded to C.L.G. by the Trustees of the Kennedy Institute of Rheumatology. Back

2 C.L.G. and A.I.R. contributed equally to this manuscript. Back

3 Address correspondence and reprint requests to Dr. Claire L. Gorman, Imperial College London, 1 Aspenlea Road, Hammersmith, London W6 8LH, U.K. E-mail address: c.gorman{at}imperial.ac.uk Back

4 Senior authors A.P.C. and T.J.V. contributed equally to this manuscript. Back

5 Abbreviations used in this paper: 3'-UTR, 3' untranslated region; ARE, adenine-uridine-rich element; Elf-1, E-74-like factor; LD, linkage disequilibrium; MFI, mean fluorescence intensity; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; SNP, single nucleotide polymorphism; TDT, transmission disequilibrium test. Back

Received for publication August 6, 2007. Accepted for publication October 24, 2007.


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 Introduction
 Materials and Methods
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 Discussion
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