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Gene in Human CD4+ T Cell Clones 1
* Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands;
Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands;
Toegepast Natuurwetenschappel
k Onderzoek Prevention and Health, Leiden, The Netherlands; and
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Abstract |
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gene in CD4+ T cells. Analysis of peripheral blood T cell clones derived from healthy individuals revealed that the IL-1 gene shows predominantly monoallelic expression. Monoallelic expression was observed in Th0, Th1, and Th2 cell clones. In addition, we demonstrate monoallelic expression in T cell clones from rheumatoid arthritis patients derived from synovial fluid of the knee joint, suggesting that the occurrence of this phenomenon is not different from that in clones derived from healthy individuals. The finding of monoallelic expression of a cytokine gene in human CD4+ T cell clones provides evidence for allele-specific silencing/activation as another layer of regulation of IL-1 gene expression. |
Introduction |
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plays an important role in the immune response that is central to pathology in many inflammatory diseases, including rheumatoid arthritis (RA)
3 (1, 2). IL-1 is produced by a variety of cells involved in immune function, including T cells (3, 4) and specifically the memory subset (CD4+CD45RO+) of human CD4+ T cells (5). IL-1 production is characterized by the tightly regulated and transient mode of expression in response to external stimuli. Transcriptional activation is a dominant mechanism in the stimulation-induced expression of cytokine genes, the basis of which is assigned to the promoter region and regulatory sequences in the proximity and/or within cytokine genes. Recently, another layer of regulation of cytokine gene expression in T cells was reported, i.e., the allele-specific expression of cytokine genes. A variety of studies have demonstrated that individual T cells may express only one, the other, or both alleles of the IL-2, IL-3, IL-4, IL-5, and IL-13 murine genes (6, 7, 8, 9, 10, 11). One study has described the monoallelic expression of IL-2 in individual human T cells (12). The monoallelic expression of cytokine genes was unexpected, and the role of this form of gene expression in the function of T cells is not obvious. Elucidation of that role may provide important insights into the development and function of T cells. Monoallelic expression is an unusual form of gene expression in which one allele is expressed, while the other remains silent. The precise role of monoallelic expression in the functioning of many genes is poorly understood (13), while in other cases the advantages of expression of a single allele are immediately obvious. Ig and TCR genes code for cell surface receptors and are capable of rearrangements that generate a wide diversity of forms, leading to a phenotypic diversity that is essential to adaptive immune function (14). Monoallelic expression may be involved in the distribution of active genes in individual cells, as seen with the NK receptors encoded by the Ly49 genes. These genes play an important role in immune function and are present in a variety of polymorphic forms, each form showing expression on a limited subset of all NK cells (15). The cell surface expression of a receptor with a single, defined specificity as a result of monoallelic expression is also seen with olfactory receptors (16).
Previous reports describing the monoallelic expression of cytokine genes used a PCR-based approach, exploiting a polymorphism to distinguish transcripts from each allele (6, 10), or generated transgenic mice in which the loss or replacement of an allele by a reporter gene (6, 8, 9) allowed the role of a single allele to be analyzed. The latter approach carries the risk that, due to modification of the structure of the gene, the maintenance of endogenous gene regulation will be disturbed. Therefore, we chose to use RT-PCR amplification and naturally existing polymorphisms that, after restriction endonuclease digestion, allow the discrimination of allele-specific transcripts. Using this approach we studied the expression of the human IL-1 genes in human T cell clones.
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Materials and Methods |
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CD45RA+ CD45RO - naive CD4 + Th cells from two healthy individuals were isolated as described previously (17), stimulated in 96-well culture plates (1 x 105 cells/well; Costar, Cambridge, MA) with immobilized anti-CD3 (CLB-T3/3; 1 µg/ml) and soluble anti-CD28 (CLB-CD28/1; 2 µg/ml; CLB, Amsterdam, The Netherlands), and cultured for 10 days in the absence (Th0 cell lines) or the presence of human rIL-4 (1000 U/ml; PBH, Hannover, Germany; sp. act., 108 U/mg) to obtain Th2 cell lines or human rIL-12 (100 U/ml; a gift from Dr. M. K. Gately, Hoffmann-La Roche, Nutley, NJ; sp. act., 1.7 x 108 U/ml) to obtain Th1 cell lines.
After 10 days cells were cloned by limiting dilution at a density of 0.3 cells/well under continued polarizing conditions (or not (Th0 cell lines)), i.e., rIL-12 (100 U/ml) and neutralizing anti-IL-4 CLB-IL-4/6 (CLB; 1 µg/ml) for Th1 cell lines, or rIL-4 (500 U/ml) and neutralizing anti-IL-12 (rabbit IgG to human IL-12 (10 µg/ml), a gift from Dr. P. H. van der Meide, U-cytech, Utrecht, The Netherlands) for Th2 cell lines. Seeded cells were stimulated with PHA and feeder cell mix as described previously (17). On day 25, 106 cells of expanded clones were stimulated with PMA (5 ng/ml) and ionomycin (250 ng/ml) for 6 h, after which cells were pelleted, lysed in 500 µl of RNAzol B (Tel-Test/Campro Scientific, Veenendaal, The Netherlands), and stored at -20°C for later analysis. All cultures were performed in IMDM (BioWhittaker, Walkersville, MD), supplemented with 10% FCS (Bio-Whittaker), gentamicin (80 µg/ml; Duchefa, Haarlem, The Netherlands), and rIL-2 (10 U/ml).
Generation of T cell clones from RA patients
T cell clones were generated from synovial fluid T cell lines of RA patients as previously described (18).
Genotyping of the IL-1 +4845 polymorphism
The +4845 polymorphism is common, with an allele frequency of 0.32 in the Dutch population (19), and is located in exon 5, allowing discrimination of RNA transcripts. Primers for the analysis of genomic DNA were as follows; sense IL1-DNA1, 5'-GAAGCACATAAGCAACAACA-3', located in intron 4; and antisense IL1-DNA2, 5'-GGATGAATTCGTATTTGATGA-3', located in exon 5. A PCR product of 287 bp was digested by the restriction enzyme HphI for between 2 and 4 h at 37°C, resulting in fragments of 221 and 66 bp (G allele) and 147, 74, and 66 bp (T allele).
Determination of allele-specific transcription by RT-PCR
Total RNA was isolated from cell lysates stored in 500 µl of RNAzol B (CAMPRO Scientific, Veenendaal, The Netherlands) according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of RNA with 500 ng of random hexamers (Promega, Madison, WI)/µg total RNA and 20 U of mouse mammary leukemia virus reverse transcriptase (Life Technologies, Breda, The Netherlands). PCR amplification of 2 µl of cDNA was performed on a PerkinElmer thermal cycler (PE 9700; PerkinElmer Cetus, Norwalk, CA) in 50-µl reaction mixtures containing 5 µl 10x AmpliTaq buffer, 2 mM MgCl2, 0.8 mM of each dNTP, 20 pmol of each primer, and 1 U of Taq polymerase (PerkinElmer Cetus). The following cycling conditions were used: 95°C for 5 min; 35 cycles of 95°C for 45 s, 55°C for 45 s, and 72°C for 45 s; followed by 72°C for 7 min. Primers for determination of the IL-1 +4845 polymorphism of cDNA were as follows; sense IL1-RNA1, 5'-TTGAGTTTAAGCCAATCCAT-3', located in exon 4; and antisense IL1-RNA2, 5'-GCATCATCCTTTGATGACTT-3', located in exon 6. A PCR product of 278 bp was digested by the restriction enzyme HphI (MBI Fermentas, St. Leon-Rot, Germany) for between 2 and 20 h at 37°C, resulting in fragments of 278 bp (G allele; not cut) or 201 and 77 bp (T allele). Using an antisense oligonucleotide located in exon 5 (IL1-RNA-4, 5'-GTATTTCACATTGCTCAGGAAGCTAAAAGcT-3') creating a G to C mismatch at position +4848 (indicated as c) together with the sense oligonucleotide located in exon 3 (IL1-RNA-3, 5'-CCATTGATCATCTGTCTCTG-3'), this generates an induced restriction site for Fnu4HI (New England Biolabs, Beverley, MA) for the +4845 G allelic product. Digestion of the G allelic product generated fragments of 200, 71, and 33 bp, whereas the T allele gave 233- and 71-bp products, as depicted in Fig. 1. Primers for determination of the IL-3 + 131 C
T polymorphism on cDNA were as follows: sense IL-3 +131RS, 5'-GTCCTGCTCCTGCTCCAACT-3', located in exon 1; and antisense IL-3 +131RAS, 5'-GCCTCCAGGTTTGGCCTTCG-3', located in exon 3. A PCR product of 296 bp was digested by the restriction enzyme PfiFI. The genotypes resulted in the following bands: TT, 175 and 121 bp; CT, 296, 175, and 121 bp; and CC, 296 bp.
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gene analysis
TCR V gene analysis was conducted as previously described (20). Briefly, a panel of 25 sense primers for V genes and a constant region antisense primer together with sense and antisense constant region primers as positive controls were used in the amplification of cDNA derived from T cell clones showing both mono- and biallelic expression of the IL-1 gene. A single dominant PCR product together with a positive control product were interpreted as evidence for clonality.
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Results |
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in T cell clones from healthy individuals
The human IL-1 gene has a common polymorphism at +4845. This single base pair G to T transition creates a recognition site for the restriction enzyme HphI (Fig. 1), which allows us to discriminate between alleles and allelic transcripts using this enzyme. We genotyped a panel of 20 healthy individuals for this polymorphism to detect differences in IL-1 allelic transcript production. Two individuals who are heterozygous were selected, and CD45RA+ CD45RO - naive CD4+ Th cells were isolated. Subsequently, T cell clones were generated under continued polarizing conditions to obtain Th0, Th1, and Th2 cell clones and were analyzed for allelic expression of the IL-1 transcript following stimulation for 6 h with PMA/ionomycin by RT-PCR. We estimated that our PCR protocol and visualization methods were capable of detecting a 16-fold difference in transcript level (data not shown). Analysis of RT-PCR products by digestion using HphI to discriminate between the IL-1 allelic products led to the surprising finding of allele-specific activation in the majority of clones tested (Fig. 2A).
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In only a few cases (Fig. 2A, e.g., lanes 7 and 11) was a residual undigested G allelic product observed beyond a dominant digested T-allelic product upon HphI digestion, an observation that was confirmed by reverse digestion with Fnu4HI. For the remainder of this report we classify the clones that have a predominant expression of one allele (between 16- and 8-fold difference) together with clones expressing monoallelic IL-1 (>16-fold difference).
From 33 clones that were tested derived from the two individuals, seven showed expression from both alleles, 20 from the G allele, and six from the T allele, as summarized in Table I. From the clones expressing either the G or the T allele, the clones expressing the G allele appeared to be more frequently expressed.
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We observed a strong predominance of monoallelic expression of the IL-1 gene, with relatively few clones showing expression from both alleles. To validate these data we performed the following control experiments. 1) We addressed the question of whether monoallelic expression of IL-1 is the norm in T cell clones. Therefore, we tested whether apparent biallelic expression was the result of more that one founder cell in a clone. To confirm the truly clonal origin of the cells being tested, we analyzed clones for the expression of TCR V genes (20). We observed one TCR V PCR product in clones tested showing both mono- and biallelic IL-1 expression, indicative of true clonal origin (data not shown).
2) A common artifact of PCR conditions where minimal amounts of starting material are used is the stochastic preferential amplification of one or the other allele during the RT-PCR reaction, creating a false impression of monoallelic expression. While this effect would not be expected with RNA derived from clones where material is not limiting, we serially diluted cDNA from a clone showing monoallelic expression up to 256-fold before PCR. We saw no emergence of an alternative allele product even at the 256-fold dilution, at which point the PCR product was barely visible. Independent preparations of cDNA from the original RNA source also gave consistent results (data not shown).
3) Feeder cells can be a source of RNA even after extended periods following irradiation. We used two separate batches of feeder cells provided by individuals genotyped as being homozygous for either the G allele or the T allele. Clones cocultured with either feeder source showed similar patterns of allelic expression excluding contribution of feeder cells (data not shown) and proved that this phenomenon is a stable feature in clonally expanded cells. 4) To rule out the possible confounder of apparent monoallelic expression due to loss of heterozygosity (21), we genotyped 21 individual clones derived from healthy individuals, of which 7 are shown in Fig. 3, and clones from RA patients (data not shown). None of these clones showed gross deletions in the IL-1 gene.
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The role of IL-1 in human T cells in unclear, although it can specifically stimulate IL-4-independent Th2 cell proliferation in the mouse (22). The autocrine and intracrine roles of IL-1 in some cell types suggest a subtle role in T cell biology. We analyzed the expression pattern and allelic expression pattern of IL-1 in human T cell clones. All possible patterns of expression are represented, with a clear predominance of monoallelic expression. IL-1 is expressed in all clones regardless of Th1 or Th2 cell polarizing conditions, and after digestion of the PCR products, there was no obvious correlation of monoallelic or biallelic expression with the polarization status of the clones (Fig. 2 and Table I). A striking feature of these data is the overrepresentation of the G allele.
Allelic expression of IL-1 in RA-derived T cell clones
To explore the phenomenon of monoallelic expression in relation to disease, we compared the allelic expression pattern of the IL-1 gene in RA patients. Three RA patients were found to be heterozygous for the +4845 IL-1 polymorphism, and clones from these individuals were analyzed. We observed constant monoallelic expression of the G allele (n = 3), predominant expression of the T allele (n = 3; T allele 8-fold higher that G allele), and biallelic expression (n = 3; data not shown). We conclude that T cell clones derived from RA patients show both monoallelic and biallelic expressions of the IL-1 gene, and therefore, RA clones are not different in this respect from clones derived from healthy individuals.
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Discussion |
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gene shows both stable monoallelic and biallelic expressions in T cell clones. This finding of monoallelic expression of a cytokine gene in cloned human Th cells provides a novel layer of regulation of IL-1 gene expression and extends this phenomenon to the field of human T cell immunology. The patterns of allelic expression revealed in our analyses are broadly consistent with dominant monoallelic expression. Our procedure allows detection of expression differences up to at least 16-fold between the allelic transcripts. Therefore, we cannot formally exclude the complete absence of the opposite allele in T cell clones expressing either of the two. Moreover, in a few cases where we observed a predominant T allelic product, we detected a residual G allelic product. This observation raises the question of whether the majority of cases where we observe absence of an allelic product absolutely reflects complete failure to express the allele, i.e., whether monoallelic expression is an absolute phenomenon.
The role of monoallelic expression in T cell biology is at present unknown, but may reveal new aspects of the maturation or functioning of these cells. As with other cytokine genes showing a pattern of monoallelic expression, it is not obvious why IL-1 does so. Murine IL-1 has been reported to be involved in an autocrine loop in which Th2 cells proliferate in an IL-4-independent manner and in which IL-1 enhances its own production. Such a loop might be sensitive to gene dosage, leading to qualitatively different outcomes in clones expressing one or both alleles. Interestingly, IL-1 can also act in an intracrine fashion through a nuclear localization signal (23) that targets the 31-kDa precursor form of the protein to the nucleus (24), where it can regulate growth, gene expression, and migration of endothelial cells (25). Similarly, in T cells and fibroblasts, the IL-1R is internalized and transported to the nucleus (26). These mechanisms of nuclear localization might be particularly sensitive to gene expression levels. These considerations fit with a model of monoallelic expression as an essentially autocrine process in which low levels of gene and protein expression influence the maturation of a T cell clone. Murine IL-1 has been shown to be involved in the maturation of early stage thymocytes, and could conceivably be playing a similar role at another critical stage in T cell differentiation.
An alternative model has been proposed by Bix and Locksley (7) in which they postulate a combinatorial assortment of cytokine gene expression profiles among the clonal progeny of individual precursor Th cells. Together with the diversity of the TCR repertoire and the discrete patterns of cytokine expression represented by the Th1/Th2 paradigm, combinations of mono- vs biallelic expression for a range of coexpressed cytokines may represent a third layer of diversity in a T cell response. This could generate an immune response in which the most effective combinations of cytokine expression enhance T cell proliferation and/or function, resulting in high specificity that matches the challenge from a specific pathogen. This may then become fixed as a heritable epigenetic trait and result in a more successful resolution of an immunological challenge.
Studies in mice have suggested that allele-specific regulation is a common expression strategy for cytokine genes (6, 7, 8, 9, 10, 11). We have studied the allelic expression of the human IL-3 gene as a control, and in contrast to the situation in murine T cell clones, we did not observe evidence of allele-specific regulation of the IL-3 gene in human T cell clones. Moreover, preliminary studies that we performed to study the IL-2 and IL-13 genes have not provided evidence for allele-specific regulation of these genes in human T cells (J.-P. Bayley et al., unpublished observations), indicating that allele-specific regulation of cytokine genes in humans may not be as common as is observed in the murine system.
It is worth noting that the +4845 polymorphism used here to discriminate between alleles of the IL-1 gene is in 100% linkage with a promoter polymorphism at position -889. However, if it is recalled that clones derived from a single individual all carry the same promoter alleles, it becomes difficult to envisage how certain clones show predominant expression of the T allele (five of the clones in donor 1). This argues against the presence of a promoter-mediated bias. Secondly, when we noted the presence of an increased number of G-expressing clones, we went back to the bulk T cell culture from which these clones were derived and analyzed the expression of the G and T alleles. If a promoter bias was driving the increased expression of the G allele, then one would also expect to observe this in bulk culture. This was not the case, and we observed balanced expression of the two allele transcripts (J. G. I. van Rietschoten, J.-P. Bayley, and C. L. Verweij, unpublished observation). This would argue against the presence of a promoter-mediated bias.
The IL-1 polymorphisms have been linked to destructive arthritis (27) in one study, although this finding was not replicated in a recent study (19). We did not observe a difference in the patterns of allelic expression in clones derived from healthy individuals or from subjects with RA, indicating that monoallelic expression of the IL-1 gene occurs in both joint-derived as well as naive T cells of RA patients and healthy controls that have undergone a regimen of in vitro polarization. There were no obvious differences in patterns of monoallelic expression between these groups of clones, providing no indication of an influence attributable to joint inflammation.
Further investigation of this phenomenon should help to define the precise role of the monoallelic expression of cytokine genes in the functioning of the immune system. This will undoubtedly provide insights into both the regulatory mechanisms that lead to stable monoallelic expression and the role of monoallelic expression in either the development of CD4+ T cells or the function of these cells when facing an immunological challenge.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cornelis L. Verweij, Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Room J283, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail address: c.verweij.{at}vumc.nl
3 Abbreviation used in this paper: RA, rheumatoid arthritis.
Received for publication February 28, 2003. Accepted for publication June 24, 2003.
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References |
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. Clin. Exp. Immunol. 73:449.[Medline]
is produced by T lymphocytes activated via the CD2 plus CD28 pathways. J. Immunol. 146:560.[Abstract]
production restricted to memory T cells. J. Immunol. 146:2654.[Abstract]
precursor. J. Biol. Chem. 268:22100. must be transported to the nucleus to exert its activity in human endothelial cells. Mol. Cell. Biol. 14:1845., but not mature IL-1, is able to regulate human endothelial cell migration in vitro. J. Biol. Chem. 272:28202. gene polymorphism and the severity of chronic polyarthritis. Eur. Cytokine Network 10:33.[Medline]This article has been cited by other articles:
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  D. P. Calado, T. Paixao, D. Holmberg, and M. Haury Stochastic Monoallelic Expression of IL-10 in T Cells J. Immunol., October 15, 2006; 177(8): 5358 - 5364. [Abstract] [Full Text] [PDF]
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 J. G. I. van Rietschoten, K. F. Verzijlbergen, S. I. Gringhuis, T. C. T. M. v. d. P. Kraan, J.-P. Bayley, E. A. Wierenga, P. A. Jones, J. M. Kooter, and C. L. Verweij Differentially methylated alleles in a distinct region of the human interleukin-1{alpha} promoter are associated with allele-specific expression of IL-1{alpha} in CD4+ T cells Blood, October 1, 2006; 108(7): 2143 - 2149. [Abstract] [Full Text] [PDF]
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, Site of digestion. RT-PCR products were analyzed by HphI, which digests the +4845 T allele, but not the G allele (upper part). After PCR amplification using an antisense oligonucleotide (indicated by an arrow) that contains a G to C mismatch (in lowercase bold) at position +4848, the +4845 G allele, but not the T allele, was recognized and digested by Fnu4HI (lower part).
