Haplotype 4 of the multiple sclerosis-associated interleukin-7 receptor alpha gene influences the frequency of recent thymic emigrants

Abstract

The receptor for the homeostatic T cell cytokine interleukin-7 (IL-7Rα) has recently shown genetic association to multiple sclerosis (MS). To investigate the functional contribution of IL-7Rα polymorphisms to the pathogenesis of MS, we correlated the IL-7Rα haplotypes with different T cell parameters in a group of MS patients and healthy controls. We show that carriers of one of the four IL-7Rα haplotypes (Hap4) show a higher expression of IL-7Rα (CD127) on their CD4+ T cells, compared with noncarriers (P=0.04). Moreover, Hap4 carriers possess higher frequencies of recent thymic emigrants (RTEs, CD31+) in both the regulatory T cell (Treg; P=0.007) and conventional T cell (Tconv) population (P=0.0001). This effect is most pronounced within the MS population (Treg, P=0.0077; Tconv, P=0.0007), whereas in healthy controls significance was only reached for Tconv (P=0.043; Treg, P=0.11). Because previous studies showed a decreased RTE–Treg frequency in MS patients compared to healthy subjects, we here conclude that this decrease is localized within the MS population of non-Hap4 carriers. In conclusion, our findings suggest that IL-7Rα polymorphisms can influence T cell development and homeostasis, and thereby contribute to the altered immune regulation associated with disease development in patients with MS.

Introduction

Multiple sclerosis (MS) is the most common disorder of the central nervous system in young adults. Although the pathogenesis of the disease has not yet been fully elucidated, there is increasing evidence that MS occurs in genetically predisposed persons. Many studies have already described that the human leukocyte antigen (HLA) class II genes, located on chromosome 6, represent the strongest risk for MS, accounting for 14–50% of the genetic susceptibility.1, 2 Because this locus is in strong linkage disequilibrium, the identification of the true susceptibility allele is a complex matter. Chao et al.3 recently showed that the once leading candidate, HLA-DRB1*1501, is not the susceptibility allele itself, but is probably part of a susceptibility haplotype. This haplotype includes at least HLA-DQA1*0102 and HLA-DQB1*0602, which interact with HLA-DRB1*1501 through epistatic mechanisms.4 In addition, it appears that vitamin D, through a vitamin D response element in the promoter region of HLA-DRB1*1501, interacts with this locus to influence its expression.5

Recently, other non-HLA genes have been shown to be associated with MS. One of these genes is the interleukin-7 receptor α-chain (IL-7Rα).6, 7, 8, 9 Its product forms a heterodimer with the common IL-2R γ-chain to bind IL-7, or with the thymic stromal lymphopoietin receptor (TSLPR) to bind TSLP. IL-7 is an essential cytokine in the development, survival, proliferation and differentiation of T and B lymphocytes. TSLP, an epithelial-cell-derived cytokine, facilitates the dendritic cell-mediated differentiation of Foxp3+ regulatory T cells (Tregs) in the thymus,10 and promotes T helper (Th) 2 differentiation in the periphery.11 Therefore, polymorphisms within the gene that encodes the receptor of these two cytokines could contribute to an altered T cell homeostasis.

Four haplotypes have been described within the IL-7Rα gene, all of which include multiple single nucleotide polymorphisms (SNPs) in this gene region.6 The SNP that shows the strongest linkage to MS is a nonsynonymous coding SNP in exon 6 (rs6897932).7, 8, 9 Functionally, when a C allele is present in this position, a twofold increase of the skipping of exon 6 occurs, which leads to the formation of the soluble form of the IL-7R.7 This changes the ratio of membrane bound versus soluble receptor, which could cause a reduction of CD127 (IL-7Rα) expression in T cells.

Recent reports, which show that CD127 expression is low on Tregs in comparison with activated conventional T cells (Tconv),12, 13 have triggered the discussion that polymorphisms in the IL-7Rα gene might influence Treg function. We and others have recently shown that an altered Treg homeostasis occurs in MS patients. Venken et al.14 showed that natural naive Treg development and function are disturbed in MS patients, but that the memory Treg pool recovers in the chronic phase of the disease. Also, two independent studies of Venken et al.14 and Haas et al.15 showed that CD31 (PECAM-1) expressing naive Tregs are significantly reduced in the peripheral blood of MS patients.14, 15 CD31 is a 130 kDa transmembrane glycoprotein expressed by endothelial cells, platelets, monocytes, neutrophils and T cell subsets. It was further shown that this adhesion molecule is a marker for recent thymic emigrants (RTE), which are characterized by a high number of T cell receptor excision circles and a low number of cell divisions.16, 17

To investigate the possible contribution of IL-7Rα polymorphisms to the pathogenesis of MS, we sought a possible correlation between the IL-7Rα genotypes and different T cell parameters in a group of MS patients and healthy controls (HC). We found that the haplotype that is tagged by the MS-associated SNP rs6897932 does not influence the frequency of CD4+CD25hiCD127loTregs nor affects the frequency of CD31+ T cells or expression levels of CD127 for both Tconv and Tregs in MS patients and HC. On analyzing the haplotype that has been called ‘protective’ by the Stewart and co-workers (haplotype 4 (Hap4), tagged by the promoter SNP rs11567685), we found a significantly higher expression of CD127 on total CD4+ T cells for Hap4+ MS patients (P=0.041). Furthermore, for the first time we show that the frequency of CD31+ naive (CD45RA+) Tconv and Tregs was significantly reduced in MS patients who did not express Hap4 (Tconv, P=0.0007; Tregs, P=0.0077). In HC, we could find only a statistically significant difference in the Tconv subset (P=0.043), but a trend could also be seen for Tregs (P=0.11). These results point toward a possible contribution of Hap4 to alterations in T cell development and homeostasis.

Results

Genotyping IL-7Rα polymorphisms in a population of Belgian MS patients and healthy controls

Three tagging SNPs in the promoter region of the IL-7Rα gene (rs7718919, rs11567685 and rs11567686) were analyzed using a restriction fragment length polymorphism procedure to determine the genotypes of our Belgian population of MS patients (n=65) and HC (n=33). The four haplotypes tagged by these three promoter SNPs (according to Teutsch et al.6) are summarized in Table 1. Allele and genotype frequencies in our study groups were similar to those previously reported6, 7, 8, 9 (Table 2). In addition, the allele and genotype frequencies for the MS-associated exon 6 SNP (rs6897932) were determined (Table 3). The results of our study confirm the published overexpression of the C allele in MS patients.7, 8, 9 However, no statistical significance was reached (χ2, P=0.780), as a result of the low sample size.

Table 1 The four different haplotypes of the IL-7R α gene
Table 2 IL7Ra haplotype frequencies in MS patients and HC
Table 3 Allele and genotype frequencies of the exon 6 SNP (rs6897932) in MS patients and HC

Treg frequencies in MS patients and healthy controls are not influenced by the IL-7Rα polymorphisms

In this part, we determined the effect of IL-7Rα polymorphisms on Treg frequencies in a group of untreated MS patients and HC. Because several groups reported that Foxp3+ Tregs have a low expression of the IL-7Rα chain (CD127lo),12, 13 polymorphisms within this gene may affect Treg homeostasis. We focused on two specific SNPs within the IL-7Rα locus, known to influence the CD127 expression on T cells. First, the MS-associated exon 6 SNP (rs6897932) is thought to influence the ratio of membrane-bound versus soluble-receptor isoform, because of increased skipping of exon 6 in C allele carriers.7, 9 Study groups were subdivided based on the presence of haplotype 2 (Hap2), which is tagged by the exon 6 SNP (Hap2 carriers express the T allele; Table 1). Second, the group of Stewart reported that the promoter SNP rs11567685 has an effect on the CD127 expression on T cells in primary progressive (PP)-MS patients.18 Moreover, they stated that the haplotype including the C allele at this site (Hap4; see Table 1) is protective in these patients.19 Correlation analyses were performed by stratifying our study groups based on the presence of Hap4. All analyses were performed on untreated MS patients, because it was reported that treatment strongly influences Treg function.14

Frequencies of total Tregs (CD4+CD25hiCD127lo), naive Tregs (nTregs) and memory Tregs (mTregs) (see Figure 1) were determined in our population of untreated MS patients (n=32) and HC (n=27). No significant differences in frequencies of the mentioned Treg populations were found between Hap2 and non-Hap2 carriers within the total population (totTreg, P=0.31; mTreg, P=0.45; nTreg, P=0.98), nor within the MS or HC population (Figure 2a). Moreover, Hap4 and non-Hap4 carriers did not differ in their Treg frequencies in the total population (totTreg, P=0.80; mTreg, P=0.73; nTreg, P=0.70) nor within the MS or HC population (Figure 2b).

Figure 1
figure1

Four different populations of T cells can be distinguished in peripheral blood by means of CD4, CD25, CD127 and CD45RA expression. The plot on the left is gated on CD4+CD45RA memory T cells; the plot on the right is gated on CD4+CD45RAhi naive T cells. The markers CD25 and CD127 can then discriminate between conventional T cell (Tconv, CD25CD127+) and regulatory T cells (Tregs, CD25hiCD127lo).

Figure 2
figure2

Regulatory T cell (Treg) frequencies within the CD4+ lymphocytes of multiple sclerosis (MS) patients and healthy controls. (a) Haplotype 2 (Hap2) carriers versus non-Hap2 carriers; (b) Hap4 carriers versus non-Hap4 carriers. Total Treg, CD4+CD25hiCD127lo; memory Tregs (mTreg), CD4+CD25hiCD127loCD45RA; naive Tregs (nTreg), CD4+CD25hiCD127loCD45RAhi. Differences were not significant in any of the populations (P>0.05).

Haplotype 4 carriers have a significantly higher CD127 expression on total CD4+ T cells

In the light of the functional role of the exon 6 SNP rs6897932, we analyzed the expression of CD127 on total CD4+ T cells, Tconv (CD4+CD25CD127+) and Treg (CD4+CD25hiCD127lo). The presence or absence of Hap2 had no effect (P>0.05) on the mean fluorescence intensity of CD127 on all three T cell populations in the total study group (Figure 3, upper panel). Also, no significant difference was found on analyzing MS patients (total CD4+, P=0.81; Tconv, P=0.78; Treg, P=0.29) and controls (total CD4+, P=0.74; Tconv, P=0.83; Treg, P=0.37) separately.

Figure 3
figure3

Mean fluorescence intensity (MFI) of CD127 on total CD4+ T cells (CD4+ total), CD4+CD25CD127+ T cells (naive conventional T cells, nTconv) and CD4+CD25hiCD127lo Tregs (naive Tregs, nTreg) of pooled multiple sclerosis (MS) patients and healthy controls (HC). The MFI is plotted against presence or absence of haplotype 2 (Hap2, upper panel) and the presence or absence of Hap 4 (lower panel).

When these data were analyzed based on the presence or absence of Hap4, a statistically significant difference was found on total CD4+ T cells (P=0.041) in the total study population (Figure 3, lower panel). More specifically, carriers of Hap4 showed a higher CD127 expression on total CD4+ T cells as compared to non-Hap4 carriers. For Tconv, a trend could also be observed (P=0.082), but for Tregs no difference was found (P=0.80). When individual groups were analyzed, only trends were found in the MS group (total CD4+, P=0.11; Tconv, P=0.14; Treg, P=1.00), but not in HC (total CD4+, P=0.23; Tconv, P=0.46; Treg, P=0.54).

Frequency of CD31+ naive T cells is higher in MS patients carrying haplotype 4

Interleukin-7 has a major role in lymphocyte development, as has been shown by the paucity of T and B cells in IL-7- and IL-7Rα-deficient mice.20, 21 To investigate the effect of IL-7Rα polymorphisms on T cell development, we examined the amount of RTEs present in the peripheral blood of MS patients and HC. For this purpose, CD31 was used as a marker for these RTEs.16 Two independent studies by Venken et al.14 and Haas et al.15 previously showed that RTE and Treg (CD4+CD25hiCD127loCD31+) are significantly reduced in MS patients, in comparison to HC. In this study, we reconfirmed these results (HC, 65.30±9.65%; MS, 58.93±11.35%; P=0.035). To determine the contribution of IL-7Rα polymorphisms to this difference, we analyzed four different T cell subsets in this experiment: naive Tconv (nTconv), memory Tconv (mTconv), nTreg and mTreg (see Figure 1). The memory T cell pool intrinsically possesses a low percentage of CD31-expressing cells, and this amount was not significantly different between any of the studied haplotypes (data not shown). When analyzing our data based on carriers or noncarriers of Hap2, no correlation with the amount of CD31-expressing T cells was found in the total study population (nTconv, P=0.16; nTreg, P=0.28), nor within the MS patient and HC group (Figure 4, upper panel).

Figure 4
figure4

The frequency of CD31-expressing T cells in different T cell populations in haplotype 2 (Hap2) carriers versus non-Hap2 carriers (ad) and Hap4 versus non-Hap4 carriers (eh). Naive Tregs (nTreg), CD4+CD25hiCD127loCD45RAhi; naive conventional T cells (nTconv), CD4+CD25CD127+CD45RAhi. Results are considered significant when P<0.05.

In contrast, the presence of Hap4 did have an effect on the frequency of CD31-expressing naive T cells (both nTconv and nTreg) in the total population (nTconv, P=0.0001; nTreg, P=0.005). More specifically, non-Hap4 carriers have a lower percentage of CD31+ nTregs (P=0.007) and nTconv (P=0.0001). The mean age of Hap4 carriers and noncarriers was not significantly different (39.4±10.9 versus 40.1±12.7; P=0.86), excluding the effect of age. When MS patients and controls were analyzed separately, we found that the MS groups contributed most to the significant difference found in the total study population (nTreg, P=0.0077; nTconv, P=0.0007). In HC, a significant difference was only reached in the nTconv subset (P=0.043), whereas a trend could also be seen for nTregs (P=0.11; Figure 4). Within non-Hap4 carriers, there was a significant reduction of CD31+ nTregs (P=0.041), but not of CD31+ nTconv (P=0.37) in the MS population compared with HC. Within the Hap4 carriers, no significant differences were found (nTregs, P=0.44; nTconv, P=0.62). Therefore, we show for the first time that the overall reduction of the RTE-Treg frequency in MS patients compared to controls is localized within the non-Hap4 carriers.

Analysis of CD31 expression after TCR stimulation in the presence or absence of IL-7

To gain more insight into the mechanism behind the effect of Hap4 on the frequency of CD31-expressing naive T cells, we set up an in vitro experiment using healthy Hap4 (n=4) and non-Hap4 (n=4) carriers. Peripheral blood mononuclear cell (PBMC) from these donors were cultured in the presence of anti-CD3, to induce T cell receptor (TCR) stimulation, known to induce a loss of CD31. In parallel, the effect of IL-7 on CD31 expression was determined, by culturing the cells in the presence or absence of IL-7. On days 0 and 5, flow cytometric analysis was performed. TCR stimulation of PBMC clearly resulted in a decrease of the percentage of CD4+CD31+ T cells at day 5 of culture (Hap4, 67.81±8.25%; non-Hap4, 75.24±2.28%; relative to percentage directly ex vivo). The addition of IL-7 during TCR stimulation induced an increased percentage of CD4+CD31+ T cells at day 5 of culture (Hap4, 81.79±18.07%; non-Hap4, 82.27±8.82%; relative to percentage directly ex vivo), although this increase was not significant (Figure 5). Moreover, the effect of IL-7 is more pronounced in Hap4 carriers compared with non-Hap4 carriers. Three out of four Hap4 carriers showed a significant stabilization of CD31 expression by IL-7, whereas this was only the case in one out of four non-Hap4 carriers. This observation may provide a link between the observed differences found for the CD127 and CD31 expression in Hap4 versus non-Hap4 carriers.

Figure 5
figure5

Peripheral blood mononuclear cells (PBMC) from four haplotype 4 (Hap4) and four non-Hap4 healthy donors were stimulated with anti-CD3, with or without additional interleukin-7 (IL-7). The frequency of CD4+CD31+ cells was measured on days 0 and 5. Values for every donor on day 5 of culture are shown (relative to the ex vivo measurements on day 0).

Discussion

Although the association between IL-7Rα polymorphisms and MS has been shown earlier by several groups,6, 7, 8, 9, 22 the functional relevance of this association is not clear yet. In this study, we provide evidence that polymorphisms in the IL-7R gene can have an influence on the development of naive T cells. To the best of our knowledge, we are the first to show that carriers of the previously reported protective Hap4 have a higher percentage of RTEs in both naive Treg and Tconv subsets, compared to non-Hap4 carriers. In addition, Hap4 carriers express higher levels of IL-7Rα on their CD4+ T cells. In contrast, the MS-associated exon 6 SNP did not affect Treg frequency or CD127 expression on T cells.

Recently, investigators of our14 and another group15 compared the frequency of RTE and Treg in MS patients and HC. Both studies provide evidence that this subset of naive Tregs was significantly reduced in MS patients as compared to HC. In addition, Haas et al.15 showed that CD31-expressing Tregs are mainly responsible for the functional properties of the entire Treg population. Therefore, a decrease in this subpopulation of Tregs might explain the dysfunctional immune regulation seen in MS patients.15 CD31 is an adhesion molecule that is expressed on T cells that have recently emigrated out of the thymus and entered the peripheral circulation. These RTEs are characterized by a high T cell receptor excision circle content and hardly any recent cell divisions.16 In this study, we confirm that RTE and Tregs are decreased in MS patients compared to HC, and we provide a link between the frequency of RTEs and IL-7Rα polymorphisms. More specifically, the frequency of RTE and Tregs was decreased in MS patients compared with HC in non-Hap4 carriers, but not in the Hap4 carrier population. These results suggest that the decrease in RTE–Tregs in MS patients is a consequence of a lower frequency in non-Hap4 carriers. The observation that RTEs are reduced for both the Treg and the Tconv subsets in non-Hap4 individuals may indicate that Hap4 expression has an overall effect on thymic T cell development. Early experiments with IL-7 and IL-7R knockout animals showed that IL-7 signaling is crucial for thymic T cell development.20, 21 Recently, Mazzucchelli et al.23 also discovered that Treg development is dependent on either IL-7 or TSLP. These two cytokines bind to the IL-7Rα, and their combined function leads to the normal development of Tregs. Taken together, these results link IL-7Rα polymorphisms to a possible defect in thymic T cell development.

Furthermore, we performed a functional assay to determine the contribution of these polymorphisms to CD31 kinetics in vitro. We found that, after TCR stimulation, the addition of IL-7 stabilized the CD31 phenotype in CD4+ T cells. This phenomenon was most pronounced in Hap4 carriers, thus pointing toward a possible role of IL-7Rα polymorphisms in peripheral T cell homeostasis. More importantly, this may provide us with a link between the expression levels of CD127 and CD31, which were also different between Hap4 carriers and noncarriers. A recent study by Azevedo et al.24 determined the role of IL-7 in the homeostasis of RTEs. They found that IL-7-mediated proliferation does not result in a loss of CD31 expression on naive CD4+ T cells. This suggests that IL-7 preferentially maintains RTEs during adult life. The results of our study now provide a link between the genetic variation within the IL-7Rα gene and the stabilization of the CD31 phenotype in anti-CD3-stimulated CD4+ T cells. This mechanism might be important in the peripheral T cell homeostasis in MS patients, because the differences in CD31 expression levels were more evident in MS patients compared with HC. On the basis of this, we can conclude that the observed alterations in RTE frequency are not only a result of genetic influences, but also of ongoing inflammatory processes, which are present in MS patients but not in HC. Taken together, these data suggest that IL-7Rα polymorphisms not only affect thymic T cell development, but could also be involved in peripheral RTE homeostasis.

Because we found an effect of IL-7Rα polymorphisms on the development or homeostasis of T cells, we hypothesized that this might reflect a difference on the molecular level (for example, the expression of CD127). Indeed, we observed an increased CD127 expression on total CD4+ T cells in Hap4 carriers compared with noncarriers in the total study population. This is in line with the reportings by McKay et al.19 who found that the CD127 expression on CD4+ T cells (both Treg and Tconv) was higher in Hap4 carrying compared with non-Hap4 carrying PP-MS patients. Therefore they proposed that this haplotype is protective in PP-MS patients. However, our total study group consisted of HC, as well as relapsing-remitting (RR) MS and secondary progressive (SP) MS patients. This may indicate that the protective effect of Hap4 is not restricted to PP-MS patients alone.

When subjects were subdivided in Hap2 carriers and non-Hap2 carriers, no significant differences were found for CD127 expression. This is again in line with the findings of McKay et al.18 However, a study of Gregory et al.7 who analyzed the mRNA expression of the membrane bound versus the soluble form of the receptor, reported that carriers of the T allele at the exon 6 SNP rs6897932 (which tags Hap2) show a higher mRNA expression of the membrane-bound IL-7Rα protein, compared with carriers of the C allele. Because our study cannot confirm this result on the protein level, two explanations can be given: (1) post-translational modifications, which cannot be seen by real-time PCR, bias these results, or (2) flow cytometry cannot pick up subtle differences in the expression levels of CD127. To address this issue, other and more sensitive detection techniques should be used in the near future.

The observed molecular effects in our study can have consequences on the cellular level and even on the organism in total. A deficiency in Treg frequencies (especially in RTE–Tregs) can be detrimental in patients with autoimmune diseases such as MS. Also, a decrease in the output of Tconv might have disadvantages. That is, a decrease in thymic output of Tconv might lead to homeostatic proliferation, which in turn causes dividing cells to age. Aging of the immune system, also called immunosenescence, has been found to be accelerated in patients with a diverse range of autoimmune diseases.25, 26

Given the relatively low odds ratio (1:18)8 of the IL-7Rα gene regarding its association to MS, a small effect on the MS risk could be expected. However, the results described here clearly show a significant effect of Hap4 on the frequency of RTEs. Because MS is a complex disease, with many genetic factors probably contributing to the predisposing genotype, the effect of Hap4 on T cell parameters might be the result of genetic interactions with other alleles influencing the immune system (for example, two alleles within the IL-2R8). The low odds ratio of the IL-7Rα gene also in part explains why some Hap4 carriers still develop MS. As has been stated, the strongest genetic risk is represented by the HLA class II genes, but still other, nongenetic factors contribute to the disease, such as viral load and exposure to sunlight, all of which may affect MS susceptibility.

To conclude, the results of our study provide the first evidence that IL-7Rα polymorphisms influence the frequency of RTE in the peripheral blood of MS patients, either on the level of T cell development or in the periphery. These results might therefore contribute to a better understanding of the biological role of IL-7Rα polymorphisms in complex diseases such as MS.

Materials and methods

Study subjects

Peripheral blood samples were collected from 33 HC and 65 patients with clinically definite MS (RR-MS, n=39; SP-MS, n=15; PP-MS, n=11). Of these, 32 patients were untreated in that they had not received corticosteroids or immunomodulatory drugs within at least 3 months of blood collection. The other 33 patients were treated with interferon-β (n=25), glatiramer acetate (n=7) or both (n=1) at time of blood sampling. Within our population of MS patients, a diverse range of clinical subtypes was included. The mean age of MS patients was 44.7±11.2 years (range 20–68 years), the average disease duration was 10.3±7.8 years (range of 3 months to 28 years), and Expanded Disability Status Scale scores ranged from 1 to 5.5 (median of 2±1.5). The mean age of the HC was 33.2±10.7 years (range 21–55 years). This study was approved by the local medical ethical committee of Hasselt University, and informed consent was obtained from all study subjects.

IL-7Rα genotyping

Multiple sclerosis patients and HC were genotyped for the IL-7Rα locus using a restriction fragment length polymorphism procedure, as previously described.6 Briefly, a fragment of the IL-7Rα gene promoter region, including three tagging SNPs (rs7718919, rs11567685 and rs11567686), was PCR-amplified from genomic DNA isolated from PBMC of MS patients and HC. This fragment was then cut by the restriction enzymes HphI (Fermentas, Burlington, Ontario, Canada) and PstI (New England Biolabs, Ipswich, MA, USA) to obtain banding patterns specific for each haplotype. To verify the genotyping assay, we sequenced selected samples using an ABI automated DNA sequencer (PerkinElmer, Waltham, MA, USA) to confirm the genotype.

Flow cytometric analysis

Peripheral blood mononuclear cells were isolated from whole blood by Ficoll density-gradient centrifugation (Histopaque; Sigma-Aldrich, St Louis, MO, USA). Cells were quadruple stained with the following monoclonal antibodies: anti-human CD4, CD25 (both from BD Biosciences, Franklin Lakes, NJ, USA), CD45RA, CD31 (both from ImmunoTools, Friesoythe, Germany) or CD127 (eBioscience, San Diego, CA, USA), and subsequently analyzed by flow cytometry using a FACSCanto II (BD Biosciences). Statistical analysis was performed using FACSDiva software (BD Biosciences). On the basis of four of these markers, namely CD4, CD25, CD45RA and CD127, one can distinguish four populations of T cells (see Figure 1)13, 14: nTregs (CD4+CD25hiCD127loCD45RAhi), mTregs (CD4+CD25hiCD127loCD45RA), nTconv (CD4+CD25CD127+CD45RAhi) and mTconv (CD4+CD25CD127+CD45RA). CD31 staining was performed together with CD4, CD25 and CD45RA, to determine its expression in nTregs (CD4+CD25hiCD45RAhi), mTregs (CD4+CD25hiCD45RA), nTconv (CD4+CD25CD45RAhi) and mTconv (CD4+CD25CD45RA).

Cell culture

For the analysis of the effect of IL-7 on CD31 kinetics in vitro, PBMC were cultured in the presence or absence of recombinant human IL-7 (10 ng ml−1; R&D Systems, Minneapolis, MN, USA), additionally stimulated with monoclonal anti-CD3 antibody (αCD3; 2 μg ml−1; BIOMED, Diepenbeek, Belgium). On days 0 and 5, flow cytometric analysis was performed with the following antibodies: CD4-PERCP (BD Biosciences) and CD31-FITC (ImmunoTools). Flow cytometric analyses were performed using a FACSCalibur (BD Biosciences). Statistical analysis was performed using CellQuest software (BD Biosciences). Because of high interdonor variation, values are represented as relative to day 0 (ex vivo) values (set at 100%).

Statistical analysis

Differences of genotype or allele frequencies between MS patients and HC were analyzed using χ2-test. For comparison of T cell parameters between carriers and noncarriers of certain alleles, a Mann–Whitney test was performed using Prism software version 4.0 (GraphPad Software). Results are expressed as mean values±s.e.m. Differences were considered significant when P<0.05.

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Acknowledgements

We thank patients and healthy controls for blood donations, and Tom Broekmans and Anne Bogaers for assistance in collecting the blood samples. We also thank Hanne Jongen (the hematology unit of the Virga Jesse hospital, Hasselt) for performing flow cytometric analyses on FACSCanto.

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Correspondence to P Stinissen.

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Broux, B., Hellings, N., Venken, K. et al. Haplotype 4 of the multiple sclerosis-associated interleukin-7 receptor alpha gene influences the frequency of recent thymic emigrants. Genes Immun 11, 326–333 (2010). https://doi.org/10.1038/gene.2009.106

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Keywords

  • interleukin-7 receptor
  • polymorphism
  • recent thymic emigrants
  • multiple sclerosis

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