A common intronic variant of CXCR3 is functionally associated with gene expression levels and the polymorphic immune cell responses to stimuli  Jung-Won Choi, PhD, Choon-Sik Park, MD, Minyoung Hwang, BS, Hye-Young Nam, MS, Hun Soo Chang, PhD, Seong Gyu Park, PhD, Bok-Ghee Han, PhD, Kuchan Kimm, MD, Hyung Lae Kim, MD, Bermseok Oh, PhD, Yeonjung Kim, PhD  Journal of Allergy and Clinical Immunology  Volume 122, Issue 6, Pages 1119-1126.e7 (December 2008) DOI: 10.1016/j.jaci.2008.09.026 Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 The asthma-associated intron variant rs2280964 differentially regulates CXCR3 expression levels. A, Map of the CXCR3 gene locus located on the X chromosome. The black box shows the coding region, and the white box shows the untranslated regions. The SNP is marked with an asterisk. B, Comparison of CXCR3-A and CXCR3-B splicing variant levels in LCLs from unaffected male subjects (indicated as G or A carriers for CXCR3 on the X chromosome). Columns represent mean values ± SDs of 3 independent experiments. The significance of differences in mean CXCR3-A/CXCR3-B values was calculated by using the Student t test. C, CXCR3 transcript levels in LCLs. Each dot represents a mean value of a duplicated assay. Mean values of the group are indicated with red bars. Statistical significance was calculated by using the Student t test. Journal of Allergy and Clinical Immunology 2008 122, 1119-1126.e7DOI: (10.1016/j.jaci.2008.09.026) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Test for rs2280964 regulation of gene expression. A, Schematic presentation of reporter constructs carrying only the promoter region (Pro), intron (G or A), or both (Pro-G or Pro-A) upstream of the β-lactamase–coding bla (M) gene. B, Reporter activity in transiently transfected cells. Relative activity is indicated with respect to no-DNA mock-transfected control cells (Cells only). Mean values ± SDs are shown. Statistical significance between the negative control and each construct was evaluated by using the Wilcoxon rank sum test (∗P < .0005). C, Test for protein binding of the rs2280964 intronic region by means of EMSA. Specific DNA-protein complexes are indicated with closed arrowheads. Journal of Allergy and Clinical Immunology 2008 122, 1119-1126.e7DOI: (10.1016/j.jaci.2008.09.026) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Phenotypic assessment of rs2280964G>A-CXCR3–transfected Jurkat T cells. A and B, CXCR3 expression levels. Two clones were selected from each transfection and used to assess the transcript and surface protein levels by using real-time RT-PCR (Fig 3, A) and flow cytometric analysis (Fig 3, B), respectively. Mean values ± SD are shown. The significance of differences was evaluated by using the Wilcoxon rank sum test (∗∗P = .007, ∗P < .05). C, Comparison of CXCR3 gene copy numbers. Relative DNA levels indicate the ratio of DNA in rs2280964G>A-CXCR3–transfected Jurkat cells compared with that in vector-carrying cells. Mean values ± SDs are shown. The significance between the vector cells and each CXCR3-transfected cell type was estimated by using the Wilcoxon rank sum test (∗P < .05). Journal of Allergy and Clinical Immunology 2008 122, 1119-1126.e7DOI: (10.1016/j.jaci.2008.09.026) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 The rs2280964 polymorphism induces differential immune responsiveness in Jurkat T cells in vitro. A, Ca2+ mobilization in rs2280964G>A-CXCR3 Jurkat T cells. The cells carrying pEM7 are shown as gray triangles, cells carrying rs2280964G-CXCR3 are shown as blue squares, and cell carrying rs2280964A-CXCR3 are shown as pink circles. B, Chemotactic activity. C, Cytokine mRNA expression assessed by using real-time RT-PCR. Mean values ± SDs are indicated. Statistical significance was estimated by using the Wilcoxon rank sum test (∗P < .005). Journal of Allergy and Clinical Immunology 2008 122, 1119-1126.e7DOI: (10.1016/j.jaci.2008.09.026) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Phenotypic assessment of human primary T cells. A, CXCR3 transcript levels in primary CD3+ cells isolated from unstimulated PBMCs. Each dot represents a mean value of 5 replicates for each subject. The mean value of each group is indicated with red bars. Statistical significance was calculated by using the Student t test. B, Chemotactic activities of CD3+ cells prestimulated with IFN-γ. Each dot indicates a mean value of a duplicate assay. C, Comparison of allele differential DNA-protein interaction between Jurkat and primary cell nuclear proteins. Journal of Allergy and Clinical Immunology 2008 122, 1119-1126.e7DOI: (10.1016/j.jaci.2008.09.026) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

No effect of rs2280964G>A on transcript levels for the alternatively spliced variants CXCR3-A and CXCR3-B. A and B, mRNA for both CXCR3-A and CXCR3-B is known to be expressed by either resting or activated human T cells, with consistently predominant expression of CXCR3-A in both the unstimulated and antigen-stimulated cells.11,14 We sought to assess rs2280964G>A differential expression for CXCR3-A and CXCR3-B by carrying out quantitative real-time RT-PCR (n = 5) of total RNA from the same LCLs that were used in Fig 1, B. Relative transcript levels are shown in comparison with the transcript levels of SP1. The mean value of the relative transcript levels in each group is indicated with red bars. Statistical significance of the mean differences was calculated by using the Student t test. C, The CXCR3-A/CXCR3-B ratio was similar in both the unstimulated Jurkat T cells stably expressing rs2280964G- and rs2280964A-CXCR3/pEM7. The ratio was 6.3, 6.9, or 7.4 for the pEM7-, rs2280964G-, or rs2280964A-CXCR3/pEM7–carrying cells, respectively. Data are representative of 3 independent experiments showing similar results (see also this article's online Methods section). Journal of Allergy and Clinical Immunology 2008 122, 1119-1126.e7DOI: (10.1016/j.jaci.2008.09.026) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Quantitative analysis of DNA-protein interaction shown by EMSA in Fig 2, C. Our EMSA with Jurkat nuclear proteins revealed strong protein-binding activity by the intronic SNP-carrying sequence. Three specific DNA-protein complexes were observed in our experimental binding conditions. We quantified band intensity of 2 of the complexes, I and III (Quantity one 1-D analysis software; Bio-Rad, Hercules, Calif), and presented the result as a percentage of intensity with respect to the intensity of complexes formed in the absence of competitors (“No competitor”: from lane 6 or lane 12; the bands for complex II were too weak and smeared to be quantified beyond background levels). The intensity ratios between lanes 6 and 12 for complexes I and III are 0.98 and 1.12, respectively. The analysis result indicates that complexes I and III distinctly behave in the presence of the competitors, suggesting their differences in binding affinity toward each of the SNP-bearing sequences. Further functional approaches will be needed to substantiate the in vitro DNA-protein bindings and to identify its functional relevance to the allele-differential gene expression patterns. Journal of Allergy and Clinical Immunology 2008 122, 1119-1126.e7DOI: (10.1016/j.jaci.2008.09.026) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Inhibition of in vitro transcription by rs2280964G>A intronic region. To determine whether the rs2280964G>A intronic region would inhibit a transcription reaction driven by CMV promoter sequence, we set up runoff transcription reactions by using HeLa nuclear extract (Promega no. E3110). The experiment was done according to a protocol provided by the manufacturer. Journal of Allergy and Clinical Immunology 2008 122, 1119-1126.e7DOI: (10.1016/j.jaci.2008.09.026) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Chemotactic activities of human primary CD3+ cells extracted from PBMCs of affected or unaffected subjects. The primary cells were pretreated with either IL-4 (10 ng/mL) or PMA (100 ng/mL) plus PHA (5 mg/mL) for 24 hours before being transferred into a medium that contains CXCL11 (1000 ng/mL). Each dot indicates a mean value of a duplicate assay. Mean chemotaxis values of each group are presented as red bars. The significance of differences between the affected and unaffected groups was evaluated by using the Student t test. Journal of Allergy and Clinical Immunology 2008 122, 1119-1126.e7DOI: (10.1016/j.jaci.2008.09.026) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Comparison of CXCR3-A and CXCR3-B splicing variant levels in primary CD3+ cells isolated from unaffected or affected donors. Total RNA was isolated from CD3+ cells extracted from unstimulated PBMCs and used for real-time RT-PCRs. Columns represent mean values ± SD of triplicate real-time PCRs. The significance of differences in mean CXCR3-A/CXCR3-B values was calculated by using the Student t test. Journal of Allergy and Clinical Immunology 2008 122, 1119-1126.e7DOI: (10.1016/j.jaci.2008.09.026) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions