And Maintenance of Peripheral T Cells Locus Controls IL-7 Receptor

An Enhancer of the IL-7 Receptor α-Chain Locus Controls IL-7 Receptor Expression and Maintenance of Peripheral T Cells

This information is current as Akifumi Abe, Shizue Tani-ichi, Soichiro Shitara, Guangwei of October 2, 2021. Cui, Hisataka Yamada, Hitoshi Miyachi, Satsuki Kitano, Takahiro Hara, Ryo Abe, Yasunobu Yoshikai and Koichi Ikuta J Immunol 2015; 195:3129-3138; Prepublished online 2 September 2015; doi: 10.4049/jimmunol.1302447 Downloaded from http://www.jimmunol.org/content/195/7/3129

Supplementary http://www.jimmunol.org/content/suppl/2015/09/01/jimmunol.130244 http://www.jimmunol.org/ Material 7.DCSupplemental References This article cites 46 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/195/7/3129.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

An Enhancer of the IL-7 Receptor a-Chain Locus Controls IL-7 Receptor Expression and Maintenance of Peripheral T Cells

Akifumi Abe,*,† Shizue Tani-ichi,* Soichiro Shitara,*,† Guangwei Cui,*,‡ Hisataka Yamada,x Hitoshi Miyachi,{ Satsuki Kitano,{ Takahiro Hara,* Ryo Abe,‖ Yasunobu Yoshikai,x and Koichi Ikuta*

The IL-7R plays critical roles in lymphocyte development and homeostasis. Although IL-7R expression is strictly regulated during lymphocyte differentiation and the immune response, little is known regarding its in vivo regulation. To address this issue, we established a mouse line with targeted deletion of the conserved non-coding sequence 1 (CNS1) element found 3.6 kb upstream of the IL-7Ra promoter. We report that IL-7Ra isexpressednormallyonTandBcellsinthymusandbonemarrowofCNS12/2 mice Downloaded from except for in regulatory T cells. In contrast, these mice show reduced IL-7Ra expression in conventional CD4 and CD8 T cells as well as regulatory T, NKT, and gd T cells in the periphery. CD4 T cells of CNS12/2 mice showed IL-7Ra upregulation in the absence of growth factors and IL-7Ra downregulation by IL-7 or TCR stimulation, although the expression levels were lower than those in control mice. Naive CD4 and CD8 T cells of CNS12/2 mice show attenuated survival by culture with IL-7 and reduced homeostatic proliferation after transfer into lymphopenic hosts. CNS12/2 mice exhibit impaired maintenance of Ag-stimulated T cells. Furthermore, IL-7Ra upregulation by glucocorticoids and TNF-a was abrogated in CNS12/2 mice. This work demon- http://www.jimmunol.org/ strates that the CNS1 element controls IL-7Ra expression and maintenance of peripheral T cells, suggesting differential regulation of IL-7Ra expression between central and peripheral lymphoid organs. The Journal of Immunology, 2015, 195: 3129–3138.

nterleukin-7, a cytokine essential for lymphocyte develop- IL-7 supports survival and homeostasis of naive and memory ment and homeostasis, controls the survival, proliferation, and T cells (6). IL-7 exerts its effects through interaction with the IL-7R, I differentiation of early T and B cells (1–3) as well as V(D)J which consists of the IL-7R a-chain (IL-7Ra) and a common cy- recombination of the TCRg and IgH loci (4, 5). In the periphery, tokine receptor g-chain. IL-7Ra also dimerizes with the thymic stromal lymphopoietin receptor. IL-7 binding to the IL-7R activates by guest on October 2, 2021 JAK-1 and JAK-3, which then activates STAT5 and PI3K. *Laboratory of Biological Protection, Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan; †Graduate School of Bio- IL-7Ra expression is dynamically controlled in T and B cell studies, Kyoto University, Kyoto 606-8501, Japan; ‡Graduate School of Medicine, development. During T cell differentiation, IL-7Ra is expressed Kyoto University, Kyoto 606-8501, Japan; xDivision of Host Defense, Network Center for Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka in double-negative (DN) thymocytes, downregulated in double- 812-8582, Japan; {Reproductive Engineering Team, Institute for Virus Research, Kyoto positive (DP) thymocytes, upregulated in single-positive (SP) ‖ University, Kyoto 606-8507, Japan; and Division of Immunobiology, Research Insti- thymocytes, and maintained in naive T cells in the periphery (2, tute for Biomedical Sciences, Tokyo University of Science, Chiba 278-0022, Japan 7). When naive T cells receive Ag stimuli, the IL-7Ra is down- Received for publication September 12, 2013. Accepted for publication July 28, 2015. regulated in effector T cells (6, 8, 9). However, a small subset of effector T cells, referred to as memory precursor effector cells, This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Scientific Research [C] Grants 25460589 upregulates IL-7Ra and differentiates into memory CD8 T cells [to K.I.] and 26460572 [to S.T.], Scientific Research on Innovative Areas Grants (9). During B cell development, IL-7Ra is expressed on common 25111504 and 15H01153 [to K.I.], and Young Scientists [B] Grants 24790469 [to S.T.] and 24790468 [to T.H.]). This work was also supported by the Platform Project lymphoid progenitors (CLPs) and pro– and pre–B cells and then is for Supporting Drug Discovery and Life Science Research (Platform for Dynamic downregulated during the transition from pre–B to B cells (7, 10, 11). Approaches to Living System) from the Ministry of Education, Culture, Sports, Thus, IL-7Ra expression is precisely regulated during lymphocyte Science and Technology of Japan, a grant from the Fujiwara Memorial Foundation, a grant from the Shimizu Foundation for Immunology and Neuroscience, and by the development and the immune response. BioLegend/TOMY Digital Biology Young Scientist Research Award for 2011. IL-7Ra expression on T cells is regulated at transcriptional Address correspondence and reprint requests to Dr. Koichi Ikuta, Laboratory of levels and suppressed by IL-7 and other prosurvival cytokines, Biological Protection, Department of Biological Responses, Institute for Virus Re- such as IL-2, IL-4, IL-6, and IL-15 (12). Additionally, TCR sig- search, Kyoto University, 53 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address: [email protected] naling also represses IL-7Ra transcription (13). Furthermore, both The online version of this article contains supplemental material. glucocorticoids (GC) and TNF-a upregulate IL-7Ra transcription (14–16). Additionally, several transcriptional factors interact with Abbreviations used in this article: cDC, conventional dendritic cell; CLP, common lymphoid progenitor; CNS1, conserved non-coding sequence 1; DN, CD42CD82 the IL-7Ra locus and control its transcription. Two Ets family double-negative; DP, CD4+CD8+ double-positive; ES, embryonic stem; GC, gluco- proteins, PU.1 and GA binding protein a, interact with the IL-7Ra corticoid; GR, glucocorticoid receptor; IEL, intraepithelial lymphocyte; ab IEL, TCRb+ intraepithelial lymphocyte; IHL, intrahepatic lymphocyte; IL-7Ra, IL-7R promoter and are crucial for IL-7Ra transcription in early B and a-chain; pDC, plasmacytoid dendritic cell; rLM-OVA, recombinant Listeria mono- T cells, respectively (17, 18). We previously showed that a con- + + 2 + 2 + cytogenes expressing OVA; SP, CD3 CD4 CD8 or CD3 CD4 CD8 single-posi- served non-coding sequence 1 (CNS1) element found 3.6 kb up- tive; Treg, Foxp3+CD4+ regulatory T cell. stream of the IL-7Ra promoter is a GC-responsive enhancer of the Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 IL-7Ra locus in vitro (16). Consistently, a DNase I hypersensitivity www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302447 3130 FUNCTION OF AN IL-7 RECEPTOR LOCUS ENHANCER site was identified 3.8 kb upstream of that promoter (19). Consensus BioLegend (San Diego, CA), and Wako Pure Chemical Industries: CD3ε motifs for NF-kB, the GC receptor (GR), Evi-1, and forkhead (145-2C11), TCRb (H57-597), CD4 (RM4.5), CD8a (53-6.7), CD8b transcription factors are conserved in the CNS1 element, and Foxo1 (H35-17.2), CD25 (7D4), Foxp3 (FJK-165), CD44 (IM7), gdTCR (GL3), NK1.1 (PK136), CD45R/B220 (RA3-6B2), CD43 (S7), BP-1 (6C3), CD24 and Foxp1 interact with the element to control IL-7Ra expression in (30F1), Igm (M41), CD11c (N418), CD11b (M1/70), Gr-1 (RB6-8C5), naive T and early B cells (20–22). However, it is largely unknown TER-119, Sca-1 (E13-161.7), c-Kit (2B8), Thy-1 (53-2.1), IL-7Ra how the element controls IL-7Ra expression during lymphocyte (A7R34), CD45.1 (A20), CD45.2 (104), Ly-6C (HK1.4), IFN-g (XMG1.2), 694 development and the immune response. phosphorylated STAT5 (Tyr ) (47), Bcl-2 (A19-3), annexin V, Ki-67 (SolA15). H-2Kb OVA G4 tetramer–SIIGFEKL-PE was purchased from To determine its function in vivo, we established a mouse line Medical and Biological Laboratories (Nagoya, Japan). Biotinylated mAbs carrying targeted deletion of the CNS1 element and analyzed IL-7Ra were detected with PE-, allophycocyanin-, or PE-Cy7–conjugated streptavidin expression at different differentiation stages. IL-7Ra expression (eBioscience). Viable cells were analyzed with FACSCalibur or FACSCanto was significantly reduced in peripheral T cells of CNS12/2 mice, II flow cytometers (BD Biosciences) with CellQuest and FlowJo software. and naive CNS12/2 T cells showed impaired survival and homeo- Values in quadrants, the gated area, and interval gates indicate the percentages in each population in all figures. static proliferation. IL-7Ra upregulation by GC and TNF-a was 2 2 Intracellular staining of phosphorylated STAT5 was performed as described also impaired in CNS1 / T cells. We also report that IL-7Ra (27). In brief, sorted naive CD4 (CD3+CD4+CD252CD44loCD62Lhi)and upregulation after Ag stimulation was independent of the CNS1 CD8 (CD3+CD8+CD44loCD62Lhi) T cells were stimulated for 20 min with element. Overall, this study demonstrates that the CNS1 element 5 ng/ml IL-7 (R&D Systems, Minneapolis, MN) at 37˚C, stained for surface Ags, fixed, permeabilized in ice-cold methanol, and stained with anti- controls IL-7Ra expression and maintenance of peripheral T cells. phosphorylated STAT5 Ab using a Foxp3 staining buffer set (eBioscience). For intracellular staining of Foxp3, Bcl-2, and Ki-67, cells were first stained Materials and Methods for surface Ags, fixed, permeabilized, and stained with relevant Abs using Mice a Foxp3 staining buffer set according to the manufacturer’s instructions. Downloaded from Intracellular staining of IFN-g in OT-1 T cells was carried out using the same 2 2 B6.CD45.1 congenic, B6.OT-1 TCR transgenic (23), and Rag2 / mice buffer set. In brief, lymphocytes obtained from spleen were incubated with were used. All mice were maintained under specific pathogen-free con- 1 mg/ml OVA257–264 peptide (Medical and Biological Laboratories), 2 mg/ml ditions in the Experimental Research Center for Infectious Diseases in the anti-CD28 (PV-1), and 10 mg/ml brefeldin A (Biomol, Hamburg, Germany) Institute for Virus Research, Kyoto University. All mouse protocols were for 4 h at 37˚C. Cells were stained for surface Ags, fixed, and permeabilized approved by Kyoto University. with fixation/permeabilization solution (eBioscience) for 30 min at 4˚C and

stained with FITC–anti-IFN-g Ab for 30 min at 4˚C. http://www.jimmunol.org/ Generation of IL-7Ra CNS1-deleted mice To construct a targeting vector, the following DNA fragments were assembled Cell culture in the pBluescript KS (+) vector: a diphtheria toxin A cassette, a 1822-bp T cells were cultured in RPMI 1640 medium supplemented with 10% FBS fragment of the 59 CNS1 element, a neomycin resistance gene cassette flanked and 50 mM 2-ME. The following reagents were used at the indicated by loxP sites, and a 7683-bp fragment of the 39 CNS1 element. That line- concentrations: 0.5 and 5 mg/ml anti-CD3ε (2C11), 2 mg/ml anti-CD28 arized vector was introduced into the KY1.1 embryonic stem (ES) cells (PV-1), 5 ng/ml IL-7 (R&D Systems), 1029 M dexamethasone (Sigma- 3 (C57BL/6 129S6/SvEvTac F1 background) by electroporation, and ho- Aldrich, St. Louis, MO), and 1.5 ng/ml TNF-a. For T cell stimulation, mologous recombinants were screened by PCR. Targeted ES clones were purified CD4 T cells (1 3 106 cells/ml) were stimulated with IL-7 or plate- confirmed by Southern blot analysis with 59 and 39 probes (see Fig. 1A, 1B). bound anti-CD3 in the presence of fixed doses of soluble anti-CD28 for The neomycin resistance gene cassette was removed from the recombinant

20 h, or dexamethasone or TNF-a for 12 h at 37˚C. For an in vitro survival by guest on October 2, 2021 allele by infecting targeted ES clones in vitro with adenovirus expressing Cre assay, sorted naive CD4 (CD3+CD4+CD252CD44loCD62Lhi) and CD8 recombinase (a gift of Dr. Izumu Saito of the Institute of Medical Science, (CD3+CD8+CD44loCD62Lhi) T cells (2 3 105 cells/ml) were cultured with University of Tokyo) (24). Resultant ES clones contained one copy of the loxP and without IL-7, and the proportion of propidium iodide2 living cells was site with an 839-bp deletion of the CNS1 element. ES clones were injected measured by flow cytometry. For in vitro apoptosis assay, purified T cells into ICR eight-cell embryos. Chimeric mice were bred with C57BL/6 mice, (1 3 106 cells/ml) were cultured with and without IL-7 for 24 h, and the and CNS1-deficient mice were backcrossed onto C57BL/6 mice for eight proportion of annexin V+ dead cells was measured by flow cytometry. generations. Real-time RT-PCR Cell isolation Total RNA was isolated and treated with RNase-free DNase to remove To sort thymocyte subpopulations, triple-negative and CD8SP thymocytes genomic DNA. After DNase inactivation, total RNAwas reverse transcribed were first enriched by negative separation with anti-CD4 microbeads and LS 2 2 2 using random primers. Complementary DNAs were analyzed by real-time columns (Miltenyi Biotec, Auburn, CA). DN (CD3 CD4 CD8 ), DP 2 2 RT-PCR using SYBR Green PCR Master Mix (Qiagen, Hilden, Germany) in (CD4+CD8+), CD4SP (CD3hiCD4+CD8 ), and CD8SP (CD3hiCD4 CD8+) an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA). thymocytes were sorted with a FACSAria II cell sorter (Becton Dick- PCR efficiency was normalized using cDNA made from whole thymocytes inson, Franklin Lakes, NJ) (purity .98%). CD4 and CD8 T cells were of wild-type mice. The following primer sets were used: Il7ra,59-GGA- purified from lymph nodes using an EasySep CD4+ and CD8+ T cell en- TGGAGACCTAGAAGATG-39 and 59-GAGTTAGGCATTTCACTCGT-39; richment kits (Stemcell Technologies, Vancouver, BC, Canada; purity 2 bcl2,59-TCGCTACCGTCGTGACTTC-39 and 59-AAACAGAGGTCGCATG- .98%). Naive CD4 (CD3+CD4+CD25 CD44loCD62Lhi or CD3+CD4+ 2 2 CTG-39; bclxl,59-GGAGAGCGTTCAGTGATC-39 and 59-CAATGGTGG- CD25 CD44loNK1.1 ) T cells, naive CD8 (CD3+CD8+CD44loCD62Lhi) CTGAAGAGA-39; mcl1,59-TCAAAGATGGCGTAACA-39 and 59-CCCGTTT- T cells, and regulatory T (Treg) cells were sorted from lymph node cells or CGTCCTTACAAGAAC-39;andGapdh,59-CCTCGTCCCGTAGACAAAATG-39 enriched CD4 T cells (purity .98, .97, and .95%, respectively). In some and 59-TCTCCACTTTGCCACTGCAA-39. experiments, enriched CD8 T cells were stained with PE–anti-CD44, followed by incubation with anti-PE microbeads (Miltenyi Biotec), and CFSE dilution assay negatively separated on MS columns to purify naive CD8 T cells (CD3+ CD8+CD44lo) (purity .98%). gd T cells (CD3+gdTCR+) were sorted from Naive CD4 (CD3+CD4+CD252CD44loNK1.12) and CD8 (CD3+CD8+ lymph node cells (purity .92%). Intraepithelial lymphocytes (IELs) were CD44lo) T cells were labeled with 5 mM CFSE (Dojindo Laboratories, prepared from small intestine as described (25). Intrahepatic lymphocytes Kumamoto, Japan) in PBS at 37˚C for 10 min and washed thoroughly. (IHLs) were prepared as described with minor modification (26). Briefly, Cells were resuspended in 200 ml PBS and adoptively transferred into mice were perfused with PBS. IHLs were obtained by dispersing the liver Rag22/2 mice by i.v. injection. After 7 d, proliferation of transferred cells through a strainer, followed by centrifugation through a 33% Percoll was assessed by FACS-measured CFSE dilution. CFSE-labeled cells were gradient and erythrocyte lysis. NKT (CD3+NK1.1+)andNK(CD3–NK1.1+) divided into three populations: CFSEhi (no division), CFSEint (one to four cells were sorted from IHLs (purity .98%). divisions), and CFSElo (five or more divisions) cells. Abs and flow cytometry Adoptive transfer and immunization of OT-I cells The following fluorescent dye– or biotin-conjugated Abs were purchased Adoptive transfer and immunization of OT-I cells was performed as de- from BD Biosciences (San Jose, CA), eBioscience (San Diego, CA), scribed (28, 29). In brief, OT-I cells (1.5 3 106 cells) were purified from The Journal of Immunology 3131

B6.OT-1 CNS1+/+Rag22/2 and B6.OT-1 CNS12/2Rag22/2 mice. OT-I cells from CNS1+/+ or CNS12/2 Rag22/2 mice were suspended in 200 ml PBS and adoptively transferred into 6- to 8-wk-old B6.CD45.1 recipient mice. After 24 h, mice were immunized by i.v. injection of 200 mgOVA (Wako Pure Chemical Industries, Osaka, Japan) and 25 mg LPS (from Esherichia coli 055:B5, Sigma-Aldrich). Transferred OT-I cells were analyzed 3, 7, 15, and 25 d after the primary immunization by ﬂow cytometry.

Bacterial infection Recombinant Listeria monocytogenes expressing OVA (rLM-OVA) was previously described (30, 31). For primary infection, mice were injected i.v. with 2 3 104 CFU rLM-OVA at day 0. At days 7, 14, 21, 28, and 35, CD8 T cells were analyzed. To examine memory T cells, the recipient mice were rechallenged with 2 3 105 CFU rLM-OVA at day 35. After 5 d, CD8 T cells were analyzed. Flow cytometry was performed after ﬁxing surface-stained cells with 0.1% paraformaldehyde-PBS (2) for 20 min.

Statistical analysis An unpaired two-tailed Student t test was used for all statistical analysis. A p value ,0.05 was considered statistically significant. Downloaded from Results The CNS1 element controls IL-7Ra expression in thymic Treg cells To investigate the function of the CNS1 element in IL-7R expression in vivo, we established a mutant mouse line with targeted deletion of that element at the IL-7Ra locus (CNS12/2 mice) http://www.jimmunol.org/ (Fig. 1A, 1B). Initially, we used flow cytometry to compare thymocytes between CNS1+/+ and CNS12/2 mice. Thymocyte numbers were unchanged between wild-type and mutant mice (Fig. 1C). Additionally, both the proportion and absolute numbers of DN1, DN2, DN3, DN4, DP, CD4SP, and CD8SP thymocytes were unchanged between genotypes (Fig. 2A, 2B). We next analyzed surface IL-7Ra expression and found that each thymocyte population showed comparable levels of IL-7Ra ex- by guest on October 2, 2021 pression in CNS1+/+ and CNS12/2 mice(Fig.2C).Toassess regulation at the transcriptional level, we compared Il7ra transcripts in thymocyte subpopulations using real-time RT-PCR and found that transcript levels were also comparable between wild- FIGURE 1. Generation of CNS12/2 mice. (A) Schematic illustration of type and mutant mice (Fig. 2D). These results demonstrate that the IL-7Ra locus, targeting vector, and CNS1-targeted and -deleted alleles. the CNS1 element is dispensable for IL-7Ra expression during Boxes and the oval indicate exons and CNS1, respectively. Triangles in- thymocyte development. dicate loxP sequences. Probes for Southern blot analysis are shown as We next compared numbers of Treg, gd T, NKT, and NK cells horizontal bars. E, EcoRV; S, SphI. (B) Southern blot analysis of targeted between CNS1+/+ and CNS12/2 thymocytes using flow cytom- ES clones. Genomic DNA from three ES clones was digested with EcoRV or SphI and hybridized with probe A or B. (C) Number of thymus, bone etry. The proportion and absolute numbers of all of these cells +/+ were unchanged between CNS1+/+ and CNS12/2 mice (Fig. 2E, marrow, lymph node, spleen, and IHL cells from 4- to 7-wk-old CNS1 and CNS12/2 mice was determined. *p , 0.05. 2F). Whereas gd T and NKT cells showed comparable levels of surface IL-7Ra expression between wild-type and mutant mice, Treg cells showed significantly lower levels of IL-7Ra ex- a pression in CNS12/2 compared with wild-type mice (Fig. 2G). The CNS1 element controls IL-7R expression in peripheral These results strongly suggest that CNS1 controls IL-7Ra ex- CD4 and CD8 T cells pression in Treg cells but not in gd T, NKT, and NK cells in We previously showed that GR interacts with the CNS1 element thymus. and enhances Il7ra transcription in vitro (16). Foxo1 and Foxp1 also reportedly interact with the element to control IL-7Ra ex- The CNS1 element is dispensable for B cell development in pression in naive T cells (20, 21). To determine the function of this bone marrow element in peripheral CD4 and CD8 T cells, we first analyzed Next, we asked whether the CNS1 element functions during B cell lymph node cells in CNS1+/+ and CNS12/2 mice using flow development in bone marrow. We observed that the numbers of cytometry. The numbers of lymph node cells were slightly de- bone marrow cells were comparable in CNS12/2 and CNS1+/+ creased in CNS12/2 compared with CNS1+/+ mice (Fig. 1C). The mice (Fig. 1C). The proportion and absolute numbers of CLPs proportion and absolute numbers of naive CD4 and CD8 T cells and cells in Hardy fractions A–F were also unchanged (Fig. 3A, were moderately reduced in CNS12/2 compared with wild-type 3B). Furthermore, surface IL-7Ra expression was comparable in mice (Fig. 4A, 4B). Surface IL-7Ra expression in peripheral naive each of these B cell fractions in wild-type and mutant mice (Fig. CD4 and CD8 T cells was significantly reduced in mutant versus 3C), indicating that the CNS1 element is dispensable for IL-7Ra wild-type mice (Fig. 4C). Il7ra transcripts were decreased by 3.5- expression during B cell development in bone marrow. fold in CD4 and CD8 T cells in mutant compared with wild-type 3132 FUNCTION OF AN IL-7 RECEPTOR LOCUS ENHANCER Downloaded from

FIGURE 3. The CNS1 element is dispensable for IL-7Ra expression during B cell development in bone marrow. Bone marrow cells of 4- to 5- wk-old CNS1+/+ and CNS12/2 mice were analyzed by ﬂow cytometry (n = 3–5). (A) Flow cytometric analysis of B cell fractions. (B) Cell numbers of B cell fractions: CLP (Lineage (B220, Gr-1, CD11b, CD3, and Ter-119)2 http://www.jimmunol.org/ Sca-1intc-Kitint), fraction A (B220+CD43+CD242BP-12), fraction B (B220+CD43+CD24+BP-12), fraction C (B220+CD43+CD24+BP-1+), fraction D (B220+CD432Igm2), fraction E (B220loCD432Igm+), and fraction F (B220hiCD43–Igm+) (mean 6 SEM). (C) Surface IL-7Ra expression in B cell fractions.

in lymph nodes, we found that their absolute numbers and proportion were slightly reduced in CNS12/2 compared with CNS1+/+ by guest on October 2, 2021 mice (Fig. 5A, 5B). Additionally, the proportion of gd Tcells FIGURE 2. The CNS1 element is required for IL-7Ra expression in 2/2 2 2 showing low levels of IL-7Ra expression increased in CNS1 thymic Treg cells. Thymocytes of 4- to 5-wk-old CNS1+/+ and CNS1 / A mice (Fig. 5C). Furthermore, Il7ra transcript levels were slightly mice were analyzed by flow cytometry. ( ) Flow cytometric analysis of 2/2 +/+ thymocyte subsets. (B) Cell numbers of thymocyte subsets: DN1 (CD44+ decreased in gd T cells of CNS1 compared with CNS-1 mice CD252CD32CD42CD82), DN2 (CD44+CD25+CD32CD42CD82), DN3 (Fig. 5D). When we analyzed NKT cells in liver, we found that 2/2 (CD442CD25+CD32CD42CD82), DN4 (CD442CD252CD32CD42 numbers of IHLs moderately decreased in CNS1 compared with CD82), DP (CD4+CD8+), 4SP (CD3+CD4+CD82), and 8SP (CD3+CD42 CNS1+/+ mice (Fig. 1C). Although the proportion of NKT cells was CD8+) (mean 6 SEM, n = 7–9). (C) Surface IL-7Ra expression in thy- unchanged (Fig. 5A), the number of NKT cells decreased slightly in mocyte subsets. (D) Real-time RT-PCR analysis of Il7ra transcripts in CNS12/2 compared with CNS1+/+ mice (Fig. 5B), and those cells thymocyte subsets (mean 6 SEM, n = 3). (E) Flow cytometric analysis of displayed relatively lower levels of surface IL-7Ra expression Treg, gd T, NKT, and NK cells. (F) Cell numbers of Treg (Foxp3+CD3+ + + + + + + (Fig. 5C). Furthermore, Il7ra transcripts in NKT cells decreased 2- CD4 ), gd T(gdTCR CD3 ), NKT (NK1.1 CD3 ), and NK (NK1.1 2/2 2 fold in CNS1 mice (Fig. 5D). CD3 ) cells (mean 6 SEM, n = 7–9). (G) Surface IL-7Ra expression in Treg, gd T, and NKT cells. We next analyzed NK cells in liver. Absolute numbers and proportion of NK cells were slightly reduced in CNS12/2 compared with CNS1+/+ mice (Fig. 5A, 5B). Additionally, the pro- + 2/2 mice (Fig. 4D), strongly suggesting that the CNS1 element con- portion of IL-7Ra NK cells was reduced in CNS1 mice trols IL-7Ra expression in these cell populations. (Fig. 5C). Furthermore, Il7ra transcript levels in NK cells decreased by 5-fold in CNS12/2 mice (Fig. 5D). a The CNS1 element controls IL-7R expression in multiple Because IL-7 plays a critical role in conventional and plasma- immune cell populations cytoid dendritic cell (cDC and pDC) precursors (34), we analyzed We next asked whether the CNS1 element was important for IL-7Ra DCs in bone marrow and spleen of CNS1+/+ and CNS12/2 mice. expression in other T cell subpopulations in the periphery. To do so, Numbers of pDCs and cDCs were unchanged between CNS1+/+ we first analyzed Treg cells in CNS1+/+ and CNS12/2 lymph nodes. and CNS12/2 mice (Fig. 5A, 5B) as was cell surface IL-7Ra ex- The absolute number of Treg (Foxp3+CD4+) cells was unchanged pression in these cells (Fig. 5C). between genotypes (Fig. 5A, 5B). Additionally, Treg cells displayed Because IL-7 is indispensable for IEL development (35), we significantly reduced levels of IL-7Ra (Fig. 5C). Furthermore, analyzed IL-7Ra expression in IELs of CNS1+/+ and CNS12/2 Il7ra transcript levels were decreased by 3.5-fold in Treg cells of from the small intestine. The absolute numbers and proportion of CNS12/2 relative to wild-type mice (Fig. 5D). ab IELs were unchanged in CNS12/2 compared with CNS1+/+ Both gd T and NKT cells reportedly required IL-7 and IL-15 for mice (Supplemental Fig. 1A, 1B). However, the proportion of maintenance in periphery (32, 33). When we analyzed gd T cells CD4+ ab IELs was significantly reduced in CNS12/2 mice, The Journal of Immunology 3133

FIGURE 4. The CNS1 element controls IL-7Ra expression and maintenance in naive T cells. (A–C) Lymph node cells of 6- to 7-wk-old CNS1+/+ and CNS12/2 mice were analyzed by ﬂow cytometry (n =12). A B ( ) Flow cytometric analysis of CD4 and CD8 T cells. ( ) Numbers of Downloaded from naive CD4 and CD8 T cells: naive CD4 T (CD3+CD4+CD252CD44lo CD62Lhi) and naive CD8 T (CD3+CD8+CD44loCD62Lhi) cells (mean 6 SEM). (C) Surface IL-7Ra expression in naive CD4 and CD8 T cells: naive CD4 T (TCRb+CD4+CD252CD44loCD62Lhi) and naive CD8 T(TCRb+CD8+CD44loCD62Lhi)cells.(D) Real-time RT-PCR analysis of Il7ra transcripts in CD4 and CD8 T cells from lymph nodes: CD4 + + + + T(CD3CD4 )andCD8T(CD3CD8 ) cells (mean 6 SEM, n =3). http://www.jimmunol.org/ *p , 0.05, **p , 0.01, ***p , 0.005.

FIGURE 5. The CNS1 element controls IL-7Ra expression in periph- ab eral Treg, NKT, and NK cells. Lymphoid cells from lymph nodes, liver, whereas the proportion of other IELs remained unchanged +/+ 2/2 (Supplemental Fig. 1C). Additionally, surface IL-7Ra expression bone marrow, and spleen of 4- to 7-wk-old CNS1 and CNS1 mice + + 2/2 were analyzed by ﬂow cytometry. (A) Flow cytometric analysis of lymph in CD4 and CD8ab ab IELs was slightly reduced in CNS1 + + + + +/+ node cells: Treg (Foxp3 CD4 , n = 6) and gd T cells (gdTCR CD3 , n = compared with CNS1 mice (Supplemental Fig. 1D). IL-7Ra + + + 2 + 9); IHLs (n = 7): NKT (NK1.1 CD3 ) and NK (NK1.1 CD3 ) cells; and expression was signiﬁcantly decreased in CD8aa ab and gd + +

bone marrow and spleen cells (n = 4): pDCs (CD11c B220 ) and cDCs by guest on October 2, 2021 IELs in mutant mice. However, each IEL subpopulation displayed (CD11c+B2202). (B) Numbers of Treg cells, gd T cells, NKT cells, NK comparable levels of surface IL-2/15Rb expression in mutant and cells, pDCs, and cDCs (mean 6 SEM). (C) Surface IL-7Ra expression in wild-type mice (Supplemental Fig. 1D). Overall, these analyses Treg cells (CD3+CD4+CD25+), gd T cells, NKT cells, NK cells, pDCs, show that the CNS1 element controls IL-7Ra expression primarily and cDCs. (D) Real-time RT-PCR analysis of Il7ra transcripts (mean 6 in Treg cells, gd T cells, NKT cells, some NK cells, and some ab SEM, n = 2). *p , 0.05. IELs but not in DCs.

The CNS1 element alters survival of naive T cells The CNS1 element controls homeostatic proliferation of naive Because IL-7 controls survival of T cells, we first analyzed STAT5 T cells phosphorylation and cell survival of peripheral CD4 and CD8 Because IL-7 is essential for homeostatic proliferation of naive T cells after IL-7 stimulation in vitro. STAT5 phosphorylation T cells (6), we analyzed expansion of naive T cells transferred levels were moderately reduced in naive CD4 and CD8 T cells of into lymphopenic hosts. Naive CD4 and CD8 T cells were puri- CNS12/2 compared with wild-type mice (Fig. 6A, 6B). Addition fied from CNS1+/+ and CNS12/2 mice, labeled with CFSE, of IL-7 only weakly rescued naive CNS12/2 CD4 and CD8 and transferred into Rag22/2 recipients. After 7 d, proliferation of T cells from cell death (Fig. 6C). Consistent with the cell sur- transferred T cells was assessed using a CFSE dilution assay. vival results, CNS12/2 CD4andCD8Tcellsshoweda2-to3- Naive T cells transferred into lymphopenic hosts are classified into fold increase in the proportion of annexin V+ dead cells after rapidly and slowly dividing populations. Rapidly dividing cells culture with IL-7 (Supplemental Fig. 2A). These results suggest are cross-reactive with environmental Ags undergoing IL-7–inde- that CNS1 element affects IL-7 signaling and survival of naive pendent spontaneous proliferation, whereas slowly dividing cells T cells. require IL-7 for homeostatic proliferation (6, 39). As previously Because IL-7 induces Bcl-2 in T cells, we next analyzed ex- reported, transferred T cells split into rapidly and slowly dividing pression of antiapoptotic proteins. Both CD4 and CD8 T cells populations by day 7 (Fig. 6D). The proportion of CFSE+ cells in showed comparable levels of Bcl-2 protein in CNS1+/+ and the slowly dividing population tended to be low in CNS12/2 CNS12/2 mice (Supplemental Fig. 2B). Given that IL-7 induces T cells, although that tendency was more prominent in CD4 than bcl2 transcription in CD4 and CD8 T cells (12), we measured bcl2 in CD8 T cells. Absolute numbers of cells in the CFSE+ slowly transcripts and found that they were comparable between CNS1+/+ dividing population were significantly decreased in CD4 and CD8 and CNS12/2 mice (Supplemental Fig. 2C). Because T cell sur- T cells of CNS12/2 relative to wild-type mice (Fig. 6E). We hi vival also depends on Bcl-xL and Mcl-1 (36–38), we also mea- further divided slowly dividing cells into three fractions: CFSE sured bclxl and mcl1 transcripts but did not detect significant (no division), CFSEint (one to four divisions), and CFSElo (five or changes in their levels (Supplemental Fig. 2C). more divisions). The proportion of CFSEhi cells was significantly 3134 FUNCTION OF AN IL-7 RECEPTOR LOCUS ENHANCER

increased in CD4 and CD8 T cells of CNS12/2 mice (Fig. 6F), indicating that homeostatic proliferation was partially impaired. Consistently, the absolute numbers of CFSEint and CFSElo cells were significantly reduced in CNS12/2 relative to wild-type T cells (Fig. 6G). These results demonstrate that the CNS1 element controls homeostatic proliferation of naive T cells. The CNS1 element responds to GC and TNF-a in T cells in vitro Removal of T cells from their in vivo environment mark- edly increases IL-7Ra expression, whereas IL-7 and TCR signals downregulate it (12, 13). We next sought to determine how IL-7Ra expression is regulated by the CNS1 element in peripheral T cells. To do so, we first analyzed IL-7Ra upregulation in the absence of growth factors. CD4 T cells cultured without IL-7 showed an ∼3- fold increase in Il7ra transcripts in a time-dependent manner in CNS1+/+ and CNS12/2 CD4 T cells, although absolute levels were much lower in CNS12/2 than in CNS1+/+ cells (Fig. 7A). We next analyzed IL-7Ra downregulation by IL-7 or anti-CD3ε plus anti- CD28 Abs. Both stimuli significantly reduced levels of Il7ra tran- Downloaded from scripts in CNS1+/+ and CNS12/2 CD4 T cells (Fig. 7B, 7C). Both GC and TNF-a reportedly enhance IL-7Ra transcription (14–16). Therefore, we analyzed the function of the CNS1 element on IL-7Ra upregulation by dexamethasone and TNF-a. Both stimuli induced Il7ra transcripts by 6- to 8-fold in CNS1+/+ CD4

T cells compared with the cells grown in medium alone (Fig. 7D). http://www.jimmunol.org/ However, this induction was completely lost in CNS12/2 CD4 T cells. Overall, these results demonstrate that CNS1 is a GC- and TNF-a–responsive enhancer of the IL-7Ra locus in T cells. Changes in IL-7Ra expression in CD8 T cells during the immune response are independent of the CNS1 element To determine the function of CNS1 element in IL-7Ra expression during the immune response, we used an OT-I TCR transgenic mouse model. To do so, we transferred CD8 T cells from CNS1+/+ by guest on October 2, 2021 or CNS12/2 OT-I Rag22/2 mice into CD45.1+ recipients and then immunized them with OVA and LPS. After immunization, transferred OT-I cells were assessed by ﬂow cytometry. Absolute

FIGURE 6. The CNS1 element controls survival and homeostatic proliferation of naive T cells. (A) Sorted naive CD4 (CD3+CD4+CD252 CD44loCD62Lhi) and CD8 (CD3+CD8+CD44loCD62Lhi) T cells were stimulated with IL-7, and intracellular expression of p-STAT5 was measured by flow cytometry. (B) Mean fluorescence intensity (MFI) of p- STAT5 is shown (mean 6 SEM, n = 3). (C) Sorted naive CD4 and CD8 T cells were cultured with or without IL-7 at indicated time points, and the proportion of living cells (propidium iodide [PI]2) was measured by flow cytometry (mean 6 SEM, n = 3). (D) Sorted naive CD4 (CD44loCD3+ CD4+CD252NK1.12) or CD8 (CD44loCD3+CD8+) T cells were labeled with CFSE and adoptively transferred into Rag22/2 mice. After 7 d, proliferation of transferred T cells in lymph nodes was assessed by a CFSE FIGURE 7. The CNS1 element is a GC- and TNF-a–responsive element. E + 2 6 2 2 dilution assay. ( ) Cell numbers of CFSE and CFSE populations (mean Purified CD4 T cells from CNS1+/+ and CNS1 / mice were incubated in F + SEM, n =4).() CFSE cells were divided into three fractions: medium (A), stimulated with IL-7 (B), stimulated with plate-bound anti- high int lo CFSE (no division), CFSE (one to four divisions), and CFSE (five CD3 plus soluble anti-CD28 Abs (C), or treated with dexamethasone (Dex) G 6 or more divisions). ( ) Cell numbers of each fraction (mean SEM, n = or TNF-a (D). Il7ra transcripts were measured by real-time RT-PCR , , , 4). *p 0.05, **p 0.01, ***p 0.005. (mean 6 SEM, n = 2–3). *p , 0.05, **p , 0.01. The Journal of Immunology 3135

over time in CNS1+/+ OT-I cells at later time points (from days 7 to 25). CNS12/2 OT-I cells completely lost IL-7Ra expression by day 3, suggesting that the CNS1 element is dispensable for IL-7Ra downregulation by Ag stimulation. After the peak of expansion, CNS12/2 OT-I cells showed IL-7Ra upregulation (from days 7 to 15). To evaluate the frequency of memory phenotype effector CD8 T cells, we next analyzed Ly-6C expression in OT-I cells (40). Both mutant and wild-type OT-I cells showed comparable frequencies of Ly-6C+ cells during the immune response (Fig. 8D). These results demonstrate that although IL-7Ra expression is controlled by the CNS1 element, changes in IL-7Ra expression that occur during the immune response are independent of the CNS1. The CNS1 element controls survival of effector T cells Given the relative reduction of CNS12/2 OT-I cells compared with wild-type OT-I cells, we next compared the function of these cells. To do so, we first evaluated their cytokine production upon restimulation. Three days after immunization, spleen cells from recipients transferred with CNS1+/+ or CNS12/2 OT-I cells were Downloaded from restimulated with OVA peptide, and IFN-g production was assessed by flow cytometry. Both CNS1+/+ and CNS12/2 OT-I cells produced IFN-g at similar levels (Fig. 8E). Next, we compared Bcl-2 protein expression. At the peak of the primary response (day 3), Bcl-2 was expressed at comparable levels between 2 2 CNS1+/+ and CNS1 / OT-I cells (Fig. 8F). However, afterward http://www.jimmunol.org/ (by day 7), Bcl-2 expression significantly declined in CNS12/2 effector OT-I cells, suggesting that the CNS1 element is crucial for induction of Bcl-2 expression in effector T cells. To evaluate proliferation of Ag-specific CD8 T cells, we next analyzed Ki-67 expression. Whereas Ki-67 expression was comparable between CNS1+/+ and CNS12/2 OT-I cells at day 3, the proportion of Ki- 67hi cells significantly increased in CNS12/2 OT-I cells after the peak of expansion (Fig. 8G). These results suggest that the CNS1 by guest on October 2, 2021 element may control survival of effector T cells. FIGURE 8. The CNS1 element controls maintenance of Ag-stimulated Changes in IL-7Ra expression in CD8 T cells during L. +/+ 2/2 CD8 T cells. Purified T cells from B6.OT-I CNS1 Rag2 and B6.OT-I monocytogenes infection are independent of the CNS1 element CNS12/2Rag22/2 mice were adoptively transferred into naive B6.CD45.1 recipient mice. After 24 h, mice were immunized with OVA and LPS (day To determine the function of CNS1 element during the immune 0). Transferred OT-I cells were analyzed at 3–25 d after primary immuni- response after infection and to exclude any possibility of rejection 2 2 zation. (A) Flow cytometric analysis of transferred OT-I cells. (B) Numbers of transferred T cells by host, we infected CNS1+/+ or CNS1 / of transferred OT-I cells (mean 6 SEM, n = 4). (C) Surface IL-7Ra ex- mice with rLM-OVA and analyzed the OVA-specific CD8 T cell pression in transferred OT-I cells. (D) Surface Ly-6C expression in trans- response. At the peak of the primary response (day 7), both ferred OT-I cells. (E) Three days postimmunization, spleen cells were +/+ 2/2 + b + CNS1 and CNS1 CD44 OVA2572264/K MHC tetramer restimulated with OVA peptide and analyzed for intracellular expression of CD8 T cells had drastically expanded (Fig. 9A). Indeed, CNS12/2 6 F IFN-g in transferred OT-I cells (mean SEM, n =3).() Bcl-2 ex- cells proliferated more extensively compared with CNS1+/+ cells pression in transferred OT-I cells. (G) Ki-67 expression in transferred 2 2 at day 7 (Fig. 9B). Whereas both CNS1+/+ and CNS1 / OVA- OT-I cells. *p , 0.05, **p , 0.01, ***p , 0.005. specific CD8 T cells were reduced during contraction, CNS12/2 cells tended to decrease more rapidly than did CNS1+/+ cells. We numbers and the proportion of CNS12/2 OT-I cells were slightly next compared surface expression of IL-7Ra, Ly6C, and KLRG1 +/+ reduced compared with CNS1+/+ OT-I cells before immunization in OVA-specific CD8 T cells during the infection. CNS1 and 2/2 (∼70% of control) (day 0, Fig. 8A, 8B). At the peak of the primary CNS1 OVA-specific CD8 T cells showed similar time course response (day 3), the population of CNS1+/+ OT-I cells had of IL-7Ra, Ly6C, and KLRG1 expression, although the proportion lo drastically expanded, whereas the absolute numbers and propor- of KLRG1 memory precursor effector T cells tended to be lower 2/2 +/+ tion of CNS12/2 OT-I cells were significantly reduced compared in CNS1 cells compared with CNS1 cells at days 14 and 21 with controls (∼50% of control). After the peak, the number of (Fig. 9C). These results suggest that the changes of IL-7Ra ex- 2/2 CNS12/2 OT-I cells substantially declined compared with con- pression during the infection are not impaired in CNS1 mice. trols during contraction (∼17 and ∼7% of control at days 7 and 15, respectively) and were hardly detectable at day 25. Discussion We next compared surface IL-7Ra expression in Ag-specific In this study, we determined the function of the CNS1 element in CD8 T cells during the immune response. Whereas naive IL-7Ra expression during lymphocyte development and immune CNS1+/+ OT-I cells expressed high levels of IL-7Ra at day 0, most response. We first showed that IL-7Ra expression was unchanged effector cells slightly downregulated IL-7Ra at the peak of ex- in thymocytes of CNS12/2 mice, except that Treg cells showed pansion (day 3) (Fig. 8C). IL-7Ra expression slightly increased significantly lower IL-7Ra expression. We also found that IL-7Ra 3136 FUNCTION OF AN IL-7 RECEPTOR LOCUS ENHANCER

CNS12/2 naive T cells. As reported, Bcl-2 expression is not completely abrogated in IL-7Ra–deficient mice (6). Thus, these prosurvival proteins may be maintained via the activity of other cytokines present in the normal lymphoid microenvironment. Al- ternatively, decreased levels of these prosurvival proteins may lead to rapid cell death. Deletion of CNS1 element did not alter regulation of IL-7Ra by IL-7 and TCR signals: IL-7Ra transcription was induced by growth factor withdrawal and repressed by IL-7 and TCR signals in CNS12/2 T cells, albeit at lower expression levels than seen in wild-type T cells. In contrast to CNS12/2 T cells, Foxo1-deficient T cells failed to up-regulate IL-7Ra after growth factor withdrawal (20). IL-7 signaling activates the PI3K/Akt pathway, which then inhibits Foxo1 activity by phosphorylation (41). Foxo1 reportedly binds to the CNS1 element and enhances IL-7Ra expression in peripheral T cells (20). Although lack of Foxo1 binding to the CNS1 contributes in part to IL-7Ra reduction in CNS12/2 T cells, Foxo1 and/or other transcription factors may further modulate IL- 7Ra transcription through different control elements. CNS12/2 T cells failed to induce IL-7Ra transcription in re- Downloaded from sponse to GC and TNF-a in vitro, suggesting a potential role for these factors in controlling IL-7Ra expression in peripheral T cells. Although GR-deficient mice show normal cellular composition of thymus and peripheral lymphoid organs (42, 43), little is known about IL-7Ra expression in GR-deficient T cells. Following infection with lymphocytic choriomeningitis virus, effector CD8 http://www.jimmunol.org/ T cells reportedly express the IL-7Ra at lower levels in TNF-a– deficient compared with control mice (44). The NF-kB consensus motif found in the CNS1 element may function in IL-7Ra induction by TNF-a. Further studies should elucidate how GC and TNF-a FIGURE 9. IL-7Ra expression during Listeria infection is independent regulate IL-7Ra. 2 2 of the CNS1 element. (A) CNS1+/+ or CNS1 / mice were infected with Treg cells are highly dependent on the CNS1 element for IL- b 2/2 LM-OVA. Flow cytometric analysis of CD8 T cells positive for OVA257–264/K 7Ra expression. Compared to wild-type cells, CNS1 Treg tetramer. Spleen cells were isolated from infected mice at 7, 14, 21, cells completely lost IL-7Ra expression in thymus and lymph 28, 35, and 40 d after primary infection. Numbers in quadrants indicate the by guest on October 2, 2021 b + nodes. Treg cells receive stronger agonistic TCR signals than do percentages of CD44 and OVA257–264/K tetramer cells in CD8 T cells. + b + conventional CD4 T cells. Therefore, agonistic TCR signaling (B) Kinetics of the ratio of knockout CD44 OVA257–264/K tetramer CD8 T cells to wild-type cells. (C) Expression of IL-7Ra, Ly6C, and KLRG1 on may exert effects through the CNS1 element in this cell type. In b + D contrast, anti-CD3 and anti-CD28 stimulation downregulated IL- CD44 and OVA257–264/K tetramer CD8 T cells. ( ) Kinetics of the ratio 2 2 + b + lo / of knockout CD44 OVA257–264/K tetramer KLRG1 CD8 T cells to wild- 7Ra in CNS1 T cells in vitro. Such stimulation might be type cells. KO, knockout; WT, wild-type. much stronger than agonistic TCR signals in Treg cells and exert effects through control elements other than the CNS1. Further- more, because IL-7Ra–deficient Treg cells show defects in expression was unchanged during B cell development in the suppressive activity (2, 45), CNS12/2 Treg cells may show weaker bone marrow of CNS12/2 mice. In contrast, IL-7Ra expression suppression than CNS1+/+ Treg cells. was significantly reduced in peripheral T cell subpopulations of The CNS1 element plays a role in clonal expansion of naive CD8 CNS12/2 mice. Naive T cells of CNS12/2 mice showed signifi- T cells. OT-I cells from CNS12/2 mice showed impaired expan- cantly reduced survival and homeostatic proliferation. Addition- sion after Ag stimulation compared with cells from CNS1+/+ mice. ally, IL-7Ra upregulation by GC and TNF-a was completely Although OT-I cells from wild-type mice downregulated IL-7Ra inhibited in naive CNS12/2 T cells in vitro. Clonal expansion of after Ag stimulation, they expressed IL-7Ra at substantial levels naive T cells and maintenance of memory T cells were significantly at the peak of the immune response (day 3). In contrast, OT-I cells impaired in CNS12/2 CD8 T cells. Although IL-7Ra expression from CNS12/2 mice completely lost IL-7Ra expression by day 3. was significantly lower in CNS12/2 CD8 T cells, regulation of IL- Therefore, IL-7 signaling may transmit a competence signal for 7Ra expression during the immune response was retained. Thus, this efficient clonal expansion. In agreement with this idea, we pre- study demonstrates that the CNS1 element is an enhancer essential viously observed that naive T cells from CD4-Cre IL-7Raflox/flox for IL-7Ra expression in peripheral T cells and suggests a potential mice proliferate less efficiently than do T cells from control mice role for GC and TNF-a in controlling IL-7Ra expression and im- after anti-CD3 and anti-CD28 stimulation (2). mune homeostasis. The CNS1 element may be important for survival of Ag- The CNS1 element controls survival and proliferation by regu- stimulated effector T cells. CNS12/2 Ag-specific CD8 T cells lating IL-7Ra expression in peripheral T cells. CNS1-deficient mice showed a substantial reduction in effector cells, which was as- represent a new model: they exhibit a profound defect in IL-7Ra sociated with low levels of IL-7Ra and Bcl-2 protein. Con- expression in peripheral T cells but do not show complications in sistently, the number of CNS12/2 memory CD8 T cells was T cell development associated with deficiencies in IL-7 or IL-7Ra. substantially reduced. Despite loss of the CNS1 element, we Although naive CNS12/2 T cells showed defective cell survival observed relatively normal alterations in IL-7Ra expression 2/2 in vitro, Bcl-2, Bcl-xL, and Mcl-1 expression was unchanged in in CNS1 CD8 T cells. These data indicate that the CNS1 The Journal of Immunology 3137 element may control survival and proliferation of effector cells 14.Chen,X.,L.Fang,S.Song,T.B.Guo,A.Liu,andJ.Z.Zhang.2009.Thymic + by regulating IL-7Ra expression. regulation of autoimmune disease by accelerated differentiation of Foxp3 regulatory T cells through IL-7 signaling pathway. J. Immunol. 183: 6135– The CNS1 element is essential for IL-7Ra expression in pe- 6144. ripheral T cells but not thymocytes. One explanation for this 15. Franchimont, D., J. 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