Of Antigen Receptor-Driven T Cells Hypoxia-Inducible Factor Regulates Survival

Hypoxia-Inducible Factor Regulates Survival of Antigen Receptor-Driven T Cells Yuichi Makino, Hiroshi Nakamura, Eiji Ikeda, Kei Ohnuma, Kenji Yamauchi, Yutaka Yabe, Lorenz Poellinger, Yasunori This information is current as Okada, Chikao Morimoto and Hirotoshi Tanaka of September 28, 2021. J Immunol 2003; 171:6534-6540; ; doi: 10.4049/jimmunol.171.12.6534 http://www.jimmunol.org/content/171/12/6534 Downloaded from

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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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Hypoxia-Inducible Factor Regulates Survival of Antigen Receptor-Driven T Cells1

‡,Kenji Yamauchi ء,Eiji Ikeda,† Kei Ohnuma ء,Hiroshi Nakamura ء,Yuichi Makino and ء,Yutaka Yabe,‡ Lorenz Poellinger,§ Yasunori Okada,† Chikao Morimoto ءHirotoshi Tanaka2

Peripheral T lymphocytes undergo activation by antigenic stimulation and function in hypoxic areas of inflammation. We demonstrated in CD3-positive human T cells accumulating in inflammatory tissue expression of the hypoxia-inducible factor-1␣ (HIF-1␣), indicating a role of hypoxia-mediated signals in regulation of T cell function. Surprisingly, accumulation of HIF-1␣ in human T cells required not only hypoxia but also TCR/CD3-mediated activation. Moreover, hypoxia repressed activation-induced cell death (AICD) by TCR/CD3 stimulation, resulting in an increased survival of the cells. Microarray analysis suggested the involvement of HIF-1 target gene product adrenomedullin (AM) in this process. Indeed, AM receptor antagonist abrogated Downloaded from hypoxia-mediated repression of AICD. Moreover, synthetic AM peptides repressed AICD even in normoxia. Taken together, we propose that hypoxia is a critical determinant of survival of the activated T cells via the HIF-1␣-AM cascade, defining a previously unknown mode of regulation of peripheral immunity. The Journal of Immunology, 2003, 171: 6534–6540.

successful immune response is achieved by rapid mo- different compartments of the body, T cells are likely to encounter bilization of circulating peripheral T cells and antigen- significant differences in oxygen tension, i.e. ϳ100 mm of Hg http://www.jimmunol.org/ driven expansion of a certain fraction of the cells in situ. (14% O ) in arterial blood and 40 mm of Hg (5Ð6% O ) or less in A 2 2 Subsequently, accumulated T cells may need to be cleared away to the tissue interstitium (9). Moreover, activated T cells accumulate prevent a harmful over-response or for preservation of homeostasis and function in an area of inflammation or tumor growth, both of within the T cell compartment of peripheral immunity. An apo- which are known to be hypoxic (10). Responses of T cells to hyp- 3 ptotic process termed activation-induced cell death (AICD) trig- oxia, therefore, may be essential not only for adaptation but also gered by repeated Ag challenge via the TCR/CD3 complex, is for their functional performance. believed to be a mechanism for efficient elimination of activated T Cellular adaptation to hypoxic conditions involves a transcrip- cells (1, 2). Dysregulation of AICD in T cells has been shown to tional response pathway mediated by the hypoxia-inducible factor by guest on September 28, 2021 result in autoimmune diathesis (3), failure in transplantation tol- (HIF)-1, a heterodimeric complex of the basic helix-loop-helix erance (4), or immunosuppression by an environmental toxin such (bHLH) PAS (Per, Arnt, Sim) domain proteins HIF-1␣ and as dioxin (5). AICD in T cells has been suggested to be mediated HIF-1␤ (Arnt) (11Ð13). Two distinct mechanisms are important mainly by Fas (CD95)/Fas-ligand (Fas-L) interaction (6), and also for regulation of HIF-1 activity by oxygen. Under normoxic con- has been modulated by environmental constituents (7, 8). The ␣ mechanism of regulation of AICD in situ, however, remains ditions, HIF-1 is targeted by the von Hippel-Lindau protein largely unknown. It should be noted that during traffic through (pVHL) for ubiquitylation and rapid proteasomal degradation (14, 15). pVHL binding is mediated through hydroxylation of specific prolyl residues within the N-terminal transactivation domain

␣ ء Division of Clinical Immunology, Advanced Clinical Research Center, Institute of (TAD) of HIF-1 by a set of dioxygenases that have an absolute Medical Science, University of Tokyo, †Department of Pathology, Keio University requirement of dioxygen, iron, and 2-oxoglutarate as cosubstrates School of Medicine, ‡Department of Orthopedics, Kyosai Tachikawa Hospital, To- kyo, Japan; and ¤Department of Cell and Molecular Biology, Medical Nobel Institute, (16Ð18). Upon a decrease in available oxygen levels, there is a Karolinska Institutet, Stockholm, Sweden corresponding decrease in prolyl hydroxylation of HIF-1␣, result- Received for publication July 9, 2003. Accepted for publication October 13, 2003. ing in release of pVHL and dramatic stabilization of HIF-1␣ pro- The costs of publication of this article were defrayed in part by the payment of page tein. In addition, hypoxia induces interaction between the C-ter- charges. This article must therefore be hereby marked advertisement in accordance minal TAD (C-TAD) of HIF-1␣ and transcriptional coactivators with 18 U.S.C. Section 1734 solely to indicate this fact. (19Ð21). Under normoxic conditions, this interaction is blocked by 1 This work was supported by Japan Society for the Promotion of Science, Kanagawa Academy of Science and Technology, The Vehicle Racing Commemorative Foun- hydroxylation of an asparagine residue within the C-TAD of dation, the Cell Science Research Foundation, and the Ministry of Education, Culture, HIF-1␣ (22). HIF-1␣-mediated signaling serves as a master regu- Sports, Science, and Technology of Japan. lator in oxygen homeostatic processes, from sensing to responding 2 Address correspondence and reprint requests to Dr. Hirotoshi Tanaka, Division of to changes in environmental oxygen tension. Expression of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Sci- ence, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639 Japan. HIF-1␣ has been documented in a wide variety of mammalian E-mail address: [email protected] cells including immune cells (23). A recent study has indicated that 3 Abbreviations used in this paper: AICD, activation-induced cell death; Fas-L, Fas genetic disruption of HIF-1␣ expression resulted in abnormal B ligand; HIF, hypoxia-inducible factor; bHLH, basic helix-loop-helix; PAS, Per-Arnt- Sim; pVHL, von Hippel-Lindau; TAD, transactivation domain; C-TAD, C-terminal cell development and autoimmunity (24), and that selective dele- TAD; AM, adrenomedullin; CGRP, calcitonin gene-related peptide; PI, propidium tion of HIF-1␣ in granulocytes and macrophages/monocytes leads iodide; VEGF, vascular endothelial growth factor; CRLR, calcitonin receptor-like receptor; RAMP, receptor activity-modifying protein; HRE, hypoxia response ele- to impairment of inﬂammatory responses such as motility and in- ment; GLUT, glucose transporter. vasiveness of, and bacterial killing by, those cells (25). The role of

HIF-1␣ in regulation of function of peripheral T cells, however, is RT-PCR analysis largely unknown. Total RNA was isolated from T cells by guanidine isothiocyanate lysis/ In this study, we described that AICD of human peripheral T phenol chloroform extraction, followed by removal of contaminating cells by TCR/CD3 engagement was suppressed under hypoxic DNA. First-strand cDNA was synthesized using 2 ␮g of DNase-treated conditions and HIF-1 target gene product adrenomedullin (AM) total RNA as a template in 20 ␮l of reaction mixture containing 50 mM Tris-HCl (pH 8.3), 3 mM MgCl , 10 mM DTT, 75 mM KCl, 1 mM dNTPs, increased survival of T cells in an autocrine fashion. Therefore, 2 ␣ 0.1 mM oligo dT (25) primer, and 50 U Superscript II (Invitrogen, Carls- HIF-1 -AM cascade-mediated control of T cell survival may con- bad, CA) at 42¡C for 50 min. PCR was carried out in a total volume of 30 stitute a novel milieu for regulation of T cell-mediated immune ␮l in a mixture composed of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 ␮ ␮ response in situ. mM MgCl2, 200 M dNTPs, 0.25 M each of the sense and antisense primers, 1 U ExTaq DNA polymerase (TaKaRa, Ohtsu, Japan). The amount of cDNA, as judged by the intensity of the amplified ␤-actin signal, Materials and Methods was comparable among the preparations. Amplification by 27 cycles of Abs and reagents 94¡C for 30 s, 50¡C for 30 s, and 72¡C for 1 min was performed after 3 min of denaturing of the samples at 94¡C, and shown to be within linear range mAb against human CD3 (UCHT1) was purchased from BD PharMingen or nonsaturated conditions for ␤-actin amplification. Identities of the PCR (San Diego, CA). Anti-human Fas Ab (CH11) was from Medical & Bio- products were confirmed by sequencing. PCR primer pairs for amplifica- logical Laboratories (Nagoya, Japan). Anti-human HIF-1␣ Ab (Ab463) tion of each gene are as following; Fas, sense: 5Ј-CCAAGTGACTGACA was from Abcam (Cambridge, U.K.). Recombinant human IL-2 was ob- TCAACTC-3Ј, antisense: 5Ј-ACTCTTTGCACTTGGTGTTGC-3Ј, Fas-L, tained from PeproTech (London, U.K.) and PHA-M was from Sigma-Al- sense: 5Ј-GGAATGGGAAGACACCTATG-3Ј, antisense: 5Ј-GCACTGG drich (St. Louis, MO). Synthetic peptides of human AM and human cal- TAAGATTGAACAC-3Ј; bcl-2, sense: 5Ј-TGAACTGGGGGAGGATT Ј Ј Ј citonin gene-related peptide (CGRP) 8Ð37 were provided by Peptide GTG-3 , antisense: 5 -GCCAGGAGAAATCAAACAGA-3 ; bcl-xL sense: Downloaded from Institute (Minoh, Japan). Other chemicals were purchased from Sigma- 5Ј-CCCAGAAAGGATACAGCTGG-3Ј, antisense; 5Ј-GCGATCCGACT Aldrich unless specified. CACCAATAC-3Ј; vascular endothelial growth factor (VEGF), sense: 5Ј- TGCCTTGCTGCTCTACCTCC-3Ј, antisense: 5Ј-TCACCGCCTCGGCT Cell culture and activation TGTCAC-3Ј; glucose transporter (GLUT)-1, sense: 5Ј-CTTTCTCCAG CCAGCAATGA-3Ј, antisense: 5Ј-TGGATCCTGAGTCGAAGTCT-3Ј, Fresh PBMC were prepared from heparinized blood of healthy volunteers GLUT-3, sense: 5Ј-GATGCTGGAGAGGTTAAGGT-3Ј, antisense: 5Ј- by Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) density ACTTCCACCCAGAGCAAAGT-3Ј; AM, sense: 5Ј-AAGAAGTGGAA gradient centrifugation and suspended in RPMI 1640 medium (Sigma-Al- TAAGTGGGCT-3Ј, antisense: 5Ј-TGGCTTAGAAGACACCAGAGT-3Ј; http://www.jimmunol.org/ drich) supplemented with 10% FCS and antibiotics. Adherent cells were calcitonin receptor-like receptor (CRLR), sense: 5Ј-GATGCTCTGTGA removed by incubation on plastic dishes for1hat37¡C, and the rest of the AGGCATTT-3Ј, antisense: 5Ј-CAGAATTGCTTGAACCTCTC-3Ј, recep- cells were separated on nylon wool columns to obtain T cell-rich fraction. tor-activity-modifying protein (RAMP)1, sense: 5Ј-GAGACGCTGTGGT For activation, T cell blasts were generated by stimulation of the T cell GTGACTG-3Ј, antisense: 5Ј-TCGGCTACTCTGGACTCCTG-3Ј; RAMP2, ϫ 6 ␮ fraction (1 10 cells/ml) with 5 g/ml PHA-M for 48 h. The mitogen sense: 5Ј-GGACGGTGAAGAACTATGAG-3Ј, antisense: 5Ј-ATCATGGC was then washed out, and the cells were maintained in the medium with 10 CAGGAGTACATC-3Ј; ␤-actin, sense: 5Ј-CCTCGCCTTTGCCGATCC-3Ј, ϫ ng/ml recombinant human IL-2. TCR/CD3 engagement of the cells (1 antisense: 5Ј-GGATCTTCATGAGGTAGTCAGTC-3Ј. 106 cells/ml) was performed in the presence of 10 ng/ml IL-2 on 96-well plates coated with CD3 Ab (5 ␮g/ml). Jurkat T cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in fully DNA microarray analysis supplemented RPMI 1640 medium. A Jurkat T cell derivative stably inte- by guest on September 28, 2021 grated with cDNA encoding HIF-1␣/1-396/VP16 AD chimeric protein, DNA microarrays (human genome U95A v2 array; Affymetrix, Santa Jurkat-HIF-1␣, was established by means of G418 selection. Exposure of Clara, CA) were hybridized with labeled cRNA derived from T cells with the cells to various oxygen concentrations was carried out as described various treatments as recommended by the manufacturer. Differential ex- previously (14). pression, as analyzed by GeneChip 3.1 software (Affymetrix), was assessed by pairwise comparisons of samples from each treatment with untreated subject. The criterion for differential expression required an over 2-fold Determination of cell death and viability increase or decrease in mRNA expression levels. The cells were harvested after culture under the indicated conditions and incubated with Annexin VFITC (0.25 ␮g/ml) and propidium iodide (PI) (50 ␮g/ml) in binding buffer (10 mM HEPES, 140 mM sodium chloride, 2.5 Results mM calcium chloride) 30 min before flow cytometric analysis for detection Expression of HIF-1␣ in T cells accumulating in the interstitium of apoptotic/necrotic cells. Numbers and the ratio of the viable cells were of inflammatory tissue determined by means of trypan blue dye exclusion and counting on a hemocytometer. We first examined expression of HIF-1␣ in T cells accumulating in a hypoxic inflammatory tissue. Immunohistochemical analysis Immunoblot assay demonstrated expression of HIF-1␣ in infiltrating CD3-positive T Immunodetection of HIF-1␣ protein was performed as described previ- cells in the synovial tissue of a patient with rheumatoid arthritis ously (16). Briefly, whole cell extracts of T cells were prepared in lysis (Fig. 1). The largest portion of HIF-1␣ staining was found in the buffer (25 mM HEPES, 100 mM NaCl, 5 mM EDTA, 100 ␮M orthovana- cytoplasmic compartment of T cells, and a nuclear signal was date, and 0.5% Triton X-100, pH 7.9), followed by centrifugation for 30 only occasionally detected (Fig. 1B). The affected skin in a min at 14,000 rpm. One hundred micrograms of whole cell extract were separated by SDS-PAGE and blotted onto polyvinylidine difluoride filters. patient with dermatomyositis also revealed accumulation of The filters were incubated with anti HIF-1␣ Ab (Abcam) in TBS contain- CD3-positive T cells expressing HIF-1␣ (data not shown). ing 1% nonfat milk at 4¡C overnight, followed by anti-mouse Ig-HRP Given these results, we hypothesized that HIF-1␣ might have a conjugate (Amersham Biosciences) in the same buffer. Immunocomplexes role in regulation of T cell-mediated immune reactions in the were visualized by ECL (Amersham Biosciences). inflammatory tissues. Immunohistochemistry Hypoxia represses AICD of human peripheral T cells with Samples of the synovial tissue were fixed overnight in 4% parafolmalde- ␣ hyde at 4¡C, dehydrated in alcohol, and embedded in paraffin. Four-mi- concomitant expression of HIF-1 crometer sections were then deparaffinized and incubated with monoclonal To explore the potential role of HIF-1␣ in regulation of T cell anti-HIF-1␣ Ab and alkaline phosphatase-conjugated anti mouse Ig Ab, or polyclonal anti-human CD3 Ab (DAKO, Glostrup, Denmark) and histofine function in the microenvironment of the tissue, we investigated the simple stain MAX PO (Nichirei, Tokyo, Japan). Nonimmunized mouse Ig effect of hypoxia on AICD of peripheral T cells. PHA-activated was used as a negative control for the primary Ab. human peripheral blood T cells were maintained with IL-2 6536 HIF-1␣ REGULATES SURVIVAL OF ACTIVATED T CELLS Downloaded from FIGURE 1. Expression of HIF-1␣ in CD3-positive T cells accumulating in inflammatory tissues. Sections from the synovium from a patient with rheumatoid arthritis were stained for expression of HIF-1␣ and CD3. A, Low magnification; B, high magnification. Double staining with the Abs against HIF-1␣ (blue staining by alkaline phosphatase/fast blue) and CD3 (brown staining by peroxidase/3,3Ј-diaminobenzidine tetrahydrochloride).

Arrows indicate CD3-positive cells with cytoplasmic HIF-1␣ expression, http://www.jimmunol.org/ and the arrowhead shows CD3-positive cells with nuclear signals of HIF- 1␣. Scale bars in A, and B represent 100 and 20 ␮m, respectively. C, Staining with anti-CD3 Ab and mouse IgG as negative control for a primary Ab (scale bar, 20 ␮m). FIGURE 2. Oxygen concentration regulates survival of TCR/CD3- stimulated T cell. A, Hypoxia and TCR/CD3 stimulation repress AICD in peripheral T cells. PHA-preactivated human peripheral T cells maintained supplementation, followed by incubation with an immobilized in the medium supplemented with IL-2 (PHA-activated T cells) were cul- Ϫ ␮ mAb against CD3 either under normoxic (21% O2) or hypoxic (1% tured in the absence ( ) or presence of immobilized CD3 Abs (5 g/ml) by guest on September 28, 2021 O2) conditions. Staining with annexin V and PI revealed massive either at 21% O2 (CD3) or at 1% O2 (CD3/hypoxia). Apoptotic cell death apoptotic cell death of T cells induced by TCR/CD3-mediated ac- was detected by staining the cells with Annexin VFITC and PI followed by FITC tivation under normoxic conditions, indicating the induction of flow cytometric analysis, and percentages of Annexin V and/or PI- AICD. To our surprise, this TCR/CD3-triggered AICD of T cells positive cells are indicated. Three independent experiments were performed and representative results are shown. B, Role of oxygen concen- was dramatically attenuated by exposure of the cells to hypoxia tration in T cell survival. PHA-activated T cells were incubated in the (Fig. 2A). When the cells were incubated at various oxygen con- ■ ᮀ presence ( ) or absence ( ) of CD3 Abs at 21, 10, 7, 5, 3, and 1% O2, centrations (ranging from 21 to 1% O2), the survival rates of T respectively, for 72 h, and cell viability was determined by the trypan cells after TCR/CD3 ligation were gradually increased as oxygen blue-dye exclusion method. Cell counting was performed in triplicate, and ,p Ͻ 0.01 ,ء .tension decreased, whereas survival of T cells without TCR/CD3 means Ϯ SD of percentages of the viable cells are shown Ͻ ءء ligation showed proportional reduction (Fig. 2B). This influence of compared to the cells without CD3 stimulation at 21% O2; , p 0.01, the oxygen concentration on T cell viability became evident when compared to the cells with CD3 stimulation at 21% O2. C, Hypoxia- and the oxygen concentration was decreased to 7 or 5%, corresponding TCR/CD3 stimulation-dependent expression of HIF-1␣ protein in T cells. to that often encountered in the tissue interstitium. These results PHA-activated T cells were incubated in the absence (Ϫ) or presence of indicate that not only activation through the Ag receptor but also immobilized CD3 (CD3) or soluble Fas (Fas) Abs (100 ng/ml) at indicated oxygen concentrations, and expression of protein (top panel, filled the oxygen concentration play critical roles in determination of arrowhead) and mRNA (middle panel, open arrow head) levels of -were determined by im (ء ,peripheral T cell life. HIF-1␣ and ␤-actin mRNA (bottom panel ␣ HIF-1 mRNA was constitutively expressed in human periph- munoblot and RT-PCR, respectively. Numbers (kDa) at left side of the eral T cells (Fig. 2C). In contrast, it was not possible to detect top panel depict the molecular masses of the reference proteins run in HIF-1␣ protein by immunoblot in the extracts from unstimulated T parallel. D, Role of hypoxia in HIF-1␣ protein expression in PMA/ cells even if the cells had been exposed to hypoxia (1% O2 (Fig. ionomycin-activated T cells. PHA-activated T cells were incubated in 2C). However, Ab-mediated TCR/CD3 engagement resulted in the presence (P/I) or absence (Ϫ) of PMA (10 ng/ml)/ionomycin (1 ␮M) dramatic accumulation of HIF-1␣ protein upon exposure of the at the indicated oxygen concentrations for 24 h and cell lysates were cells to hypoxic conditions corresponding to tissue oxygen levels subjected to immunoblot analysis for HIF-1␣ expression (filled arrow- (below 7% O ), indicating that expression of HIF-1␣ protein in head). Open arrowheads indicate nonspecific bands. Numbers (kDa) 2 depict the molecular masses of the reference proteins run in parallel. peripheral T cells requires not only hypoxia but also additional Representative results of triplicated experiments are shown. E, Oxygen signals following TCR/CD3-mediated stimulation (Fig. 2C and concentration-dependent inhibition of PMA/ionomycin-induced AICD data not shown). Because Ab-cross-link of Fas molecules did of T cells. PHA-activated T cells were incubated in the presence of not induce HIF-1␣ protein accumulation either under normoxic PMA/ionomycin at 21, 10, 7, 5, 3, and 1% O2 for 24 h, cell viability was or hypoxic conditions (Fig. 2C), it may not be the cell death determined by trypan blue-dye exclusion, and results of three indepen- p Ͻ 0.01, compared to ,ء .executing components but activation pathways after TCR/CD3 dent assays were shown as described above ligation that are involved in HIF-1␣ induction. Activation of T the cells at 21% O2. The Journal of Immunology 6537

Table I. DNA microarray analysis of gene expression proﬁle in T cells with TCR/CD3 stimuli under hypoxic conditionsa

Fold Change Compared to Untreated Cells GenBank Accession Common Name Number CD3 Hypoxia CD3/Hypoxia

Apoptosis-related genes Fas X83490 2.9 Ϫ2.0 2.3 Fas-L U11821 4.6 ϽϮ2.0 3.0 bcl-2 M14745 3.5 ϽϮ2.0 3.9 Ϫ ϽϮ Ϫ Bcl-xL Z23115 7.2 2.0 6.4 TRAP-1 U12595 2.5 ϽϮ2.0 ϽϮ2.0 TRAIL U37518 ϽϮ2.0 Ϫ3.3 ϽϮ2.0 TRADD L41690 ϽϮ2.0 ϽϮ2.0 ϽϮ2.0 p53 X02469 2.3 ϽϮ2.0 ϽϮ2.0

Hypoxia-inducible genes Aldolase A X05236 3.4 ϽϮ2.0 4.1 Enolase X66610 ϽϮ2.0 ϽϮ2.0 ϽϮ2.0 GAPDH M33197 5.9 3.2 3.5 GLUT1 K03195 ϽϮ2.0 ϽϮ2.0 ϽϮ2.0 GLUT3 M20681 2.9 8.2 9.0

VEGF AF022375 2.4 7.0 8.1 Downloaded from AM D14874 ϽϮ2.0 2.5 7.2

Secreted mediators IL-2 X00695 ϽϮ2.0 ϽϮ2.0 ϽϮ2.0 IL-4 M13982 ϽϮ2.0 ϽϮ2.0 ϽϮ2.0 IL-5 X04688 Ϫ18.1 ϽϮ2.0 Ϫ8.0 IL-8 M28130 ϽϮ2.0 Ϫ5.0 Ϫ3.3 IL-13 U31120 24.5 ϽϮ2.0 12.7 http://www.jimmunol.org/ IFN-␥ X13274 44.4 Ϫ5.8 17.8 IFN-␤ X04430 ϽϮ2.0 Ϫ10.1 Ϫ6.1 TNF-␣ X02910 5.9 ϽϮ2.0 3.4 MIF L19686 3.6 2.0 3.9 GM-CSF M13207 17.8 Ϫ3.2 10.8

Cell surface molecules IL-1 receptor antagonist X52015 Ϫ2.9 Ϫ6.5 Ϫ10.5 IL-2 receptor X01057 14.9 ϽϮ2.0 7.7 IL-6 receptor X12830 ϽϮ2.0 ϽϮ2.0 Ϫ3.8 IL-15 receptor U31628 4.5 ϽϮ2.0 4.7 by guest on September 28, 2021 IL-10 receptor U00672 Ϫ3.4 ϽϮ2.0 Ϫ2.6 IL-12 receptor U64198 Ϫ41.8 ϽϮ2.0 Ϫ10.0 TNF receptor M32315 2.0 Ϫ2.1 ϽϮ2.0 LFA-1␤ M15395 2.8 ϽϮ2.0 2.0 ␣ Ϫ Ϫ Ϫ Integrin 6 X53586 12.2 2.3 11 ␤ Ϫ Ϫ Ϫ Integrin 6 S66213 7.4 3.7 7.9 CCR6 U45984 ϽϮ2.0 ϽϮ2.0 Ϫ2.7

Signaling molecules MEK 1 L11284 3.8 ϽϮ2.0 5.3 MEK 2 L11285 2.3 ϽϮ2.0 5.3 c-Src X59932 2 ϽϮ2.0 ϽϮ2.0 MAPKK K05624 4.1 ϽϮ2.0 ϽϮ2.0 MAPKKK5 U67156 Ϫ2.4 Ϫ2.8 Ϫ6.1 T cell-speciﬁc tyrosine kinase L10717 2.2 Ϫ2.2 ϽϮ2.0

Transcriptional regulators NF-ATc U08015 7.1 ϽϮ2.0 8.6 NF-IL6 X52560 ϽϮ2.0 ϽϮ2.0 ϽϮ2.0 c-fos V01512 2.5 ϽϮ2.0 3.2 c-jun J04111 3.1 ϽϮ2.0 ϽϮ2.0 Jun D X56681 3.8 ϽϮ2.0 3.7 NF-␬B subunit X61498 4.9 ϽϮ2.0 3.1 I␬B␧ U91616 4.0 ϽϮ2.0 2.8 FKBP52 M88279 4.8 ϽϮ2.0 2.5 CBP U47741 ϽϮ2.0 ϽϮ2.0 Ϫ2.7

a TRAP, TNF type 1 receptor-associated protein; TRADD, TNFR-1 associated death domain protein; MIF, macrophage migration inhibitory factor; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase; MAPKK, MAP kinase kinase; MAPKKK, MAP kinase kinase kinase; NF-ATc, NF-AT cytoplasmic component; NF-IL-6, nuclear factor of IL-6; I␬B, inhibitory subunit of NF-␬B; FKBP, FK506 binding protein; CBP, CREB-binding protein.

cells by a combination of PMA and a calcium ionophore iono- the reduction of oxygen levels and expression of HIF-1␣ (Fig. mycin mimicked TCR/CD3 ligation with regard to cooperation 2E). Taken together, we may conclude that HIF-1␣ plays an with hypoxia in HIF-1␣ protein accumulation (Fig. 2D). PMA/ essential role in regulation of AICD of T cells in the hypoxic ionomycin-mediated AICD was gradually decreased along with tissue microenvironment. 6538 HIF-1␣ REGULATES SURVIVAL OF ACTIVATED T CELLS

AM suppresses AICD of peripheral T cells via autocrine regulatory mechanism To examine the mechanism of HIF-1␣-mediated attenuation of AICD, we performed gene expression proﬁling experiments using DNA microarray analysis and RT-PCR assays of peripheral T cells cultured in the absence or presence of TCR/CD3 ligation and exposure to hypoxia. As shown in Table I, a variety of changes in expression proﬁle of T cell-related genes were induced by those treatments. Among them, expression of Fas mRNA was detected irrespective of cellular treatment. Fas-L mRNA expression was induced by TCR/CD3 ligation, whereas hypoxic culture had no effect on this induction response, suggesting that hypoxia does not likely interfere with TCR/CD3-mediated activation of T cells leading to Fas-L expression. mRNA levels of Bcl-2 remained constant,

whereas Bcl-xL mRNA expression was induced by TCR/CD3 activation but not by hypoxia. These classical apoptosis-regulating factors, thus, do not show any correlation with HIF-1␣ protein levels. Expression of mRNA for GLUT-1, -3, and VEGF were induced by hypoxia but not altered after TCR/CD3 stimulation. In Downloaded from contrast, hypoxia had only a modest effect on AM mRNA induction, and the combination of hypoxia and TCR/CD3 stimulation resulted in a robust enhancement of AM gene expression in T cells (Fig. 3A and Table I). These results may indicate a close correla- FIGURE 4. Regulation of AM by HIF-1␣ mediates protection of T cells tion between AM expression and HIF-1␣ protein accumulation in against AICD. A, Schematic representation of the primary structure of the T cells. It is therefore likely that AM may represent a HIF-1␣ wild-type HIF-1␣ and chimeric HIF-1␣/1-396/VP16AD proteins. N-TAD, http://www.jimmunol.org/ N-terminal TAD; VP16 AD, VP16 TAD. B, HIF-1␣/1-396/VP16 AD is a constitutively active regulator of the hypoxia response element (HRE)- driven gene expression. HeLa cells were transfected with the expression plasmid for HIF-1␣/1-396/VP16 AD and the HRE-luciferase reporter gene ᮀ ■ and incubated at either 21% ( )or1%( )O2 for 24 h, whereafter cellular luciferase activity was determined. Experiments were performed in triplicate and means Ϯ SD of fold induction relative to the cells transfected with

only the reporter gene at 21% O2 are shown. C, Establishment of Jurkat T ␣ cell-derived cells expressing a constitutively active form of HIF-1 . Pa- by guest on September 28, 2021 rental Jurkat T cells (Jurkat), mock-transfected Jurkat cells (Jurkat-mock), and Jurkat-HIF-1␣ cells stably expressing HIF-1␣/1-396/VP16 AD were transfected with the HRE-luciferase reporter gene and cultured for 24 h at Ϯ 21% O2. Cellular luciferase activity was monitored and means SD of fold induction relative to parental cells are shown. D, Expression of AM and AM receptor in Jurkat-HIF-1␣ cells under normoxic conditions. Total RNA was prepared from the parental Jurkat T cells or Jurkat-HIF-1␣ cells, and RT-PCR analysis was performed to monitor mRNA expression of the indicated gene. A representative result is shown. E, Blocking of AM signals abrogates the survival of Jurkat T cells overexpressing the constitutively active HIF-1␣. Parental Jurkat T cells and Jurkat-HIF-1␣ cells with (■) or without (ᮀ) CD3 stimuli (5 ␮g/ml) were cultured in the presence or ␮ absence of 10 M CGRP 8Ð37 at 21% O2 for 48 h whereafter cell viability was examined. Results from three independent experiments are presented FIGURE 3. AM mediates the effect of hypoxia to protect T cells from .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .as described in Fig. 2B AICD through an autocrine regulatory loop. A, AM represents an HIF-1␣ target gene in TCR/CD3-activated T cells. PHA-activated T cells were incubated in the presence (CD3) or absence (Ϫ) of immobilized CD3 Abs ␮ target gene involved in determination of cell survival of activated (5 g/ml) at 21 or 1% O2 for 24 h, and mRNA expression of the indicated genes was examined by RT-PCR. Three independent experiments have T cells. In fact, RT-PCR analysis demonstrated mRNA expression shown almost identical results and representative results are shown. B, of the components of the receptor complex for AM, CRLR and TCR/CD3-activated T cells express components of the AM receptor com- RAMP 1 and 2 (26) in T cells (Fig. 3B). Blocking of the AM plex. T cells incubated as described above were examined for mRNA ex- receptor by a fragment of the calcitonin gene-related peptide pression of the indicated genes by RT-PCR. C, Blockade of the AM re- CGRP 8-37 abolished the effect of hypoxia on T cell survival (Fig. ceptor abrogates hypoxia-mediated life support of T cells. PHA-activated 3C), strongly suggesting that AM receptor-mediated signal trans- T cells were incubated with immobilized CD3 Abs and 10 ␮M CGRP8-37 duction is essential for hypoxia-dependent rescue of T cells from at 21% O (ᮀ)or1%O (■) as indicated for 72 h, and cell viability was 2 2 AICD. In excellent agreement with this model, incubation of T determined. The results of triplicated experiments are presented as de- cells with synthetic AM peptides bypassed requirement of hypoxia p Ͻ 0.01. D, Synthetic AM peptides ,ءء ;p Ͻ 0.05 ,ء .scribed in Fig. 2B mimic the effect of hypoxia in protection of T cells against AICD. PHA- for survival, and inhibited AICD under normoxic conditions (Fig. ␣ activated T cells with TCR/CD3 stimulation were cultured in the absence 3D). Taken together, these results suggest that HIF-1 target gene (ᮀ) or presence (■) of the indicated concentrations of AM peptides for product AM plays an important role in T cell survival counteract- 72 h, and cell viability was determined. Experiments were repeated for ing AICD under hypoxic conditions possibly via an autocrine loop .p Ͻ 0.005. mechanism ,ءء ;p Ͻ 0.01 ,ء .three times The Journal of Immunology 6539

Constitutively active HIF-1␣ induces AM expression under demonstrated that hypoxia down-regulates Kv1.3 channels in T normoxic conditions to protect T cells from AICD cells and modulates T cell proliferation possibly via membrane- To directly monitor the involvement of HIF-1␣ mediated induction delimited mechanisms such as membrane depolarization or acti- of AM expression in the regulation of AICD in T cells, we gen- vating the calcium release-activated channel, which was seen only erated Jurkat T cells which demonstrate functional activity of when T cells are activated by membrane-associated stimulation HIF-1␣ even under normoxic conditions. To this end, we con- including TCR ligation (33). Because stimuli that bypass the mem- ␣ structed a chimeric protein, HIF-1␣/1-396/VP16AD, consisting of brane such as PMA/ionomycin (33) similarly induced HIF-1 ex- the N-terminal bHLH/PAS domain of HIF-1␣ and the constitu- pression and consequent AICD inhibition in our study, a mecha- tively active TAD of the viral transcription factor VP16 (Fig. 4A). nism independent of Kv1.3 channel-modulation might be involved Transient expression of HIF-1␣/1-396/VP16AD in Jurkat cells re- in HIF-1-AM-mediated T cell survival control under the hypoxic sulted in constitutive activation of HIF-1␣ regulated reporter gene condition. Of interest, in the myeloid lineage of the immune cells, ␣ expression (Fig. 4B). Jurkat cells stably expressing HIF-1␣/1-396/ HIF-1 has been shown to be essentially involved in the metabolic VP16AD, designated Jurkat-HIF-1␣ cells, also showed enhanced switch for glycolytic energy production, a pivotal process for my- expression of HIF-1-regulated reporter gene activity at normoxia eloid cell functions, and thus is critically implicated in regulation (Fig. 4C). In a similar fashion, Jurkat-HIF-1␣ cells expressed not of innate immune responses (25). In contrast, as demonstrated in only AM but also CRLR and RAMP2 mRNA even under nor- the present study, hypoxia-dependent regulation of apoptosis in Ag ␣ moxic conditions (Fig. 4D). In clear contrast to the parental Jurkat receptor-activated T cells via the HIF-1 -AM pathway may be a cells, Jurkat-HIF-1␣ cells showed resistance to TCR/CD3-trig- mechanism for control of acquired immune systems. Distinct use ␣ Downloaded from gered cell death at normoxia. Moreover, blocking of the AM re- of HIF-1 and its downstream signals for a different function/ ceptor by CGRP 8Ð37 impaired the survival of Jurkat-HIF-1␣ component of the immune system, thus, may constitute a critical cells (Fig. 4E). In conclusion, sequential expression of HIF-1␣ and mechanism for ﬁne tuning of local inﬂammatory responses. AM under conditions of hypoxia critically controls the viability of In mice splenic T cells, hypoxia-independent up-regulation of ␣ Ag receptor-activated T cells, participating in maintenance of T HIF-1 mRNA by TCR/CD3 stimulation has been demonstrated, cell-mediated immune reactions in the hypoxic microenvironment. which is achieved by up-regulation of the alternatively spliced

shorter isoform of HIF-1␣ mRNA (23). This shorter isoform seen http://www.jimmunol.org/ in the mice has not been found in humans, and human HIF-1␣ Discussion mRNA corresponds to a “constitutive” longer isoform in mice In this study, we presented that the hypoxic condition correspond- (34), indicating the presence of a distinct mode of HIF-1␣ inducing to the oxygen levels in the tissue microenvironment protects tion in human T cells from that in mice. In fact, we observed a peripheral T cells from Ag receptor stimulation-triggered AICD, constitutive expression of HIF-1␣ mRNA in human peripheral T indicating a possible role of local oxygen concentrations in regu- cells irrespective of TCR/CD3 stimulation, and instead, demon- lation of immune reactions. strated significant up-regulation of HIF-1␣ in protein levels upon AICD of peripheral T cells is likely to be coregulated by a va- TCR/CD3 activation under hypoxic conditions. To our surprise, in riety of factors including environmental constituents. These regu- human peripheral T cells, hypoxia is not sufficient and an addi- by guest on September 28, 2021 latory mechanisms involve costimulatory molecules (7), humoral tional TCR/CD3 stimulation was required for HIF-1␣ protein ac- factors, or chemicals (5), all of which have been shown to involve cumulation, in clear contrast to cancer-derived cell lines in which the Fas/Fas-L system or Bcl-family proteins. Similarly, but mech- HIF-1␣ can be induced by solely hypoxic treatment (15). Simi- anistically distinct from AICD, Ag receptor stimulation in naive T larly, histological analyses have revealed that the HIF-1␣ protein cells has been shown to result in apoptosis, in which NF-␬B and was seldom recovered in normal human tissues although physio- p73 play a critical role (27). In the present study, we have dem- logical tissue oxygen concentration is known to be low enough to onstrated that hypoxia attenuates AICD in peripheral T cells induce HIF-1␣ (9, 35). However, in vivo studies have demon- through HIF-1␣-mediated expression of AM possibly via an au- strated unique aspects of HIF-1␣ expression in inflammation (36), tocrine regulatory loop mechanism. Strikingly, among the hypox- ischemia (37), and during development (38), all of which are in- ia-inducible and cell death/survival-regulating genes tested, only dicated to involve additional oxygen-independent pathways of AM gene expression was correlated with protein levels of HIF-1␣ HIF-1␣ regulation. Therefore, for normal tissues including T cells, in T cells, similar to the observation that AM gene expression is not only hypoxia-dependent but also hypoxia-independent mech- strictly dependent not on the HIF-1␤ subunit but on HIF-1␣ sub- anisms might be prerequisite for induction of HIF-1␣, allowing unit expression in ES cells (28). Although the mechanism of this rationale regulation of HIF-1␣ expression and subsequent adapta- deviation in target gene induction by HIF-1␣ in peripheral T cells tion to the hypoxic microenvironment. As seen in the present is elusive, the potential role of the HIF-1-AM regulatory pathway study, disunity in HIF-1␣ appearance both in the cytoplasm and in control of T cell survival was further illustrated by experiments the nucleus of T cells in rheumatoid arthritis synovium may indi- in which a constitutively active form of HIF-1␣ induces resistance cate a possible heterogeneity of regulatory pathways leading to to AICD. In the endometrial cancer cells, induction of Bcl-2 by HIF-1␣ accumulation even in the same tissue. AM has been shown to play an important role in inhibition of There has been compelling evidence that physicochemical com- severe hypoxia-mediated apoptosis (29). In contrast, we did not ponents of the tissue microenvironment such as osmolarity and find up-regulation of Bcl-2 in the present study on T cell AICD, temperature are crucial determinants of immune reactions (39Ð indicating that the AM-Bcl-2 cascade may not be critically in- 42). Considering that immune cells often infiltrate into a hypoxic volved in regulation of peripheral T cell death triggered by Ag area, hypoxia-mediated regulation of the immune cells (43Ð46) receptor stimulation. Indeed, roles of AM and subsequent cellular may be an efficient strategy for microenvironmental control of im- signaling in controlling cell survival have been demonstrated to be mune reactions in situ. In this line, survival control of T cells by varied, either proapoptotic or antiapoptotic, between cell types and hypoxia might represent such a rational mechanism for control of cellular stimuli inducing cell death (29Ð32). In any case, further peripheral immunity. Under hypoxic conditions and in the absence studies are clearly needed for identification of a downstream signal of TCR/CD3 stimulation, peripheral T cells are indicated to be of AM for T cell survival. Recently, Conforti et al. (33) have prone to die, whereas hypoxic T cells in the presence of TCR/CD3 6540 HIF-1␣ REGULATES SURVIVAL OF ACTIVATED T CELLS stimuli might be protected from death. This mechanism may pro- 21. Kung, A. L., S. Wang, J. M. Klco, W. G. Kaelin, and D. M. Livingston. 2000. vide the basis of the scenario that Ag-specific T cells are prefer- Suppression of tumor growth through disruption of hypoxia-inducible transcription. Nat. 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