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A New IFN-Like Cytokine, Limitin Modulates the Immune Response Without Influencing Thymocyte Development

This information is current as Isao Takahashi, Hiroshi Kosaka, Kenji Oritani, William R. of October 1, 2021. Heath, Jun Ishikawa, Yu Okajima, Megumu Ogawa, Sin-ichiro Kawamoto, Masahide Yamada, Hiroaki Azukizawa, Satoshi Itami, Kunihiko Yoshikawa, Yoshiaki Tomiyama and Yuji Matsuzawa J Immunol 2001; 167:3156-3163; ; doi: 10.4049/jimmunol.167.6.3156 Downloaded from http://www.jimmunol.org/content/167/6/3156

References This article cites 68 articles, 28 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/167/6/3156.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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. A New IFN-Like Cytokine, Limitin Modulates the Immune Response Without Inﬂuencing Thymocyte Development1

Isao Takahashi,* Hiroshi Kosaka,† Kenji Oritani,2* William R. Heath,‡ Jun Ishikawa,* Yu Okajima,* Megumu Ogawa,* Sin-ichiro Kawamoto,* Masahide Yamada,* Hiroaki Azukizawa,† Satoshi Itami,† Kunihiko Yoshikawa,† Yoshiaki Tomiyama,* and Yuji Matsuzawa*

A novel IFN-like molecule, limitin, was recently identified and revealed to suppress B lymphopoiesis through the IFN-␣␤ receptor, although it lacked growth suppression on myeloid and erythroid progenitors. Here we have studied diverse effects of limitin on T lymphocytes and compared limitin with previously known IFNs. Like IFNÐ␣ and -␤, limitin modified immunity in the following responses. It suppressed mitogen- and Ag-induced T cell proliferation through inhibiting the responsiveness to exogenous IL-2 rather than suppressing the production of IL-2. In contrast, limitin enhanced cytotoxic T lymphocyte activity associated with the Downloaded from perforin-granzyme pathway. To evaluate the effect of limitin in vivo, a lethal graft-versus-host disease assay was established. Limitin-treatment of host mice resulted in the enhancement of graft-versus-host disease. Limitin did not influence thymocyte development either in fetal thymus organ cultures or in newborn mice injected with limitin-Ig, suggesting that limitin is distinguishable from IFN-␣ and -␤. From these findings, it can be speculated that the human homolog of limitin may be applicable for clinical usage because of its IFN-like activities with low adverse effects on, for example, T lymphopoiesis, erythropoiesis, and myelopoiesis. The Journal of Immunology, 2001, 167: 3156Ð3163. http://www.jimmunol.org/

nterferon (IFN) was first discovered in 1957 as a substance ing quiescence in memory T cells at the end of immune responses that induced an antiviral state in cells (1). Since then, several (13, 14). These immunomodulatory activities have been applied to I kinds of IFNs have been identified and classified based on the many clinical uses of IFN-␣ and -␤ including the treatment of cell surface receptor, primary sequence, and chromosomal local- malignancies (15Ð17). ization (2). All IFNs are now placed into two groups, type I and Recently we identified a novel IFN-like cytokine, limitin, that type II IFNs. The type I IFN family is composed of IFN-␣,-␤,-␻, has ϳ30% amino acid sequence identity with IFN-␣,-␤, and -␻ and -␶ that have homology to each other, bind to the same cell (18). Limitin displays its biological functions through the IFN-␣␤ by guest on October 1, 2021 surface receptor, and show overlapping functions. Type I IFNs are receptor, implying that limitin is likely to belong to the type I IFN known for broad biological properties including anti-proliferative, family. Like IFN-␣ and -␤, limitin suppressed the proliferation of immunomodulatory, and antiviral effects (3, 4). In the immune- pre-B cells in response to IL-7 and completely blocked the pro- surveillance system, T cells stimulated with Ag-MHC and co- duction of B lymphocytes in Whitlock-witte type long-term bone stimulatory signals proliferate and differentiate into effector cells marrow cultures (18). Moreover, administration of limitin to new- with a wide range of functions (5, 6). Type I IFNs can modulate born mice resulted in the reduction of B lineage cell populations in these responses by inhibiting T cell proliferation (7, 8), by enhanc- the bone marrow (18). In contrast with IFN-␣ and -␤, limitin did ing T and NK cell cytotoxicity (9, 10), and by enhancing the ex- not affect the responsiveness of myeloid progenitors to colony- pression of MHC class I molecules (11). Furthermore, they aug- stimulating factors or that of erythroid progenitors to erythropoi- ment the proliferation of CD44highCD8ϩ T cells and prolong their etin in vitro (18). Furthermore, treatment of newborn mice with life span in vivo (12). Activated T cells are saved from apoptosis limitin did not change the number or the proportion of CD11b- with type I IFNs and can be reactivated efficiently with IL-2, sug- positive and TER119-positive cells in bone marrow (18). Although gesting that type I IFNs are presumably important for re-establish- limitin shares the IFN-␣␤ receptor and induces expression of IFN regulatory factor-1 (IRF-1),3 it is distinct from IFN-␣ and -␤ because of its failure to suppress the growth of myeloid and erythroid Departments of *Internal Medicine and Molecular Science and †Dermatology, Grad- progenitors (18). uate School of Medicine, Osaka University, Osaka, Japan; and ‡The Water and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, To investigate this structurally and functionally unique cytokine, Australia we needed to compare the effects of limitin on various cell types Received for publication February 20, 2001. Accepted for publication July 6, 2001. to that of IFN-␣ and -␤. This study was undertaken to determine The costs of publication of this article were defrayed in part by the payment of page whether limitin has any regulatory effects on T cells in vivo and in charges. This article must therefore be hereby marked advertisement in accordance vitro. We will discuss some functional differences between limitin with 18 U.S.C. Section 1734 solely to indicate this fact. and previously known IFNs. 1 This work was supported in part by grants from the Japan Leukemia Research Foundation, the Japan Research Foundation for Clinical Pharmacology, the Osaka Medical Research Foundation for Incurable Disease, the Ministry of Education, Sci- 3 Abbreviations used in this paper: IRF-1, IFN regulatory factor-1; LN, lymph node; ence, and Culture of Japan, and the Japan Society for the Promotion of Science. TRAIL, TNF-related apoptosis-inducing ligand; GVHD, graft-versus-host disease; 2 Address correspondence and reprint requests to Dr. Kenji Oritani, Department of EL-4Ab, T lymphoma cell line (EL-4) transfected with I-Ab; EL-4AbOVA, T lym- Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka Uni- phoma cell line (EL-4) transfected with I-Ab and OVA gene; limitin-Ig, fusion protein versity, 2-2 Yamada-oka, Suita City, Osaka 565-0871, Japan. E-mail address: composed of limitin and human Ig; MMC, mitomycin C; CMA, concanamycin A; [email protected] FTOC, fetal thymus organ culture; Fas L, Fas ligand.

Materials and Methods Cross-linking of TCR by immobilized anti-CD3 Ab Mice The rabbit anti-Armenian hamster IgG (10 ␮g/ml) was coated onto 96-well OT-I mice are MHC class I-restricted OVA-specific TCR transgenic mice flat-bottom polystyrene tissue culture plates overnight at 4¡C. After three ϩ washes with PBS, anti-CD3 Ab (145-2C11 cell culture supernatant) was having CD8 T cells (19, 20). OT-II mice are MHC class II-restricted ϩ then incubated for4hatroom temperature. The culture plate was washed OVA-specific TCR transgenic mice carrying CD4 T cells (21). II-mOVA ϫ 5 transgenic mice express the membrane-bound form of OVA under the con- in PBS three times again before use. Purified T cells (2 10 /well) were trol of MHC class II (I-E) promoter. C57BL/6 mice and BALB/c mice were cultured in the anti-CD3 Ab-coated microplate for 3 days. purchased (Japan Clea, Tokyo, Japan). All mice were maintained at the Ag-specific T cell proliferation assay Institute for Experimental Animals, Osaka University (Osaka, Japan). Mice were ϳ6Ð10 wk of age at the time of use. To evaluate the OVA Ag-specific proliferation of CD8ϩ cells, bulk or purified CD8ϩV␣2ϩ populations of OT-I LN cells (2 ϫ 105/well) and Culture medium and cell lines MMC-treated EL-4Ab or EL-4AbOVA cells (2 ϫ 105/well) were mixed and cultured in flat-bottom 96-well microplates. To evaluate the OVA Ag- DMEM (Nakarai Tesque, Kyoto, Japan) supplemented with 10% heat-in- specific proliferation of CD4ϩ cells, purified CD4ϩV␣2ϩ cells of OT-II activated FCS (ICN Biomedicals, Aurora, OH), 50 ␮M 2-ME, 2 mM L- mice (4 ϫ 105/well) were stimulated with 10 ␮M class II OVA peptide and glutamine, 10 mM HEPES, and antibiotics (100 U of penicillin G, 100 irradiated spleen cells from C57BL/6 mice. Triplicate cultures were set up ␮g/ml streptomycin) were used for the in vitro culture assays. A T lym- for each experimental group. After 2Ð4 days, the proliferation of responder b phoma cell line, EL-4, transfected with I-A (EL-4Ab) or that transfected cells was evaluated by [3H]thymidine incorporation. with I-Ab and OVA gene (EL-4AbOVA) was provided by Dr. Y. Mu- rakami (Osaka University, Osaka, Japan). CTLL-2 cells were provided by IL-2 assay Dr. M. Ogata (Osaka University). 145-2C11 cells (anti murine CD3 hy- IL-2 activity was assayed by measuring the [3H]thymidine incorporation of bridoma) were cultured to obtain anti-CD3 Ab. Pam 212 cells (a sponta- 4 ϫ Downloaded from neously transformed keratinocyte cell line, H-2Dd restricted) were pro- IL-2-dependent murine T cell line CTLL-2 (24). Briefly, 1 10 /well CTLL-2 cells were cultured with 4-fold serially diluted test sample for vided by Dr. S. Yuspa (National Cancer Institute, Bethesda, MD). All cell 3 lines were maintained as previously described (22, 23). 24 h. Their proliferation was measured with an [ H]thymidine incorporation assay. One unit of IL-2 was defined as the activity contained in a 3 Reagents and Abs sample yielding a proliferation equal to 50% of the maximum [ H]thymidine incorporation obtained with the standard rIL-2 preparation. A fusion protein composed of limitin and human Ig (limitin-Ig) was purified with a protein A column (Pierce, Rockford, IL) from the supernatant Western blotting http://www.jimmunol.org/ of 293T cells transfected with the Limitin-Ig/Bos plasmid (18). CD44-Ig Immunoprecipitation, gel electrophoresis, and immunoblotting were per- was prepared in the same way, and was used as a control. Anti-limitin formed according to published methods (25). Cells were “serum-starved,” serum was obtained by immunizing rabbits against the recombinant limitin stimulated with IL-2 and/or limitin-Ig, and then lysed in lysis buffer. After protein several times at 10-day intervals. OVA-MHC-class-II peptide was insoluble material was removed by centrifugation, the lysate obtained from provided by Dr. Y. Murakami (Osaka University). FITC-anti-CD8, FITC- 2 ϫ 107 cells was incubated with 5 ␮l of anti-stat 5b Ab (Santa Cruz anti-V␣2, PE-anti-V␣2, and PE-anti-CD4 were purchased from BD b Biotechnology, Santa Cruz, CA), followed by protein A-Sepharose beads PharMingen (San Diego, CA). FITC-anti-H-2K was purchased from (Amersham). The immunoprecipitate was analyzed on SDS-PAGE, then Caltag Laboratories (Burlingame, CA). Rabbit anti-Armenian hamster IgG electrophoretically transferred to a polyvinylidene difluoride membrane ␥ and FITC-labeled goat anti-mouse IgG Fc were purchased from Jackson (Immobilon; Millipore, Bedford, MA). After residual binding sites were ImmunoResearch Laboratories (West Grove, PA). FITC-anti-CD25 was blocked on the filter, immunoblotting was accomplished using the appro- purchased from CEDARLANE Laboratories (Hornby, Ontario, Canada). by guest on October 1, 2021 d priate Abs. Immunoreactive proteins were visualized with an ECL system Anti-H-2 (34-5-8S) was provided by Dr. S. Ono (Osaka University). Con (Amersham). A was purchased from Sigma (St. Louis, MO), and mitomycin C (MMC) was purchased from Kyowahakkou (Tokyo, Japan). Concanamycin A Cytotoxic assay (CMA), an inhibitor of perforin-granzyme pathway, was purchased from 51 WAKO Pure Chemicals (Osaka, Japan). Anti-Fas Ligand (anti-FasL; A Cr-release assay was performed as described previously (23). In brief, ϫ 451 FLIM58) was purchased from Medical and Biological Laboratories 1 10 Cr-labeled target cells and effector cells were mixed in 96-well (Nagoya, Japan). round-bottom plates (Costar) at the indicated E:T ratios. After a 4-h incubation, cell-free supernatants were collected, and radioactivity was mea- Flow cytometry sured by a liquid scintillation counter (Wallac). In some experiments, the assay was performed in the absence or presence of CMA (26) or anti-FasL Ab incubations and washing steps were accomplished at 4¡C in PBS con- Ab (27). taining 1% heat-inactivated FCS and 0.1% sodium azide. The stained cells were analyzed by a FACSSort analyzer (BD Biosciences, San Jose, CA). Fetal thymus organ culture (FTOC) technique The data were analyzed with CellQuest software (BD Biosciences). Thymus lobes dissected from fetal mice at day 14 of gestation were placed ␮ 3 on the surface of polycarbonate filters (0.8- m pore size; Nuclepore, Pleas- [ H]Thymidine incorporation assay anton, CA) that were supported on blocks of surgical gelform (Yamanou- ␮ Mixed cells were incubated in flat-bottom 96-well microplates (Corning chi, Osaka, Japan) in 500 l of complete medium in Falcon 48-well plates Costar, Tokyo, Japan) for the indicated time in each individual experiment. (1 lobe/well) (28). The cultures were grown in a humidified incubator in The cells were pulsed with 0.5 ␮Ci/well [3H]thymidine (Amersham, To- 7% CO2 in air at 37¡C. Half of the culture medium was replaced every kyo, Japan) for the last4hofculture, then harvested onto glass filters other day. (Wallac, Turku, Finland) with a semiautomatic cell harvester (Pharmacia, Lethal graft-versus-host disease (GVHD) assay Piscataway, NJ), and incorporated radioactivity was measured with a liquid scintillation counter. II-mOVA mice and C57BL/6 mice were used as hosts and OT-I mice as donors. Host mice were irradiated (600 cGy) and injected i.v. with 107 LN Purification of T cell subsets cells from OT-I mice on day 0, followed by peritoneal injection with either CD44-Ig (1 ␮g/head) or imitin-Ig (1 ␮g/head) daily from day 1. C57BL/6 Purified T cells were obtained by negative selection using immunomag- mice were used as non-GVHD controls. Survival was monitored daily. netic beads coated with anti-B220, anti-CD11c, and anti-MHC class II Abs ϩ ϩ (Miltenyi Biotec, Bergisch Gladbach, Germany). Primary CD8 V␣2 T Results cells were purified from OT-I mouse lymph node (LN) cells by negative selection using magnetic beads coated with anti-B220, anti-CD11c, anti- Growth inhibitory effects of limitin on T cells ϩ ϩ MHC class II, and anti-CD4 Abs. Likewise, CD4 V␣2 T cells were In these experiments, limitin-Ig was used as a substitute for limitin, purified from OT-II mice LN cells by negative selection using magnetic beads coated with anti-B220, anti-CD11c, anti-MHC class II, and anti-CD8 because limitin-Ig was easily purified as described in Materials Abs. In our experiments, the purity of cells was as follows: T cells, Ն98%; and Methods. Previous studies revealed that limitin-Ig behaved CD8ϩV␣2ϩ cells, Ն98%; and CD4ϩV␣2ϩ cells, Ն90%. identically to limitin (18). The polyclonal proliferation of T cells 3158 LIMITIN MODIFIES T CELL RESPONSE IN VITRO AND IN VIVO was induced with Con A or with Ab-mediated cross-linking of Mechanisms of growth regulation by limitin CD3 molecules (29). As shown in Fig. 1, limitin-Ig suppressed Because IL-2 is the powerful growth factor for T cells, we tested both Con A- and anti-CD3-Ab-induced T cell proliferation. whether limitin could suppress any aspects of IL-2-mediated pro- Growth inhibition was observed even when purified T cells were liferation. When naive T cells are activated, they secrete IL-2 and cultured in these systems, suggesting that limitin-Ig could directly act upon T cells. Growth inhibition of limitin-Ig was prevented by the addition of anti-limitin polyclonal Ab, indicating that it was a specific effect of limitin. T cells are usually activated and expanded after the recognition of nominal Ags presented in the context of MHC molecules on APCs (5). To analyze the effect of limitin on Ag-specificT cell responses, we provided OVA-Ag-specific TCR transgenic mice as responders. OT-I mice are MHC class I-restricted TCR transgenic mice that produce CD8ϩ and V␣2ϩV␤5ϩ T cells (19, 20). OT-II mice are class II-restricted, OVA-Ag-specific TCR transgenic mice that generate CD4ϩ and V␣2ϩV␤5ϩ T cells (21). OT-I LN cells responded well to EL-4AbOVA cells, which express OVA Ag, but failed to respond to OVA-negative EL-4Ab cells (Fig. 2A). The proliferation of OVA Ag-specific T cells was Downloaded from inhibited by limitin-Ig in a dose-dependent manner, and the inhibitory effect of limitin-Ig was evident by 10 ng/ml and maximal at 100 ng/ml (Fig. 2, A and B). Even when purified CD8ϩ T cells derived from OT-I mice were used as responder cells, ϳ30% of their proliferation was inhibited by limitin-Ig (Fig. 2C). To evaluate the effect of limitin-Ig on CD4ϩ T cells, purified CD4ϩ T http://www.jimmunol.org/ cells derived from OT-II mice were mixed with C57BL/6 spleen cells pulsed with OVA peptide. The proliferation of CD4ϩ T cells was also decreased by ϳ30% with limitin-Ig (Fig. 2D). Based on these results, limitin down-regulates the proliferation of both CD4ϩ T cells and CD8ϩ T cells in response to mitogen and Ag stimuli. by guest on October 1, 2021

FIGURE 2. Effect of limitin on Ag-specific T cell proliferation. A and B, LN cells of OT-I mice (2 ϫ 105/well) were mixed with MMC-treated EL-4Ab or EL-4AbOVA cells (2 ϫ 105/well) and cultured in the indicated conditions for 2 days. C, Purified CD8ϩV␣2ϩ cells of OT-I mice (2 ϫ FIGURE 1. Effects of limitin on mitogen- or anti-CD3 Ab-induced T 105/well) were mixed with MMC-treated EL-4Ab or EL-4AbOVA cells cell proliferation. A and B, Spleen cells (5 ϫ 105/well) from C57BL/6 mice (2 ϫ 105/well) and cultured in the indicated conditions for 2 days. D, were stimulated with 2 ␮g/ml Con A (A), or LN cells (2 ϫ 105/well) were Purified CD4ϩV␣2ϩ cells of OT-II mice cells (2 ϫ 105/well) were mixed stimulated with coated anti-CD3 Ab (B) in the indicated culture conditions. with C57BL/6 mouse spleen cells (4 ϫ 105/well) plus 10 ␮M OVA peptide After 3-day incubation, the proliferation of cultured cells was evaluated in the indicated conditions for 4 days. The proliferation of responders was using [3H]thymidine incorporation assays. The results are shown as evaluated using [3H]thymidine incorporation assays. The results are shown means Ϯ SD of triplicate cultures. Similar results were obtained in three as means Ϯ SD of triplicate cultures. Similar results were obtained in three independent experiments. independent experiments. The Journal of Immunology 3159 up-regulate the expression of CD25, the IL-2 receptor ␣-chain (30Ð32). When OT-I T cells were cultured with EL-4AbOVA cells, the activated T cells secreted IL-2 (Fig. 3A). However, no significant difference in IL-2 secretion was observed between cultures with limitin-Ig or CD44-Ig. Next, the effect of limitin on expression of CD25 on activated T cells was tested. In contrast to IL-2 production, there was a notable augmentation of CD25 expression by limitin-Ig, compared with CD44-Ig (Fig. 3B). As shown in Fig. 3C, the proliferation of activated T cells correlated with the concentration of IL-2. However, the proliferation of activated T cells in response to exogenous IL-2 was significantly suppressed when limitin-Ig was added to the culture. To determine whether limitin-Ig regulates IL-2 signaling pathways, we exam- ined tyrosine phosphorylation of signal transducers and activators of transcription 5 (Stat 5) (Fig. 3D). LN cells from OT-I mice were activated by exposure to EL-4AbOVA cells for 7 days, and the activated T cells were then stimulated by limitin-Ig and/or IL-2. The Stat 5 molecule was significantly phosphorylated by IL-2, and addition of limitin-Ig did not affect the IL-2-induced Stat 5 Downloaded from phosphorylation.

The effect of limitin on the development of thymocytes FTOC was used to examine the effect of limitin on T cell maturation in the thymus. Fetal thymus lobes were taken from 14-day

C57BL/6 embryos and cultured in the presence of limitin-Ig or http://www.jimmunol.org/ CD44-Ig for 10 days. As shown in Fig. 4, control cultures contained CD4ϪCD8Ϫ, CD4ϩCD8ϩ, as well as single positive populations. Limitin-Ig did not change the production of each population of thymocytes determined by CD4/CD8 expression. Furthermore, TCR␤ expression was not affected (data not shown). We next injected limitin-Ig into newborn mice daily from days 3Ð8 of age. As previously reported (33), newborn mice injected with limitin-Ig showed a signiﬁcant decrease in B220ϩ B lymphocytes in the spleen when compared with control mice (Fig. 5). The by guest on October 1, 2021 inhibitory effect on B lymphopoiesis was revealed at a dose of 2 ␮g/mouse and reached the plateau level at 10 ␮g/mouse (data not shown). In contrast, limitin-Ig treatment did not affect the total number of thymocytes at a dose of 10 ␮g/mouse. Moreover, no difference was observed in the proportion of thymocyte subpopu- lations between limitin-Ig-treated mice and controls. In conclu- sion, limitin has no obvious effect on the proliferation or maturation of thymocytes in vivo or in vitro.

The effect of limitin on T cell functions One of the major T cell functions is CTL activity. When OT-I T cells are cultured with EL-4AbOVA cells for 5 days, effectors with FIGURE 3. Mechanisms of the growth inhibition of limitin. A, LN cells OVA-Ag-speciﬁc CTL activity were generated. Addition of lim- ϫ 5 ϳ of OT-I mice (2 10 /well) and MMC-treated EL-4Ab or EL-4AbOVA itin-Ig to cultures resulted in an 2-fold increase in cytotoxicity cells (2 ϫ 105/well) were mixed and cultured for 24 h. Their culture su- when compared with that of CD44-Ig and medium control (Fig. pernatants were collected and subjected to IL-2 assay. B, Responding cells 6A). The cytotoxic activity was suppressed by the addition of were stained with FITC-anti-CD25 and PE-anti-V␣2, and CD25 expression CMA, an inhibitor of the perforin-granzyme pathway, but not anti- on V␣2-positive cells was measured by using FACSSort cytometry. The FasL Ab (Fig. 6B). The cytotoxicity enhanced by limitin-Ig was results are shown as means Ϯ SD of triplicate cultures. Similar results were also completely abrogated with CMA treatment, implying that it obtained in three independent experiments. C and D, Bulk LN cells derived ﬁnally depends on the perforin-granzyme pathway rather than the from OT-I mice were stimulated with MMC-treated EL4-OVA cells. After FasL-Fas pathway. 7-day incubation, the cells were harvested and washed twice in HBSS. C, Because the expression of MHC class I Ag on target cells is Collected cells were incubated with the indicated concentration of IL-2 in the absence or presence of 100 ng/ml CD44-Ig or 100 ng/ml limitin-Ig. required for the recognition of CTLs, we tested whether limitin After 3-day incubation, the proliferation of cells was then evaluated using could up-regulate class I on Pam 212 cells, a mouse keratinocyte- [3H]thymidine incorporation assays. Each dot is shown as means of trip- derived cell line that has been used to examine the induction of licate cultures. Similar results were obtained in three independent experi- MHC class I Ag with other IFNs (34, 35). As shown in Fig. 7, ments. D, Collected cells were deprived of growth factor and then incu- H-2Dd expression was induced by limitin-Ig. Similar enhancement bated in the absence or presence of 10 U/ml IL-2 or 100 ng/ml limitin-Ig of MHC class I expression by limitin-Ig was also observed when for 10 min. Stat 5 immunoprecipitates were blotted with anti-Stat 5b or spleen cells were treated with limitin-Ig. Therefore, limitin-Ig also anti-phosphotyrosine Abs as indicated. 3160 LIMITIN MODIFIES T CELL RESPONSE IN VITRO AND IN VIVO

tion, and the transferred mice were injected with either CD44-Ig or limitin-Ig daily from day 1. Although C57BL/6 mice used as non- GVHD controls were alive during the observation time, all II- mOVA mice transplanted with OT-I LN cells died by lethal GVHD within 15 days (Fig. 8). GVHD mortality was signiﬁcantly enhanced in limitin-Ig-treated mice (mean survival time ϭ 6.2 days) when compared with that of CD44-Ig (mean survival time ϭ 8.3 days) ( p Ͻ 0.01 using the Mann-Whitney U test). Thus we see that limitin functions as an enhancer of immune response in vivo in our GVHD model.

FIGURE 4. Effect of limitin on the development of thymocytes in Downloaded from FTOC. Thymuses from day-14 C57BL/6 mouse embryos were put into organ cultures for 10 days with medium alone, 100 ng/ml limitin-Ig, or 100 ng/ml CD44-Ig. Thymocytes derived from FTOC were counted (A) and subjected to ﬂow cytometry analysis (B). Representative ﬂow cytometry results are shown for one of triplicate cultures. Total cell numbers of the cultured thymuses are represented as means Ϯ SD of triplicate cultures.

Similar results were observed in four independent experiments. http://www.jimmunol.org/ augments MHC class I expression that may be related to the enhancement of CTL activity.

Limitin enhances immune response in vivo As described above, limitin had some apparently contradictory effects in that it suppressed the proliferation of T cells but aug-

mented their CTL activity. To study how limitin regulated immune by guest on October 1, 2021 responses in vivo, a murine model of lethal GVHD directed toward the OVA Ag (OT-I3II-mOVA) was established (see Materials and Methods). Bulk populations of LN cells derived from OT-I mice were engrafted into II-mOVA mice after sublethal irradia-

FIGURE 6. Effect of limitin on CTL activity. A, Bulk LN cells from OT-1 mice (1 ϫ 106/well) were mixed with MMC-treated EL-4AbOVA cells (1 ϫ 106/well) and then cultured in the absence or presence of either 100 ng/ml CD44-Ig or 100 ng/ml limitin-Ig. After 5 days, their CTL activity was tested on 51Cr-labeled EL-4AbOVA cells as target cells. For FIGURE 5. In vivo effect of limitin on thymus. Peritoneal injections of each E:T ratio, the mean percent specific lysis for duplicate cultures is CD44-Ig or limitin-Ig (10 ␮g/head) were given daily from 3 to 8 days of shown. The specificity for OVA Ag was confirmed by the fact that the age and the mice were killed at 9 days of age. Cell numbers of thymus were percent specific lysis against EL-4Ab cells was Ͻ10% in any E:T ratio. counted by hemocytometer (A), and their surface phenotypes were ana- Similar results were obtained in three independent experiments. For clarity, lyzed with flow cytometry (B). Total cell numbers of thymocytes of the SD bars were omitted from the graph, but were B, CTL activities of OT-1 injected mice are represented as means Ϯ SD from nine mice per group. LN cells stimulated as above against EL-4AbOVA cells were tested in the Representative results of the flow cytometry are shown for one of nine absence or presence of 50 nM CMA or 10 ␮g/ml anti-FasL mAb in a 4-h injected mice. Similar results were observed in three independent 51Cr release assay at an E:T ratio of 1. Similar results were obtained in two experiments. independent experiments. The Journal of Immunology 3161

press high-affinity IL-2 receptors after stimulation with some mitogens or nominal Ags (40, 41). We showed that although limitin reduced the response of activated T cells to IL-2, it did not change IL-2 production and actually enhanced the expression of high-affinity IL-2 receptors. Limitin failed to block the activation of Stat 5 that up-regulated the expression of CD25 on activated T cells (42). It is still unclear how limitin regulates T cell proliferation, but we think that there are two possibilities. First, limitin may block a common replication as IFN-␣ and -␤ do. IFN treatment has been shown to interfere with S-phase entry that is accompanied by several changes in cell-cycle molecules, e.g., the reduction in expression of cyclin D and cyclin E (43Ð46), hypophosphorylation of retinoblastoma protein (43), suppression of E2F DNA-binding activity (43, 45), inhibition of cdk 2 activity (43, 44), and abrogation of IL-2-induced reduction of p27 protein levels (44, 46). Alterna- tively, limitin may modify some aspects of IL-2 signals other than the Stat 5 pathway. Because the cell-type specificity of limitin is more restricted than that of IFN-␣ and -␤ (18), limitin might selectively work on signaling pathways of particular growth factors Downloaded from such as IL-2. Limitin enhanced CTL activity through at least two mechanisms. First, limitin augments perforin-granzyme activity. We FIGURE 7. Effect of limitin on MHC class I expression of PAM212 showed that CMA, an inhibitor of the perforin-granzyme pathway, cells. PAM212 cells were cultured in 24-well plates for 2 days in the completely cancelled the enhanced cytotoxicity by limitin. The indicated conditions. MHC class I Ag expression was quantified by im- second mechanism involves induction of MHC class I expression

d http://www.jimmunol.org/ munofluorescence staining with anti-H-2D and then with the secondary on target cells and APCs. Because limitin induced expression of ␥ goat anti-mouse IgG Fc conjugated with FITC. Likewise, spleen cells IRF-1 (data not shown), which is upstream of MHC class I, the derived from C57BL/6 mice were cultured for 2 days in the indicated induction of MHC class I by limitin is likely to be mediated by conditions. MHC class I Ag expression was quantified by immunofluores- IRF-1 (47). Besides these mechanisms, limitin may augment CTL cence staining with FITC-anti-H-2Kb. These experiments were performed three times. activity through multiple alternative mechanisms including in- creasing the number of active CTLs, enhancing target binding, or even by lowering TCR signaling thresholds. T cell cytotoxicity is also reported to be mediated by some cell surface proteins such as

Discussion FasL and TNF-related apoptosis-inducing ligand (TRAIL) (48, by guest on October 1, 2021 Although limitin recognizes the IFN-␣␤ receptors and inhibits B 49). In the human, FasL and TRAIL are expressed on activated T lymphopoiesis, it does not affect either myelopoiesis or erythro- cells and NK cells, and IFN-␣ and -␤ can enhance their expression poiesis, indicating that biological effects of limitin are not identical (50, 51). Although limitin did not change TRAIL expression on T with those of IFN-␣ and -␤ (18). Thus, analysis of the functions of cells in our Ag-speciﬁc T cell stimulation system (data not shown), limitin on different cell types, compared with previously known it remains to be determined whether limitin can enhance FasL and IFNs, provides understanding of this structurally and functionally TRAIL expression for other types of T cell stimuli. unique IFN-like cytokine. Here, we examine the effects of limitin on T lymphocytes, which are a major target of IFN-␣ and -␤. Limitin suppressed the proliferation of T cells and enhanced the induction of CTL activity. In this regard, limitin resembles IFN-␣ and -␤, and seems to be a potent immune response modulator. However, limitin had no effect on the development of thymocytes in vitro and in vivo, whereas injection of newborn mice with the active IFN-␣1/␣2 hybrid protein has been shown to impair T cell maturation in thymus (33). The dose of limitin-Ig injected into newborn mice should be enough to work in this system because it inhibited B lymphopoiesis at an almost maximal level (data not shown). T cells, when stimulated with Ag-MHC complexes and costimu- latory signals from APCs, proliferate and differentiate into effector cells with multiple functions. CD8ϩ T cells develop into highly cytolytic effector cells (36), and CD4ϩ T cells develop into either Th1 subsets that predominantly produce IFN-␥ and lymphotoxin or Th2 subsets that produce IL-4 (37). These responses are mod- FIGURE 8. Limitin enhances GVHD after transplantation. II-mOVA mice and C57BL/6 mice (wild type) were irradiated and engrafted with ulated by cytokines as well as cell surface proteins. IL-12 posi- 7 ␥ bulk population of LN cells (10 cells/head) from OT-1 mice on day 0. tively regulates Th1 differentiation and IFN- production (38), C57BL/6 mice were used as non-GVHD controls (n ϭ 10) for the transfer. whereas IL-4 promotes Th2 cell differentiation (39). In addition, The transplanted II-mOVA mice were injected peritoneally either 1 ␮g/ IL-2 is a powerful growth factor for T cells. Although naive T cells head CD44-Ig or limitin-Ig daily from day 1 (n ϭ 15, n ϭ 16). Their secrete few cytokines and express only low afﬁnity of IL-2 recep- survival was monitored daily. Similar results were obtained from three tors composed of the ␤- and ␥-chains, they produce IL-2 and ex- independent experiments. 3162 LIMITIN MODIFIES T CELL RESPONSE IN VITRO AND IN VIVO

Limitin suppressed Ag-induced T cell proliferation but en- IFN-␣ and -␻ are produced by leukocytes, whereas IFN-␤ is hanced their CTL activity, indicating that limitin displays opposite derived from fibroblasts. IFN-␥ is classified as a type II IFN and a effects in immune response. We evaluated the effect of limitin on product of activated T lymphocytes and NK cells (67, 68). Like in vivo immunity using a lethal GVHD assay (OT-I mice3II- IFN-␣ and -␤, the limitin gene is active in normal lymphohemo- mOVA mice). Because T cells of OT-I mice express OVA-specific poietic organs. Our preliminary immunohistological analysis has TCR and II-mOVA mice express the OVA gene under the MHC revealed that limitin protein is produced by some cells in the thy- class II promoter, aggressive GVHD ensued leading to the death of mus as well as LNs (K. Oritani, unpublished data). Although all hosts. In this system, all processes of immune responses such as IFN-␣ and -␤ can be markedly increased by viral infection or T cell growth and CTL induction as well as enhanced MHC ex- exposure to double-stranded nucleic acids (3), we have not as- pression are required for the onset and progression of GVHD. Lim- sessed this for limitin. itin-Ig injection resulted in the enhancement of GVHD mortality. We now know that limitin enhances immune responses without Although we do not know the dose-dependent effects of limitin in suppressing T lymphopoiesis, myelopoiesis, or erythropoiesis. GVHD, limitin is likely to enhance immune responses in vivo Thus, a human homolog of limitin is likely to be superior to IFN-␣ under some circumstances. These observations suggest that limitin and -␤ in the clinic because of its lack of adverse effects such as could be postulated as a new immune modulator with similar spec- myelosuppression. In addition, study of this functionally and struc- ificity to IFN-␣ and -␤. turally unique IFN, limitin, is likely to be useful for clarification of Our characterization of limitin suggests that limitin has unique the complex interactions between IFNs and their receptors. functional activities despite its recognition of the IFN-␣␤ receptor.

Our observation that limitin has no effect on thymocyte develop- Acknowledgments Downloaded from ment is in contrast with that of an active IFN-␣1/␣2 hybrid protein We thank Dr. Shigekazu Nagata (Osaka University, Osaka, Japan), Dr. reported by Lin and his colleagues (33) to affect thymocyte mat- Yoshiko Murakami, and Dr. Hideo Yagita (Juntendo University, Tokyo, uration. They documented a great reduction in the total number of Japan) for technical advice. thymocytes in IFN-␣1/␣2-treated mice, along with suppression of T lymphocyte progenitors at the pro-T cell stage. We previously References ␣ ␤ 1. Isaacs, A., and J. Lindenmann. 1957. Virus interference: the interferon. Proc. reported that limitin is distinguishable from IFN- and - in that http://www.jimmunol.org/ R. Soc. London Ser. B147:258. limitin has little or no influence on myelopoiesis and erythropoi- 2. Diaz, M. O., H. M. Pomykala, S. K. Bohlander, E. Maltepe, K. Malik, esis (18). 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