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The Journal of Immunology, 2001, 167: 3156-3163.
Copyright © 2001 by The American Association of Immunologists

A New IFN-Like Cytokine, Limitin Modulates the Immune Response Without Influencing Thymocyte Development1

Isao Takahashi*, Hiroshi Kosaka{dagger}, Kenji Oritani2,*, William R. Heath{ddagger}, Jun Ishikawa*, Yu Okajima*, Megumu Ogawa*, Sin-ichiro Kawamoto*, Masahide Yamada*, Hiroaki Azukizawa{dagger}, Satoshi Itami{dagger}, Kunihiko Yoshikawa{dagger}, Yoshiaki Tomiyama* and Yuji Matsuzawa*

Departments of * Internal Medicine and Molecular Science and {dagger} Dermatology, Graduate School of Medicine, Osaka University, Osaka, Japan; and {ddagger} The Water and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A novel IFN-like molecule, limitin, was recently identified and revealed to suppress B lymphopoiesis through the IFN-{alpha}{beta} 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 IFN–{alpha} and -{beta}, 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 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-{alpha} and -{beta}. 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferon (IFN) was first discovered in 1957 as a substance that induced an antiviral state in cells (1). Since then, several kinds of IFNs have been identified and classified based on the cell surface receptor, primary sequence, and chromosomal localization (2). All IFNs are now placed into two groups, type I and type II IFNs. The type I IFN family is composed of IFN-{alpha}, -{beta}, -{omega}, and -{tau} that have homology to each other, bind to the same cell surface receptor, and show overlapping functions. Type I IFNs are known for broad biological properties including anti-proliferative, immunomodulatory, and antiviral effects (3, 4). In the immune-surveillance system, T cells stimulated with Ag-MHC and costimulatory signals proliferate and differentiate into effector cells with a wide range of functions (5, 6). Type I IFNs can modulate these responses by inhibiting T cell proliferation (7, 8), by enhancing T and NK cell cytotoxicity (9, 10), and by enhancing the expression of MHC class I molecules (11). Furthermore, they augment the proliferation of CD44highCD8+ T cells and prolong their life span in vivo (12). Activated T cells are saved from apoptosis with type I IFNs and can be reactivated efficiently with IL-2, suggesting that type I IFNs are presumably important for re-establishing quiescence in memory T cells at the end of immune responses (13, 14). These immunomodulatory activities have been applied to many clinical uses of IFN-{alpha} and -{beta} including the treatment of malignancies (15, 16, 17).

Recently we identified a novel IFN-like cytokine, limitin, that has ~30% amino acid sequence identity with IFN-{alpha}, -{beta}, and -{omega} (18). Limitin displays its biological functions through the IFN-{alpha}{beta} receptor, implying that limitin is likely to belong to the type I IFN family. Like IFN-{alpha} and -{beta}, limitin suppressed the proliferation of pre-B cells in response to IL-7 and completely blocked the production of B lymphocytes in Whitlock-witte type long-term bone marrow cultures (18). Moreover, administration of limitin to newborn mice resulted in the reduction of B lineage cell populations in the bone marrow (18). In contrast with IFN-{alpha} and -{beta}, limitin did not affect the responsiveness of myeloid progenitors to colony-stimulating factors or that of erythroid progenitors to erythropoietin in vitro (18). Furthermore, treatment of newborn mice with limitin did not change the number or the proportion of CD11b-positive and TER119-positive cells in bone marrow (18). Although limitin shares the IFN-{alpha}{beta} receptor and induces expression of IFN regulatory factor-1 (IRF-1),3 it is distinct from IFN-{alpha} and -{beta} because of its failure to suppress the growth of myeloid and erythroid progenitors (18).

To investigate this structurally and functionally unique cytokine, we needed to compare the effects of limitin on various cell types to that of IFN-{alpha} and -{beta}. This study was undertaken to determine whether limitin has any regulatory effects on T cells in vivo and in vitro. We will discuss some functional differences between limitin and previously known IFNs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

OT-I mice are MHC class I-restricted OVA-specific TCR transgenic mice having CD8+ T cells (19, 20). OT-II mice are MHC class II-restricted OVA-specific TCR transgenic mice carrying CD4+ T cells (21). II-mOVA transgenic mice express the membrane-bound form of OVA under the control of MHC class II (I-E) promoter. C57BL/6 mice and BALB/c mice were purchased (Japan Clea, Tokyo, Japan). All mice were maintained at the Institute for Experimental Animals, Osaka University (Osaka, Japan). Mice were ~6–10 wk of age at the time of use.

Culture medium and cell lines

DMEM (Nakarai Tesque, Kyoto, Japan) supplemented with 10% heat-inactivated FCS (ICN Biomedicals, Aurora, OH), 50 µM 2-ME, 2 mM L-glutamine, 10 mM HEPES, and antibiotics (100 U of penicillin G, 100 µg/ml streptomycin) were used for the in vitro culture assays. A T lymphoma cell line, EL-4, transfected with I-Ab (EL-4Ab) or that transfected with I-Ab and OVA gene (EL-4AbOVA) was provided by Dr. Y. Murakami (Osaka University, Osaka, Japan). CTLL-2 cells were provided by Dr. M. Ogata (Osaka University). 145-2C11 cells (anti murine CD3 hybridoma) were cultured to obtain anti-CD3 Ab. Pam 212 cells (a spontaneously transformed keratinocyte cell line, H-2Dd restricted) were provided by Dr. S. Yuspa (National Cancer Institute, Bethesda, MD). All cell lines were maintained as previously described (22, 23).

Reagents and Abs

A fusion protein composed of limitin and human Ig (limitin-Ig) was purified with a protein A column (Pierce, Rockford, IL) from the supernatant of 293T cells transfected with the Limitin-Ig/Bos plasmid (18). CD44-Ig was prepared in the same way, and was used as a control. Anti-limitin serum was obtained by immunizing rabbits against the recombinant limitin protein several times at 10-day intervals. OVA-MHC-class-II peptide was provided by Dr. Y. Murakami (Osaka University). FITC-anti-CD8, FITC-anti-V{alpha}2, PE-anti-V{alpha}2, and PE-anti-CD4 were purchased from BD PharMingen (San Diego, CA). FITC-anti-H-2Kb was purchased from Caltag Laboratories (Burlingame, CA). Rabbit anti-Armenian hamster IgG and FITC-labeled goat anti-mouse IgG Fc{gamma} were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). FITC-anti-CD25 was purchased from CEDARLANE Laboratories (Hornby, Ontario, Canada). Anti-H-2d (34-5-8S) was provided by Dr. S. Ono (Osaka University). Con A was purchased from Sigma (St. Louis, MO), and mitomycin C (MMC) was purchased from Kyowahakkou (Tokyo, Japan). Concanamycin A (CMA), an inhibitor of perforin-granzyme pathway, was purchased from WAKO Pure Chemicals (Osaka, Japan). Anti-Fas Ligand (anti-FasL; FLIM58) was purchased from Medical and Biological Laboratories (Nagoya, Japan).

Flow cytometry

Ab incubations and washing steps were accomplished at 4°C in PBS containing 1% heat-inactivated FCS and 0.1% sodium azide. The stained cells were analyzed by a FACSSort analyzer (BD Biosciences, San Jose, CA). The data were analyzed with CellQuest software (BD Biosciences).

[3H]Thymidine incorporation assay

Mixed cells were incubated in flat-bottom 96-well microplates (Corning Costar, Tokyo, Japan) for the indicated time in each individual experiment. The cells were pulsed with 0.5 µCi/well [3H]thymidine (Amersham, Tokyo, Japan) for the last 4 h of culture, then harvested onto glass filters (Wallac, Turku, Finland) with a semiautomatic cell harvester (Pharmacia, Piscataway, NJ), and incorporated radioactivity was measured with a liquid scintillation counter.

Purification of T cell subsets

Purified T cells were obtained by negative selection using immunomagnetic beads coated with anti-B220, anti-CD11c, and anti-MHC class II Abs (Miltenyi Biotec, Bergisch Gladbach, Germany). Primary CD8+V{alpha}2+ T cells were purified from OT-I mouse lymph node (LN) cells by negative selection using magnetic beads coated with anti-B220, anti-CD11c, anti-MHC class II, and anti-CD4 Abs. Likewise, CD4+V{alpha}2+ T cells were 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 Abs. In our experiments, the purity of cells was as follows: T cells, >=98%; CD8+V{alpha}2+ cells, >=98%; and CD4+V{alpha}2+ cells, >=90%.

Cross-linking of TCR by immobilized anti-CD3 Ab

The rabbit anti-Armenian hamster IgG (10 µg/ml) was coated onto 96-well flat-bottom polystyrene tissue culture plates overnight at 4°C. After three washes with PBS, anti-CD3 Ab (145-2C11 cell culture supernatant) was then incubated for 4 h at room temperature. The culture plate was washed in PBS three times again before use. Purified T cells (2 x 105/well) were cultured in the anti-CD3 Ab-coated microplate for 3 days.

Ag-specific T cell proliferation assay

To evaluate the OVA Ag-specific proliferation of CD8+ cells, bulk or purified CD8+V{alpha}2+ populations of OT-I LN cells (2 x 105/well) and MMC-treated EL-4Ab or EL-4AbOVA cells (2 x 105/well) were mixed and cultured in flat-bottom 96-well microplates. To evaluate the OVA Ag-specific proliferation of CD4+ cells, purified CD4+V{alpha}2+ cells of OT-II mice (4 x 105/well) were stimulated with 10 µM class II OVA peptide and irradiated spleen cells from C57BL/6 mice. Triplicate cultures were set up for each experimental group. After 2–4 days, the proliferation of responder cells was evaluated by [3H]thymidine incorporation.

IL-2 assay

IL-2 activity was assayed by measuring the [3H]thymidine incorporation of IL-2-dependent murine T cell line CTLL-2 (24). Briefly, 1 x 104/well CTLL-2 cells were cultured with 4-fold serially diluted test sample for 24 h. Their proliferation was measured with an [3H]thymidine incorporation assay. One unit of IL-2 was defined as the activity contained in a sample yielding a proliferation equal to 50% of the maximum [3H]thymidine incorporation obtained with the standard rIL-2 preparation.

Western blotting

Immunoprecipitation, gel electrophoresis, and immunoblotting were performed according to published methods (25). Cells were "serum-starved," stimulated with IL-2 and/or limitin-Ig, and then lysed in lysis buffer. After insoluble material was removed by centrifugation, the lysate obtained from 2 x 107 cells was incubated with 5 µl of anti-stat 5b Ab (Santa Cruz Biotechnology, Santa Cruz, CA), followed by protein A-Sepharose beads (Amersham). The immunoprecipitate was analyzed on SDS-PAGE, then electrophoretically transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore, Bedford, MA). After residual binding sites were blocked on the filter, immunoblotting was accomplished using the appropriate Abs. Immunoreactive proteins were visualized with an ECL system (Amersham).

Cytotoxic assay

A 51Cr-release assay was performed as described previously (23). In brief, 1 x 104 51Cr-labeled target cells and effector cells were mixed in 96-well round-bottom plates (Costar) at the indicated E:T ratios. After a 4-h incubation, cell-free supernatants were collected, and radioactivity was measured 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 (27).

Fetal thymus organ culture (FTOC) technique

Thymus lobes dissected from fetal mice at day 14 of gestation were placed on the surface of polycarbonate filters (0.8-µm pore size; Nuclepore, Pleasanton, CA) that were supported on blocks of surgical gelform (Yamanouchi, Osaka, Japan) in 500 µl of complete medium in Falcon 48-well plates (1 lobe/well) (28). The cultures were grown in a humidified incubator in 7% CO2 in air at 37°C. Half of the culture medium was replaced every other day.

Lethal graft-versus-host disease (GVHD) assay

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 cells from OT-I mice on day 0, followed by peritoneal injection with either CD44-Ig (1 µg/head) or imitin-Ig (1 µg/head) daily from day 1. C57BL/6 mice were used as non-GVHD controls. Survival was monitored daily.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth inhibitory effects of limitin on T cells

In these experiments, limitin-Ig was used as a substitute for limitin, because limitin-Ig was easily purified as described in Materials and Methods. Previous studies revealed that limitin-Ig behaved identically to limitin (18). The polyclonal proliferation of T cells was induced with Con A or with Ab-mediated cross-linking of CD3 molecules (29). As shown in Fig. 1Go, limitin-Ig suppressed both Con A- and anti-CD3-Ab-induced T cell proliferation. Growth inhibition was observed even when purified T cells were 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.



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  		FIGURE 1. Effects of limitin on mitogen- or anti-CD3 Ab-induced T cell proliferation. A and B, Spleen cells (5 x 105/well) from C57BL/6 mice were stimulated with 2 µg/ml Con A (A), or LN cells (2 x 105/well) were stimulated with coated anti-CD3 Ab (B) in the indicated culture conditions. After 3-day incubation, the proliferation of cultured cells was evaluated using [3H]thymidine incorporation assays. The results are shown as means ± SD of triplicate cultures. Similar results were obtained in three independent experiments.

 
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-specific T 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 CD8+ and V{alpha}2+V{beta}5+ T cells (19, 20). OT-II mice are class II-restricted, OVA-Ag-specific TCR transgenic mice that generate CD4+ and V{alpha}2+V{beta}5+ 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. 2GoA). The proliferation of OVA Ag-specific T cells was 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. 2Go, A and B). Even when purified CD8+ T cells derived from OT-I mice were used as responder cells, ~30% of their proliferation was inhibited by limitin-Ig (Fig. 2GoC). To evaluate the effect of limitin-Ig on CD4+ T cells, purified CD4+ T cells derived from OT-II mice were mixed with C57BL/6 spleen cells pulsed with OVA peptide. The proliferation of CD4+ T cells was also decreased by ~30% with limitin-Ig (Fig. 2GoD). Based on these results, limitin down-regulates the proliferation of both CD4+ T cells and CD8+ T cells in response to mitogen and Ag stimuli.



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  		FIGURE 2. Effect of limitin on Ag-specific T cell proliferation. A and B, LN cells of OT-I mice (2 x 105/well) were mixed with MMC-treated EL-4Ab or EL-4AbOVA cells (2 x 105/well) and cultured in the indicated conditions for 2 days. C, Purified CD8+V{alpha}2+ cells of OT-I mice (2 x 105/well) were mixed with MMC-treated EL-4Ab or EL-4AbOVA cells (2 x 105/well) and cultured in the indicated conditions for 2 days. D, Purified CD4+V{alpha}2+ cells of OT-II mice cells (2 x 105/well) were mixed with C57BL/6 mouse spleen cells (4 x 105/well) plus 10 µM OVA peptide in the indicated conditions for 4 days. The proliferation of responders was evaluated using [3H]thymidine incorporation assays. The results are shown as means ± SD of triplicate cultures. Similar results were obtained in three independent experiments.

 
Mechanisms of growth regulation by limitin

Because IL-2 is the powerful growth factor for T cells, we tested whether limitin could suppress any aspects of IL-2-mediated proliferation. When naive T cells are activated, they secrete IL-2 and up-regulate the expression of CD25, the IL-2 receptor {alpha}-chain (30, 31, 32). When OT-I T cells were cultured with EL-4AbOVA cells, the activated T cells secreted IL-2 (Fig. 3GoA). 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. 3GoB). As shown in Fig. 3GoC, 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 examined tyrosine phosphorylation of signal transducers and activators of transcription 5 (Stat 5) (Fig. 3GoD). 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 phosphorylation.



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  		FIGURE 3. Mechanisms of the growth inhibition of limitin. A, LN cells of OT-I mice (2 x 105/well) and MMC-treated EL-4Ab or EL-4AbOVA cells (2 x 105/well) were mixed and cultured for 24 h. Their culture supernatants were collected and subjected to IL-2 assay. B, Responding cells were stained with FITC-anti-CD25 and PE-anti-V{alpha}2, and CD25 expression on V{alpha}2-positive cells was measured by using FACSSort cytometry. The results are shown as means ± SD of triplicate cultures. Similar results were obtained in three independent experiments. C and D, Bulk LN cells derived from OT-I mice were stimulated with MMC-treated EL4-OVA cells. After 7-day incubation, the cells were harvested and washed twice in HBSS. C, 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. After 3-day incubation, the proliferation of cells was then evaluated using [3H]thymidine incorporation assays. Each dot is shown as means of triplicate cultures. Similar results were obtained in three independent experiments. D, Collected cells were deprived of growth factor and then incubated in the absence or presence of 10 U/ml IL-2 or 100 ng/ml limitin-Ig for 10 min. Stat 5 immunoprecipitates were blotted with anti-Stat 5b or anti-phosphotyrosine Abs as indicated.

 
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 CD44-Ig for 10 days. As shown in Fig. 4Go, control cultures contained CD4-CD8-, CD4+CD8+, 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{beta} expression was not affected (data not shown).



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  		FIGURE 4. Effect of limitin on the development of thymocytes in 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 ± SD of triplicate cultures. Similar results were observed in four independent experiments.

 
We next injected limitin-Ig into newborn mice daily from days 3–8 of age. As previously reported (33), newborn mice injected with limitin-Ig showed a significant decrease in B220+ B lymphocytes in the spleen when compared with control mice (Fig. 5Go). The inhibitory effect on B lymphopoiesis was revealed at a dose of 2 µg/mouse and reached the plateau level at 10 µg/mouse (data not shown). In contrast, limitin-Ig treatment did not affect the total number of thymocytes at a dose of 10 µg/mouse. Moreover, no difference was observed in the proportion of thymocyte subpopulations between limitin-Ig-treated mice and controls. In conclusion, limitin has no obvious effect on the proliferation or maturation of thymocytes in vivo or in vitro.



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  		FIGURE 5. In vivo effect of limitin on thymus. Peritoneal injections of CD44-Ig or limitin-Ig (10 µg/head) were given daily from 3 to 8 days of age and the mice were killed at 9 days of age. Cell numbers of thymus were counted by hemocytometer (A), and their surface phenotypes were analyzed with flow cytometry (B). Total cell numbers of thymocytes of the injected mice are represented as means ± SD from nine mice per group. Representative results of the flow cytometry are shown for one of nine injected mice. Similar results were observed in three independent experiments.

 
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 OVA-Ag-specific CTL activity were generated. Addition of limitin-Ig to cultures resulted in an ~2-fold increase in cytotoxicity when compared with that of CD44-Ig and medium control (Fig. 6GoA). The cytotoxic activity was suppressed by the addition of CMA, an inhibitor of the perforin-granzyme pathway, but not anti-FasL Ab (Fig. 6GoB). The cytotoxicity enhanced by limitin-Ig was also completely abrogated with CMA treatment, implying that it finally depends on the perforin-granzyme pathway rather than the FasL-Fas pathway.



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  		FIGURE 6. Effect of limitin on CTL activity. A, Bulk LN cells from OT-1 mice (1 x 106/well) were mixed with MMC-treated EL-4AbOVA cells (1 x 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 each E:T ratio, the mean percent specific lysis for duplicate cultures is shown. The specificity for OVA Ag was confirmed by the fact that the percent specific lysis against EL-4Ab cells was <10% in any E:T ratio. Similar results were obtained in three independent experiments. For clarity, SD bars were omitted from the graph, but were <10\% of the value of all points. >B, CTL activities of OT-1 LN cells stimulated as above against EL-4AbOVA cells were tested in the absence or presence of 50 nM CMA or 10 µg/ml anti-FasL mAb in a 4-h 51Cr release assay at an E:T ratio of 1. Similar results were obtained in two independent experiments.

 
Because the expression of MHC class I Ag on target cells is required for the recognition of CTLs, we tested whether limitin could up-regulate class I on Pam 212 cells, a mouse keratinocyte-derived cell line that has been used to examine the induction of MHC class I Ag with other IFNs (34, 35). As shown in Fig. 7Go, H-2Dd expression was induced by limitin-Ig. Similar enhancement of MHC class I expression by limitin-Ig was also observed when spleen cells were treated with limitin-Ig. Therefore, limitin-Ig also augments MHC class I expression that may be related to the enhancement of CTL activity.



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  		FIGURE 7. Effect of limitin on MHC class I expression of PAM212 cells. PAM212 cells were cultured in 24-well plates for 2 days in the indicated conditions. MHC class I Ag expression was quantified by immunofluorescence staining with anti-H-2Dd and then with the secondary goat anti-mouse IgG Fc{gamma} conjugated with FITC. Likewise, spleen cells derived from C57BL/6 mice were cultured for 2 days in the indicated conditions. MHC class I Ag expression was quantified by immunofluorescence staining with FITC-anti-H-2Kb. These experiments were performed three times.

 
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 augmented their CTL activity. To study how limitin regulated immune responses in vivo, a murine model of lethal GVHD directed toward the OVA Ag (OT-I->II-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 irradiation, 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. 8Go). GVHD mortality was significantly enhanced in limitin-Ig-treated mice (mean survival time = 6.2 days) when compared with that of CD44-Ig (mean survival time = 8.3 days) (p < 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.



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  		FIGURE 8. Limitin enhances GVHD after transplantation. II-mOVA mice and C57BL/6 mice (wild type) were irradiated and engrafted with bulk population of LN cells (107 cells/head) from OT-1 mice on day 0. C57BL/6 mice were used as non-GVHD controls (n = 10) for the transfer. The transplanted II-mOVA mice were injected peritoneally either 1 µg/head CD44-Ig or limitin-Ig daily from day 1 (n = 15, n = 16). Their survival was monitored daily. Similar results were obtained from three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although limitin recognizes the IFN-{alpha}{beta} receptors and inhibits B lymphopoiesis, it does not affect either myelopoiesis or erythropoiesis, indicating that biological effects of limitin are not identical with those of IFN-{alpha} and -{beta} (18). Thus, analysis of the functions of limitin on different cell types, compared with previously known IFNs, provides understanding of this structurally and functionally unique IFN-like cytokine. Here, we examine the effects of limitin on T lymphocytes, which are a major target of IFN-{alpha} and -{beta}. Limitin suppressed the proliferation of T cells and enhanced the induction of CTL activity. In this regard, limitin resembles IFN-{alpha} and -{beta}, 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-{alpha}1/{alpha}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 costimulatory signals from APCs, proliferate and differentiate into effector cells with multiple functions. CD8+ T cells develop into highly cytolytic effector cells (36), and CD4+ T cells develop into either Th1 subsets that predominantly produce IFN-{gamma} and lymphotoxin or Th2 subsets that produce IL-4 (37). These responses are modulated by cytokines as well as cell surface proteins. IL-12 positively regulates Th1 differentiation and IFN-{gamma} production (38), whereas IL-4 promotes Th2 cell differentiation (39). In addition, IL-2 is a powerful growth factor for T cells. Although naive T cells secrete few cytokines and express only low affinity of IL-2 receptors composed of the {beta}- and {gamma}-chains, they produce IL-2 and express 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-{alpha} and -{beta} 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 (43, 44, 45, 46), 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). Alternatively, 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-{alpha} and -{beta} (18), limitin might selectively work on signaling pathways of particular growth factors such as IL-2.

Limitin enhanced CTL activity through at least two mechanisms. First, limitin augments perforin-granzyme activity. We showed that CMA, an inhibitor of the perforin-granzyme pathway, completely cancelled the enhanced cytotoxicity by limitin. The second mechanism involves induction of MHC class I expression on target cells and APCs. Because limitin induced expression of IRF-1 (data not shown), which is upstream of MHC class I, the induction of MHC class I by limitin is likely to be mediated by IRF-1 (47). Besides these mechanisms, limitin may augment CTL activity through multiple alternative mechanisms including increasing 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 FasL and TNF-related apoptosis-inducing ligand (TRAIL) (48, 49). In the human, FasL and TRAIL are expressed on activated T cells and NK cells, and IFN-{alpha} and -{beta} can enhance their expression (50, 51). Although limitin did not change TRAIL expression on T cells in our Ag-specific T cell stimulation system (data not shown), it remains to be determined whether limitin can enhance FasL and TRAIL expression for other types of T cell stimuli.

Limitin suppressed Ag-induced T cell proliferation but enhanced their CTL activity, indicating that limitin displays opposite effects in immune response. We evaluated the effect of limitin on in vivo immunity using a lethal GVHD assay (OT-I mice->II-mOVA mice). Because T cells of OT-I mice express OVA-specific TCR and II-mOVA mice express the OVA gene under the MHC class II promoter, aggressive GVHD ensued leading to the death of all hosts. In this system, all processes of immune responses such as T cell growth and CTL induction as well as enhanced MHC expression are required for the onset and progression of GVHD. Limitin-Ig injection resulted in the enhancement of GVHD mortality. Although we do not know the dose-dependent effects of limitin in GVHD, limitin is likely to enhance immune responses in vivo under some circumstances. These observations suggest that limitin could be postulated as a new immune modulator with similar specificity to IFN-{alpha} and -{beta}.

Our characterization of limitin suggests that limitin has unique functional activities despite its recognition of the IFN-{alpha}{beta} receptor. Our observation that limitin has no effect on thymocyte development is in contrast with that of an active IFN-{alpha}1/{alpha}2 hybrid protein reported by Lin and his colleagues (33) to affect thymocyte maturation. They documented a great reduction in the total number of thymocytes in IFN-{alpha}1/{alpha}2-treated mice, along with suppression of T lymphocyte progenitors at the pro-T cell stage. We previously reported that limitin is distinguishable from IFN-{alpha} and -{beta} in that limitin has little or no influence on myelopoiesis and erythropoiesis (18). Although limitin inhibits B lymphopoiesis and modifies immune responses similarly to IFN-{alpha} and -{beta}, cellular targets of limitin are more restricted than IFN-{alpha} and -{beta}, and the pattern of cellular responses also distinguishes it from other immunomodulatory molecules, including IL-4, IL-12, IFN-{gamma}, and TGF-{beta} (52, 53).

It will be particularly important to learn why mature T cells are sensitive to limitin while thymocytes are resistant despite bearing the IFN-{alpha}{beta} receptor. T cell proliferation following Ag stimulation is mainly mediated by IL-2, whereas expansion of thymocytes in their steady state is supported by several membrane proteins and cytokines such as IL-1, IL-2, IL-4, IL-7, and thymic stromal lymphopoietin (54, 55). The different cytokine requirements for proliferation may explain why limitin acts differently on thymocytes and T cells. Alternatively, different environmental conditions between thymocytes and T cells may cause the different response to limitin. In contrast, there are several reports illustrating differences between IFN-{alpha} and -{beta}, and even between different subtypes of IFN-{alpha}. For example, antiviral activities and antiproliferative effects reportedly vary among subtypes of IFN-{alpha} (56, 57). The {beta}-R1 mRNA is induced by IFN-{beta}, but not IFN-{alpha} (58, 59). Association of a 95- to 100-kDa tyrosine-phosphorylated protein with the IFN-{alpha}{beta} receptor was found in an IFN-{beta}-, but not IFN-{alpha}-treated myeloma cell line (60). The biological diversity among type I IFNs including limitin seems to be dependent on some cellular circumstance and might be explained by differences in their affinity for the IFN-{alpha}{beta} receptor or by differences in their sites that bind to the receptor. In this regard, recent advances in the structural and functional analysis of type I IFNs helps us to understand the complex IFN system. IFN-{alpha} and -{beta} have a globular structure composed of five {alpha} helices (61, 62, 63). In addition, experiments using mAbs against IFN-{alpha} and -{beta}, site-directed mutagenesis, and the constructed hybrid IFNs revealed functionally important binding sites of IFNs (64, 65, 66). Because limitin has only 30% amino acid sequence identity with IFN-{alpha} and -{beta} (18), it probably differs from the other IFNs in structure, and this may explain why limitin has restricted cellular targets compared with IFN-{alpha} and -{beta}.

IFN-{alpha} and -{omega} are produced by leukocytes, whereas IFN-{beta} is derived from fibroblasts. IFN-{gamma} is classified as a type II IFN and a product of activated T lymphocytes and NK cells (67, 68). Like IFN-{alpha} and -{beta}, the limitin gene is active in normal lymphohemopoietic organs. Our preliminary immunohistological analysis has revealed that limitin protein is produced by some cells in the thymus as well as LNs (K. Oritani, unpublished data). Although IFN-{alpha} and -{beta} can be markedly increased by viral infection or exposure to double-stranded nucleic acids (3), we have not assessed this for limitin.

We now know that limitin enhances immune responses without suppressing T lymphopoiesis, myelopoiesis, or erythropoiesis. Thus, a human homolog of limitin is likely to be superior to IFN-{alpha} and -{beta} in the clinic because of its lack of adverse effects such as myelosuppression. In addition, study of this functionally and structurally unique IFN, limitin, is likely to be useful for clarification of the complex interactions between IFNs and their receptors.


   Acknowledgments
 
We thank Dr. Shigekazu Nagata (Osaka University, Osaka, Japan), Dr. Yoshiko Murakami, and Dr. Hideo Yagita (Juntendo University, Tokyo, Japan) for technical advice.


   Footnotes
 
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, Science, and Culture of Japan, and the Japan Society for the Promotion of Science. Back

2 Address correspondence and reprint requests to Dr. Kenji Oritani, Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita City, Osaka 565-0871, Japan. E-mail address: oritani{at}imed2.med.osaka-u.ac.jp Back

3 Abbreviations used in this paper: IRF-1, IFN regulatory factor-1; LN, lymph node; TRAIL, TNF-related apoptosis-inducing ligand; GVHD, graft-versus-host disease; EL-4Ab, T lymphoma cell line (EL-4) transfected with I-Ab; EL-4AbOVA, T lymphoma cell line (EL-4) transfected with I-Ab and OVA gene; limitin-Ig, fusion protein composed of limitin and human Ig; MMC, mitomycin C; CMA, concanamycin A; FTOC, fetal thymus organ culture; Fas L, Fas ligand. Back

Received for publication February 20, 2001. Accepted for publication July 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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