Constitutive OX40/OX40 Ligand Interaction Induces Autoimmune-Like Diseases¹ Free

Full activation of naive T cells requires not only the interaction between the TCR and Ag/MHC complexes, but also costimulation provided by accessory molecules expressed on APCs (1, 2, 3, 4). In addition to the well-known accessory molecules, CD80 and CD86, both of which bind to CD28 on T cells, several members of the TNF superfamily, including OX40 ligand (OX40L),³ CD70, 4-1BBL, and LIGHT, have been shown to induce costimulatory signals upon binding to their cognate receptors on T cells (5, 6), indicating that the TNF/TNFR superfamily members play major costimulatory roles in the T cell-APC interaction.

OX40L, a molecule that we originally identified as human gp34 whose expression is induced by the tax gene of human T cell leukemia virus (HTLV)-I (7), is expressed on activated T cells (7, 8, 9, 10, 11), activated B cells (12, 13), dendritic cells (14, 15, 16), and endothelial cells (17), while its receptor, OX40, is primarily known to be a T cell activation marker (18, 19). We and others independently generated OX40L-deficient mice, in which significant impairment of APC functions, leading to reduction in T cell proliferative responses and production of both Th1 and Th2 cytokines, were revealed (16, 20). An important avenue in which OX40/OX40L may participate is in regulating the development and survival of memory CD4⁺ T cells (21, 22, 23). Recently, we have shown that after adoptive transfer of encephalitogenic wild-type (WT) T cells to OX40L-deficient mice, the mice were apparently unable to sustain disease progression despite showing comparable onset and severity of disease to WT recipient mice (24). Furthermore, OX40 stimulation has been reported to prevent peripheral tolerance of CD4⁺ T cells (25), suggesting a possible involvement of the OX40/OX40L system in regulating autoimmunity. Indeed, expression of OX40 and/or OX40L has been demonstrated in the tissues of several immune disorders such as experimental allergic encephalitis (24, 26, 27, 28), experimental inflammatory bowel diseases (IBDs) (29, 30), graft-vs-host disease (31, 32, 33), human proliferative lupus nephritis (34), rheumatoid arthritis (35, 36), human IBD (37, 38), human inflammatory muscle diseases (39), and in thymoma of patients with myasthenia gravis (40). In vivo blockade of OX40/OX40L interaction has been described not only to suppress ongoing experimental allergic encephalitis (28) and graft-vs-host disease (32, 41), but also to ameliorate ongoing colitis in murine models of IBD (29, 30), asthma (42), and collagen-induced arthritis (35). These data suggest that OX40/OX40L interaction may play a definitive role in the immune regulation of various immune-related diseases.

To evaluate the influence of constitutive interaction between OX40 and OX40L during immune regulation, we constructed mice stably expressing OX40L on T cells, in which sustained OX40 signaling can occur. In this paper, we demonstrate convincing evidence showing the direct involvement of OX40/OX40L interaction in the development of autoimmunity.

The transgene was generated by inserting the cDNA encoding mouse OX40L into the T lineage-specific vector p1017 (43) that carries the mouse lck proximal promoter. The vector was released as a NotI fragment encompassing the tissue-specific transcription unit. The fragment, purified by standard protocols, was microinjected into the pronuclei of (C57BL/6 × DBA/2)F₁ fertilized eggs. We obtained three independent founders, in which the presence of the transgene was confirmed by PCR. The founders were then mated with C57BL/6 mice to generate OX40L-Tg offspring, and germline transmission was verified either by PCR with DNA from tail biopsy specimens or by flow cytometric analysis with peripheral blood from the tail vein. The OX40L-Tg mice were backcrossed at least 12 times onto the C57BL/6 and 9 times onto the BALB/c backgrounds.

OX40L-deficient mice were described previously (16) and had been back-crossed onto the C57BL/6 strains at least 12 times. Age and sex-matched WT littermates of the OX40L-Tg mice or OX40L-deficient mice were used as control mice. Recombination-activating gene (RAG)2-deficient mice on the C57BL/6 background, lacking B and T cells, were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan). The appropriate strain combinations were taken into account in all experiments. All the mice were kept under specific pathogen-free conditions.

Preincubation with normal rat serum was conducted to prevent labeled mAbs from nonspecific association. The cells were incubated with labeled mAbs for 30 min at 4°C. The samples were then washed with PBS and analyzed with a FACSCalibur flow cytometer (BD Immunocytometry Systems, Mountain View, CA). The analyses were conducted using the CellQuest program (BD Immunocytometry Systems). CD3, CD8, CD4, CD44, and L-selectin (CD62L) were obtained from BD PharMingen (San Diego, CA). MGP34 mAb specific for mouse OX40L was described previously (16). The cells labeled with the biotinylated mAb were visualized by streptavidin-allophycocyanin (BD PharMingen, San Diego, CA), and subjected to flow cytometric analysis. Cells, which were preincubated with unlabeled MGP34 to abolish any specific staining by the biotinylated MGP34, were used as a negative control for OX40L expression.

OX40L-Tg mice or WT littermates were immunized with 100 μg keyhole limpet hemocyanin (KLH) in the presence of CFA to each hind footpad. Alternatively, these mice were i.p. immunized with 1 mg KLH in PBS in the absence of adjuvant. Fourteen to 30 days afterwards, CD4⁺ T cells (1 × 10⁵, purity >98%) purified from the popliteal lymph node or spleen using autoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) were incubated with the indicated dose of KLH in the presence of APCs (5 × 10⁵) at 37°C for 48 h. The APCs used were irradiated (3000 rad) splenocytes derived from unimmunized WT littermates. The cultured cells were assayed for [³H]thymidine uptake and cytokine production as described previously (16). For cytokine production, culture supernatants were collected at 48 h for IL-2 and IL-4, or at 72 h for IL-5, IL-10, Il-13, and IFN-γ, and subjected to ELISA.

Cytokine levels in tissue culture supernatants or in mice sera were assayed using ELISA kits for IL-2, IL-4, IL-5, IL-10, IFN-γ (BD PharMingen), and IL-13 (R&D Systems, Minneapolis, MN) according to the manufacturer^’s recommendations.

A total of 100 μg of SEB (Toxin Technology, Sarasota, FL) was injected i.p. into 8- to 10-wk-old mice. Peripheral blood from the tail vein was analyzed by flow cytometry on the indicated days. Cells were stained with CD4-PE in combination with anti-Vβ8-FITC (BD PharMingen) or anti-Vβ6-FITC (BD PharMingen). The ratios of Vβ8⁺CD4⁺ and Vβ6⁺CD4⁺ cell populations to whole CD4⁺ cell populations in the peripheral blood were monitored.

The levels of the different Ig subclasses in the various mice sera were assayed as previously described (16). In brief, diluted sera from OX40L-Tg or WT mice were added to 96-wells coated with either goat anti-mouse Ig Ab (Southern Biotechnology Associates, Birmingham, Al), salmon sperm dsDNA (Sigma-Aldrich, St. Louis, MO), or ssDNA and incubated for 2 h at room temperature. The ssDNA was prepared by boiling salmon sperm dsDNA for 5 min. After washing, bound Abs were detected by incubation with either goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA conjugated to alkaline-phosphatase (Southern Biotechnology Associates). Color reactions using alkaline-phosphatase substrate (Sigma-Aldrich) were then evaluated by reading OD₄₅₀. This was demonstrated to be well within the linear part of the titration curve.

Animals were sacrificed at the indicated age. The main organs and tissues that included the heart, lung, aorta, liver, pancreas, submandibular glands, stomach, small and large intestines, spleen, thymus, cervical lymph nodes, tibial bones, kidneys, and ankle joints were processed for histopathological examination. Tissues were next fixed in 10% formalin in 0.001 M phosphate buffer (pH 7.2) and embedded in paraffin (Wako Pure Chemical, Osaka, Japan). In the case of tibial bones and ankle joints, these tissues were decalcified in 10% formic acid. Two- to 4-μm thick sections were prepared and stained with H&E using standard techniques and examined by light microscopy.

Single-cell suspension was prepared from the spleens of the OX40L-Tg or WT littermates, and incubated with mouse CD4 MicroBeads (Miltenyi Biotec) for 20 min at 4°C and subjected to an autoMACS (Miltenyi Biotec). The purified WT or OX40L-Tg CD4⁺ T cells (purity >99%) were i.v. injected (1–5 × 10⁶) into the syngenic RAG2-deficient mice. Between 2 and 5 wk after the transfer, some recipient mice were sacrificed, and selected tissues were extracted for histopathological analysis. The body weights of the recipient mice were monitored after inoculation.

Three independent OX40L-Tg mouse lines were generated by using the T cell-specific p1017 expression vector that carries the mouse lck proximal promoter (43). Flow cytometric analysis of whole thymocyte populations derived from the three OX40L-Tg lines, but not from the WT mice, expressed OX40L (Fig. 1). Two of the three transgenic lines (OX40L-Tg1 and OX40L-Tg2) exhibited appreciable expression of OX40L in splenic T cells, whereas the OX40L-Tg3 line showed comparable OX40L expression to WT splenic T cells (Fig. 1). Furthermore, the level of OX40L expression on OX40L-Tg1 splenic T cells was three times higher than that on OX40L-Tg2 splenic T cells (Fig. 1). Founders of the OX40L-Tg mice on the (C57BL/6 × DBA/2)F₁ strain developed normally and exhibited no readily detectable abnormalities. However, when these mice were back-crossed onto the C57BL/6 strain, we noticed weight loss accompanied with diarrhea in the OX40L-Tg1 and -Tg2 mice.

On gross examination of the lymphoid organs of OX40L-Tg1 and OX40L-Tg2 mice, massive splenomegaly and marked lymphadenopathy were observed. All other organs appeared normal including the thymus when compared with WT littermates. We next determined by flow cytometry whether the OX40L transgene in mice resulted in any intrinsic changes in the overall composition of the lymphocyte populations in the thymus, spleen, and lymph nodes. All three OX40L-Tg lines did not display any apparent abnormality in the numbers or subpopulations of T cells in the thymus as assessed by CD4⁺ and CD8⁺ expression (data not shown). However, the total number of lymphocytes in the spleen (Fig. 2,A) and lymph nodes (data not shown) were significantly increased in OX40L-Tg1 and OX40L-Tg2 mice as compared with the WT littermates. More precisely, a 2-fold increase of splenic CD4⁺ T cells but not CD8⁺ T cells was detected in these two OX40L-Tg lines at 7 wk of age (Fig. 2 A), while the numbers of splenic IgM⁺B220⁺ B cells in all the three OX40L-Tg lines were comparable to WT littermates (data not shown). By analyzing the TCR Vβ usage, no skewing of TCR repertoires of the splenic CD4⁺ T cells in both the OX40L-Tg1 and -Tg2 mice were found (data not shown), indicating the absence of oncogenic features.

Because the interaction between OX40 and OX40L is reportedly implicated in the generation or survival of the memory CD4⁺ T cell pool (21, 22, 23), we speculated that the elevated CD4⁺ T cell numbers observed in the OX40L-Tg mice mainly reflect an increase of memory CD4⁺ T cells. Thus, the phenotypic effector memory CD4⁺ T cell population (CD62L^lowCD44^high) was next evaluated by flow cytometric analysis in the OX40L-Tg mice. As predicted, a marked increase in the number of splenic effector memory CD4⁺ T cells in the OX40L-Tg1 mice was evident when compared with the WT mice at 4 wk after birth. This increase was age-dependent up to at least 7 wk of age (Fig. 2,B). Similarly, this remarkable increase in the memory CD4⁺ T cell population was observed in the OX40L-Tg2 mice; however, this was not evident in the OX40L-Tg3 mice (data not shown). Conversely, a significant reduction in this population of CD4⁺ T cells was evident in the OX40L-deficient mice as compared with the WT mice (Fig. 2 B). The results thus far reveal that OX40L-Tg mice harbor a markedly elevated memory T cell subpopulation, illustrating the importance of OX40/OX40L interaction in the generation or/and maintenance of memory CD4⁺ T cells in vivo.

To address the mechanism responsible for the elevated phenotypic memory CD4⁺ T cell population seen of the OX40L-Tg1 and -Tg2 mice, we conducted a series of Ag-specific recall proliferation assays of CD4⁺ T cells. OX40L-Tg CD4⁺ T cells had a markedly enhanced proliferative recall response to KLH (Fig. 3,A), while productions of IL-2, IFN-γ, IL-4, and IL-5 were also significantly enhanced when compared with the WT CD4⁺ T cells (Fig. 3,B). However, secretion of the regulatory cytokine, IL-10, by OX40L-Tg CD4⁺ T cells was reduced in relation to WT CD4⁺ T cells (Fig. 3,B). Next, mice were administrated with KLH in the absence of adjuvant. As expected, a limited proliferative response by the CD4⁺ T cells derived from the WT mice administered with either no Ag or soluble KLH was seen (Fig. 3,C). In contrast, soluble KLH administration induced an obvious dose-dependent proliferative response by the CD4⁺ T cells derived from the OX40L-Tg mice (Fig. 3,C). Furthermore, productions of IL-4, IL-5, IL-10, and IL-13, but not IFN-γ, by OX40L-Tg CD4⁺ T cells were readily detectable (Fig. 3,D), while no marked production of these cytokines in the WT CD4⁺ T cells were observed (Fig. 3 D). These findings indicate that OX40L-Tg mice are able to generate highly functional Ag-specific memory CD4⁺ T cell responses even in the absence of adjuvant.

In view of the extraordinary elevated number and functional capacity of the CD4⁺ memory T cells in the OX40L-Tg mice, we examined the in vivo clonal T cell deletion of these mice during activation-induced cell death (AICD), a well-known mechanism of peripheral T cell tolerance. Because clonal T cell deletion induced by superantigen, such as S. aureus enterotoxin A and SEB, mimics T cell AICD (44, 45), we examined whether SEB administered to OX40L-Tg mice would induce the deletion of the peripheral Vβ8⁺ T cell repertoire. Interestingly, the Vβ8⁺CD4⁺ T cell population in the OX40L-Tg mice remained markedly elevated up to at least 14 days after the SEB injection (Fig. 4,A), while the WT Vβ8⁺CD4⁺ T cell population, despite peaking on day-2 postinjection, promptly decreased by day 7 (Fig. 4,A). Control Vβ6⁺CD4⁺ peripheral T cell populations from OX40L-Tg and WT CD4⁺ T cells, TCRs of which are not recognized by SEB, remained unchanged (Fig. 4 B). Vβ8⁺ T cell deletion induced by SEB treatment is thus defective in OX40L-Tg mice, suggesting that constitutive OX40/OX40L interactions prevents clonal deletion of CD4⁺ T cells during AICD.

We next determined the levels of all the Ig isotypes, and of various Th1 and Th2 cytokines in the sera of OX40L-Tg1 mice. OX40L-Tg1 mice apparently had markedly elevated serum levels of all Ig classes except IgG3 when compared with WT littermates (Fig. 5,A). Notably, anti-DNA Abs to ssDNA and dsDNA were elevated in the sera of OX40L-Tg1 mice (Fig. 5,B). These data suggest that the OX40L-Tg1 mice develop polyclonal activation of B cells accompanied with production of autoantibodies specific for DNA. We next attempted to determine whether the serum levels of particular cytokines that affect Ig secretion of B cells were altered. Serum levels of IL-5 and IL-13 were four and seven times higher, respectively, in the OX40L-Tg1 mice in comparison to the WT littermates (Fig. 5 C). In contrast, the cytokine levels of IL-2, IL-4, IL-10, and IFN-γ were undetectable in the sera of both the OX40L-Tg1 mice and WT littermates (data not shown). Similar results to the OX40L-Tg1 mice were also seen among the OX40L-Tg2 mice (data not shown).

In view of the weight loss, symptoms of diarrhea, and the detection of elevated serum autoantibody levels in both OX40L-Tg1 and OX40L-Tg2 C57BL/6 mice, we conducted histopathological analyses of a broad spectrum of tissues in these mice. Cervical lymph node sections from the OX40L-Tg1 mice showed increased numbers of plasma cells when compared with WT mice (data not shown). Furthermore, numerous Mott cells containing Russell bodies, intracellular acidophilic deposits often found in mice with autoimmune diseases (46), were visible in sections derived from the OX40L-Tg1 mice (data not shown).

A remarkable appearance of inflammatory cells, consisting of lymphocytes, neutrophils, and macrophages were seen in the alveolar septa of lung sections obtained from OX40L-Tg1 mice (Fig. 6, B and D). These features were consistent with an acute interstitial pneumonia presentation. Infiltration of lymphocytes also into the peribronchiolar regions in OX40L-Tg1 lung sections (Fig. 6,B) appeared to closely resemble bronchiolitis. These lung histological features were consistently absent in the WT littermates (Fig. 6, A and C).

In colon sections obtained from OX40L-Tg1 mice, marked mucosal hypertrophy was evident. This was accompanied with severe hyperplasia of the lymphoid tissues in the lamina propria (Fig. 6,F), all of which were visibly absent in the WT colon sections (Fig. 6,E). Further magnification of the OX40L-Tg1 colon sections revealed the presence of a mixed inflammatory cell infiltrate in the submucosal regions, consisting of lymphocytes, plasma cells, and neutrophilic granulocytes (Fig. 6,G). In the mucosal epithelium, typical crypt abscesses were occasionally identified in the OX40L-Tg1 colon sections (Fig. 6 H). Taken together, this presentation appears to be reminiscent of IBD.

All these histopathological findings appeared 3 mo postnatal in the OX40L-Tg1 and OX40L-Tg2 and not the OX40L-Tg3 line (Table I). This was a significant finding since OX40L-Tg3 mice have marked expression of OX40L only in thymic and not splenic tissues (Fig. 1). Therefore, it is likely that overexpression of OX40L in the periphery may mediate the pathogenesis of these inflammatory diseases. Additional results show that the development of interstitial pneumonia and IBD is age-dependent with the incidence of the diseases increasing with age (Table I). Other tissue sections obtained from all the three OX40L-Tg lines, including heart, lung, aorta, liver, pancreas, submandibular glands, stomach, small intestines, tibial bones, kidneys, and ankle joints were histologically normal (data not shown). Furthermore, all three OX40L-Tg lines back-crossed onto the BALB/c background up to at least 20 mo of age, presented with no symptoms of disease or histological abnormalities in lung or colonic tissues (Table I), indicating the genetic background specificity in disease development.

Although mice were housed under specific pathogen-free conditions, to exclude the possibility of pathogenic microorganisms in the colony as being responsible for the interstitial pneumonia or IBD observed in the OX40L-Tg1 and -Tg2 mice, we conducted numerous tests to identify any contagious microorganisms such as Pneumocystis carinii and Helicobacter species, among others. No known pathogens responsible for interstitial pneumonia or colitis in the mice were found (data not shown). Taken together, these results strongly suggest that the diseases seen in the OX40L-Tg mice on the C57BL/6 background are mediated by an autoimmune mechanism.

Immunohistochemical analysis of the infiltrating lymphocytes in both the peribronchiolar regions of the lung and the intestinal lamina propria of the OX40L-Tg1 mice had a CD4⁺ phenotype (data not shown), demonstrating the possible participation of CD4⁺ T cells in the pathogenesis of these organ-specific autoimmune-like diseases. To determine whether OX40L-Tg CD4⁺ T cells mediate the pathogenesis of the diseases seen in the OX40L-Tg mice, we conducted a series of transfer experiments, in which CD4⁺ T cells derived from OX40L-Tg1 or WT mice were transferred i.v. into C57BL/6 RAG2-deficient mice. All the reconstituted RAG2-deficient mice displayed signs of sickness that included wasting and diarrhea 8 days after inoculation of OX40L-Tg1 CD4⁺ T cells (Fig. 7,A and data not shown). Between 2 and 5 wk posttransfer, lung histological sections revealed inflammatory features consistent with severe interstitial pneumonia (Fig. 7,C), while colon sections showed massive infiltration of lymphocytes in the intestinal lamina propria (Fig. 7,E). These histological features were reproduced in RAG2-deficient mice reconstituted with splenic CD4⁺ T cells derived from OX40L-Tg2 mice (Table I). In contrast, RAG2-deficient mice inoculated with CD4⁺ T cells from the WT mice had no symptoms of disease (Fig. 7,A and data not shown) or histological abnormalities in lung or colon tissue sections (Table I and Fig. 7, D and F). Interestingly, administration of an inhibitory anti-OX40L mAb, MGP34, along with CD4⁺ T cells from OX40L-Tg1 mice into RAG2-deficient mice completely prevented the development of the diseases (Table I, Fig. 7, G and H). These results not only demonstrate that OX40L-Tg CD4⁺ T cells are the responsible pathogenic effector cells in disease generation, but highlight the critical involvement of OX40/OX40L interaction in directly mediating the pathogenesis of these diseases.

The present study documents two important findings; first, OX40L-Tg mice have hyperproliferative CD4⁺ T cells and markedly elevated memory CD4⁺ T cell numbers, probably mediated by a disruption in AICD. Secondly, OX40L-Tg mice spontaneously develop organ-specific autoimmune-like diseases, namely interstitial pneumonia and colonic IBD, both of which can be reproduced in RAG2-deficient mice reconstituted with pathogenic OX40L-Tg CD4⁺ T cells. Furthermore, administration of an inhibitory anti-OX40L mAb prevented the development of these diseases in the reconstituted RAG2-deficient mice. This report demonstrates the first direct evidence linking the OX40/OX40L signaling pathway to the development of autoimmune-like diseases. Many previous reports have suggested that T cell costimulatory molecules (CD28, inducible costimulator, LFA-1, TNFR family molecules including OX40, etc.) may be involved in the development of autoimmune diseases (6, 47, 48, 49). However, to our knowledge, no report has revealed that constitutive costimulatory signaling in T cells lead to autoimmune/allergic diseases. In this context, our present observations should provide us with important information to understand T cell-mediated organ-specific autoimmunity.

OX40 has been reported to be expressed on autoreactive or effector T cells in patients with autoimmune diseases (34, 36, 40) and inflammatory diseases linked to immune disorders (37, 38, 39). The OX40/OX40L system may thus play a potential key role in the immune regulation of various autoimmune diseases. One mechanism to limit autoimmunity is by AICD, which has been considered to limit accumulation of autoantigen-specific T cells (50). Although a previous report demonstrated that treatment with an agonistic anti-OX40 mAb alone was not able to prevent S. aureus enterotoxin A-induced AICD (21), AICD triggered by SEB administration was markedly impaired in OX40L-Tg mice (Fig. 4 A). Because AICD plays an important role in regulating autoimmunity, defective AICD seen in OX40L-Tg mice may cause a marked elevation of effector memory CD4⁺ T cells and lead to autoimmune-like disease development. In addition, a recent report suggested a possible involvement of OX40 signaling in breaking peripheral CD4⁺ T cell tolerance (25). However, to further clarify the plausible association between the defective AICD, probably resulting in a break in peripheral tolerance and the autoimmune-like diseases seen in the OX40L-Tg mice, studies using Ag-specific T cells derived from a TCR transgenic mouse intercrossed to an OX40L-Tg mouse are required.

The spontaneous development of autoimmune-like diseases of the lung and colon in OX40L-Tg mice may provide us with the first evidence demonstrating the direct link of OX40 signaling to the development of immune disorder-associated diseases. This association is strongly supported by our results showing the effectiveness of an inhibitory anti-OX40L mAb in completely preventing these diseases in syngenic RAG2-deficient mice reconstituted with pathogenic OX40L-Tg CD4⁺ T cells. Although elevated serum autoantibody levels were seen in the OX40L-Tg mice, it is clear that B cells are not essential in the pathogenesis of interstitial pneumonia or IBD, since the reconstituted RAG2-deficient mice which lack B cells developed these diseases. However, this finding does not rule out the possibility that autoantibodies might lead to autoimmune manifestations in OX40L-Tg mice.

The Th2-biased responses observed in OX40L-Tg mice may be compatible with previous in vitro studies demonstrating that OX40-cross-linking on T cells upon Ag- or TCR-stimulation induces enhanced IL-4 production, but not IFN-γ (51, 52, 53). Interestingly, when immunizing Ag into OX40L-Tg mice in the absence of adjuvant, only Th2 cytokines were produced by CD4⁺ T cells (Fig. 3,D), although Ag immunized with adjuvant induced higher cytokine production in both Th1 and Th2 responses in OX40L-Tg mice (Fig. 3,B). In addition, OX40L-Tg mice exhibited higher serum levels of IL-5 and IL-13 (Fig. 5 C). These results clearly indicate that constitutive OX40/OX40L interaction, probably leading to excessive OX40 signaling in T cells, induces Th2 responses in vivo. In contrast, transgenic mice for CD70 (CD27 ligand), which is also a T cell costimulatory molecule belonging to TNF family members, have reportedly demonstrated Th1-dominant phenotypes, such as high IFN-γ production (54), suggesting distinct costimulatory functions on Th responses of TNFR family molecules on T cells.

Pulmonary abnormalities and ulcerative colitis are found in patients with HTLV-I-associated diseases (55, 56, 57). Expression of OX40L, which was initially described on HTLV-I-infected human T cell lines (7, 8), was detected on activated T cells (9, 10) and long-term cultured cytotoxic T cell clones (11). The interaction between OX40 and OX40L via T cell-T cell interactions may possibly have implications in the pathogenesis of these HTLV-I-associated diseases.

The onset of most human autoimmune diseases is apparently dependent on some specific genetic backgrounds such as HLA, sex-chromosomal genes, etc. The observation that the autoimmune-like diseases seen in the OX40L-Tg mice are mouse strain-dependent leads us to the conclusion that further studies by chromosomal linkage analysis of possible causative genes using resistant and susceptible strains will be required. OX40L-Tg mice may thus be a useful tool in evaluating the pathogenesis of interstitial pneumonia and IBD.

We thank Drs. M. Ito, K. Hioki, and T. Ito (Central Institute for Experimental Animals) for providing RAG2-deficient mice and screening for pathogens in the OX40L-Tg mice.