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创建帐户 Anti-CD81 antibodies reduce migration of activated T lymphocytes and attenuate mouse experimental colitis AbstractInflammatory bowel disease (IBD) is an immunological disease associated with CD4+ T cell activation in the intestines. CD81 is a regulator of the immune system with multiple biological functions. Therefore, in this study, we assessed the contribution of CD81 to IBD pathophysiology and the therapeutic efficacy of anti-CD81 antibodies. Expression of CD81 was increased on activated T cells in vitro and in colitic mice in vivo. Therapeutic effects of anti-CD81 antibodies on colitic symptoms and inflammation were evaluated in mice with colitis, including long-term effects of the antibodies. Treatment with anti-CD81 antibodies improved colitis scores, reduced colon shortening, decreased loss of body weight, and resulted in fewer pathological changes of the colon in colitic mice. Moreover, the increased inflammatory markers in the blood of colitic mice were decreased by anti-CD81 antibodies. The anti-CD81 antibody treatment had long-lasting therapeutic effects on colitic mice, even after cessation of treatment. Two different clones of the anti-mouse CD81 antibody were also effective in mice with colitis. Furthermore, anti-CD81 antibodies reduced migration of CD4+ T cells both in colitic mice and in vitro. Thus, CD81 contributes to IBD pathology and treatment with anti-CD81 antibodies may be a potential novel therapy for IBD patients. IntroductionInflammatory bowel disease (IBD), including Crohn鈥檚 disease and ulcerative colitis, is a group of diseases with chronic and relapsing intestinal inflammation. Current therapies focus on controlling inflammation using immunosuppressants, steroids, or biopharmaceuticals against proinflammatory cytokines and lymphocytes. However, the clinical benefits of current medical treatments are limited and many patients live long-term with the disease after onset at a young age1,2. IBD is an immunological disease associated with activation of CD4+ T cells in the intestines3,4 and the expression of multiple proinflammatory chemokines, including C-X-C chemokine receptor type 4 (CXCR4), which attract leukocytes into inflamed intestines in both IBD mouse models1 and patients2.CD81 is a cell surface protein belonging to the tetraspanin superfamily. It has been identified as a component of the B lymphocyte receptor and a host entry factor for the hepatitis C virus5. Tetraspanins increase the formation and stability of biologically functional receptors consisting of tetraspanins and other molecules3. CD81 associates with various immune molecules on T and B lymphocytes as well as other cell types to facilitate cell-to-cell communication at the immune synapse interface between antigen-presenting cells (APCs) and T lymphocytes6. We examined the contribution of CD81 to the pathology of IBD using anti-mouse CD81 antibodies and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis to determine its therapeutic potential for IBD. Targeting cell migration is considered as one of the most promising therapeutic approaches for IBD, because mice with TNBS-induced colitis have inflamed colons in which activated CD4+ T cells accumulate7. The present study aimed to determine the role of CD81 in the pathophysiology of IBD and the therapeutic potential of anti-CD81 antibodies for IBD.ResultsCD81 is increased on activated T cells and in mice with TNBS-induced colitisTo examine CD81 expression on activated T cells, peripheral blood mononuclear cells (PBMCs) from SJL/J mice were cultured with phytohemagglutinin (PHA) and IL-2 for 0, 24, 48, and 72鈥塰. CD69 on T cells was maximally increased at 24鈥塰 after stimulation, while CD81 was increased from 24 to 72鈥塰 (Fig.聽1A). CD81+ T cells among lymphocytes of the Peyer鈥檚 patches and mesenteric lymph nodes of mice with TNBS-induced colitis were increased compared with those of untreated mice (Fig.聽1B). Moreover, overall, CD81+ cells in the colons of mice with TNBS-induced colitis were increased compared with those of untreated mice (Fig.聽1C). Thus, CD81 was increased on activated T cells in mice with colitis.Figure 1Expression of CD81 in mouse PBMCs stimulated with PHA and IL-2, and in mice with TNBS-induced colitis. (a) PBMCs were collected from SJL/J mice and stimulated with PHA and human IL-2 for 0, 24, 48, and 72鈥塰. PBMCs were stained with anti-CD69, anti-CD81 (clone Eat2), and anti-CD3蔚 (n鈥?鈥? per group) antibodies. Then, cell surface markers were analysed using a FACSCanto II. (b) Cells in mesenteric lymph nodes (MLNs) from mice with TNBS-induced colitis and untreated mice were stained with anti-CD3e and anti-CD81 antibodies, and then analysed using the FACSCanto II (n鈥?鈥? per group). Statistical significance was determined using the Student鈥檚 t-test (*p鈥?lt;鈥?.05). Data are representative of three independent experiments. (c) Representative immunohistochemical staining of colons from mice with TNBS-induced colitis and untreated mice. Colons were removed on day 4, fixed with paraformaldehyde, embedded in paraffin, and sectioned. Immunostaining was performed with biotin-labelled hamster IgG or the anti-CD81 antibody.Full size imageAnti-CD81 antibody has short-term effects on TNBS-induced colitic miceThe effect of an anti-CD81 antibody on acute intestinal inflammation was examined in mice with acute colitis. Mice with established TNBS-induced colitis were administered the anti-mouse CD81 antibody (clone 2F7) and histopathological changes were examined for 7 days (Supplementary Fig.聽1 and Table聽1). Treatment with the anti-CD81 antibody on days 0, 2, and 4 and daily administration of sulfasalazine (SSZ) attenuated the colitis score. Notably, the colitis score on day 7 was significantly reduced by both the anti-CD81 antibody and SSZ compared with the vehicle group (Fig.聽2A). Although there were eight mice with established colitis per group, one to three mice in each group died despite the reduction of the colitis score by anti-CD81 antibody and SSZ treatments. Mice with established TNBS-induced colitis treated with the anti-CD81 antibody had increased body weights compared with vehicle-treated mice, and the difference in the body weight change over 7 days was statistically significant (Fig.聽2B). The length of the colons of mice with TNBS-induced colitis was shortened compared with that of untreated mice, and treatment with the anti-CD81 antibody and SSZ significantly improved the colon length (Fig.聽2C). TNBS-induced colitis mice had inflamed and thickened colons, and anti-CD81 antibody treatment improved these pathological changes (Fig.聽2D and Supplementary Fig.聽2). Although the anti-CD81 antibody slightly decreased epithelial cell apoptosis (TdT-mediated dUTP Nick End Labelling+; TUNEL+) in the colon, it was not statistically significant (Supplementary Fig.聽3). Inflammatory markers, including C-reactive protein (CRP), lymphotactin, monocyte chemoattractant protein-5, and macrophage inflammatory protein-2, were increased in the serum of mice with TNBS-induced colitis compared with untreated mice. The anti-CD81 antibody reduced the levels of these markers (Table聽1). Thus, the anti-CD81 antibody had therapeutic effects on acute intestinal inflammation.Figure 2Short-term effects of the anti-CD81 antibody (2F7) on TNBS-induced colitis (7 days). Mice with established TNBS-induced colitis were divided into four groups on day 0. Colitic mice were injected intraperitoneally with hamster IgG and the anti-CD81 antibody. SSZ was administered orally. Open circle, untreated (n鈥?鈥?); closed circle, TNBS-induced colitic mice treated with hamster IgG at 0.1鈥塵g/mouse (n鈥?鈥?) on days 0, 2, and 4; open and closed square, TNBS-induced colitic mice treated with the anti-CD81 antibody at 0.02鈥塵g/mouse (n鈥?鈥?) and 0.1鈥塵g/mouse (n鈥?鈥?) on days 0, 2, and 4; open triangle, TNBS-induced colitic mice treated with SSZ at 200鈥塵g/kg (n鈥?鈥?) orally administered from day 0 to 7. Data are representative of at least three independent experiments. (a) Colitis scores were evaluated daily (line graph). The bar graph is the average colitis score on day 7. Statistical significant was determined using Wilcoxon鈥檚 test [##p鈥?lt;鈥?.01, untreated vs hamster IgG; a: p鈥?lt;鈥?.05, hamster IgG vs anti-CD81 antibody (0.02); b: p鈥?lt;鈥?.05, hamster IgG vs anti-CD81 antibody (0.1); c: p鈥?lt;鈥?.05, hamster IgG vs SSZ]. (b) Body weights were measured daily (line graph). The bar graph shows the body weight change (% of day 0) on day 7. Statistical significance was determined using the Student鈥檚 t-test [*p鈥?lt;鈥?.05, hamster IgG vs anti-CD81 antibody (0.1); +p鈥?lt;鈥?.05, hamster IgG vs anti-CD81 antibody (0.02)]. (c) Representative macroscopic images of the colon and colon length. Colons were removed on day 7 and their lengths measured. Statistical significance was determined using the Student鈥檚 t-test (##p鈥?lt;鈥?.01, untreated vs hamster IgG; **p鈥?lt;鈥?.01, hamster IgG vs anti-CD81 antibody, SSZ)). (d) Representative immunohistochemical staining of colons from treated and untreated mice with TNBS-induced colitis (脳40). Hamster IgG or the anti-CD81 antibody was intraperitoneally injected into TNBS-induced colitic mice on day 0 and 2. Colons were removed on day 7, and sections were prepared for haematoxylin-eosin staining.Full size imageTable 1 Inflammation markers in serum from SJL/J mice with TNBS-induced colitis and untreated after 3 days of treatment with the anti-CD81 antibody (2F7, 0.5鈥塵g/mouse) or control antibody.Full size tableAnti-CD81 antibody has long-lasting effects on TNBS-induced colitis in miceTo further examine the effect of the anti-CD81 antibody on intestinal inflammation, mice with established TNBS-induced colitis were intraperitoneally injected with the anti-mouse CD81 antibody (clone 2F7) once a day for 6 days, and changes in the colitis score and body weight were examined for 28 days until colitis symptoms had diminished (Fig.聽3, Supplementary Fig.聽4a and Supplementary Table聽1). Because colitis scores were reduced after 1 week, all mice were fasted on day 18 and then intrarectally re-administered the TNBS solution to induce a flare of colitis on day 21. Treatment with the anti-CD81 antibody significantly attenuated the colitis score on days 7, 23, and 25. Moreover, the anti-CD81 antibody attenuated the colitis score significantly from day 23 to 28 compared with SSZ. In contrast, SSZ reduced the colitis score on day 7, but not from day 23 to 28 (Fig.聽3A,聽B). This experimental condition was milder than the previous experiment in Fig.聽2, because 8-week body weights of mice with established colitis ranged from 16.9 to 23.0鈥塯 and all mice were still alive at day 21. The body weight change of mice with established TNBS-induced colitis treated with the anti-CD81 antibody did not reach statistical significance from day 0 to 7, but the body weight change from day 0 to 18 was increased significantly. In contrast, SSZ treatment did not influence the body weight change from day 0 to 7 or from day 0 to 18 (Fig.聽3C,聽D). In addition, the anti-CD81 antibody improved the colon length compared with hamster IgG (Fig.聽3E). Therefore, after stopping anti-CD81 antibody treatment, its therapeutic effect was maintained.Figure 3Anti-CD81 antibody (2F7) attenuates TNBS-induced colitis in the long term (28 days). Mice with established TNBS-induced colitis were divided into three groups on day 0. Colitic mice were injected intraperitoneally with hamster IgG or the anti-CD81 antibody. SSZ was administered orally. Mice were intrarectally infused with a TNBS solution on day 21. Closed circle, TNBS-induced colitic mice treated with hamster IgG at 0.1鈥塵g/mouse for 6 days (day 0鈥?); closed square, TNBS-induced colitic mice treated with the anti-CD81 antibody at 0.1鈥塵g/mouse for 6 days (day 0鈥?); open triangle, TNBS-induced colitic mice treated with SSZ at 200鈥塵g/kg from day 0 to 21. (n鈥?鈥? per group). Data are representative of at least three independent experiments. (a,聽b) Colitis scores were evaluated daily (a, line graph). Colitis score on day 7 (b, left graph) and days 23鈥?8 (b, right graph). Statistical significance was determined using Wilcoxon鈥檚 test (*p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01). (c,聽d) Body weight was measured daily (c, line graph). Body weight changes (% of day 0) on day 7 (d, left graph) and day 18 (d, right graph) are shown. Statistical significance was determined using the Student鈥檚 t-test (*p鈥?lt;鈥?.05). (e) Colons were removed and their lengths measured on day 28. Statistical significance was determined using the Student鈥檚 t-test (**p鈥?lt;鈥?.01).Full size imageTwo different clones of the anti-mouse CD81 antibody are effective against TNBS-induced colitisTo compare the effectiveness of different clones (2F7 and Eat2) of the anti-mouse CD81 antibody on intestinal inflammation, mice with TNBS-induced colitis were administered the clones only once on day 0. Clone Eat2 significantly attenuated colitis scores at doses of 0.1 and 0.04鈥塵g/mouse. In contrast, the effect of clone 2F7 on colitis score was not significant when administered only once (Fig.聽4A, Supplementary Fig.聽4b, and Table聽1). Both clones significantly improved the colon length. Mice treated with hamster IgG, 0.04鈥塵g/mouse 2F7, 0.1鈥塵g/mouse 2F7, 0.04鈥塵g/mouse Eat2, or 0.1鈥塵g/mouse Eat2 had colons with average lengths of 99.3鈥壜扁€?.9鈥塵m, 112.5鈥壜扁€?.9鈥塵m, 105.6鈥壜扁€?鈥塵m, 113.0鈥壜扁€?.1鈥塵m, and 112.8鈥壜扁€?.9鈥塵m, respectively (Fig.聽4B). In addition, another anti-CD81 antibody, Eat1, ameliorated TNBS-induced colitis (Supplementary Figs.聽4c and 5 and Supplementary Table聽1). Thus, multiple anti-mouse CD81 antibody clones had therapeutic effects on intestinal inflammation.Figure 4Therapeutic effects of clone Eat2 of the anti-mouse CD81 antibody on TNBS-induced colitis are superior to those of clone 2F7. Mice with established TNBS-induced colitis were divided into five groups on day 0 (n鈥?鈥? per group). Colitic mice were injected intraperitoneally with hamster IgG or the anti-CD81 antibody on day 0. Closed circle, TNBS-induced colitic mice treated with hamster IgG at 0.1鈥塵g/mouse; open and closed square, TNBS-induced colitic mice treated with the anti-CD81 antibody (2F7) at 0.04 and 0.1鈥塵g/mouse respectively; open and closed triangle, TNBS-induced colitic mice treated with the anti-CD81 antibody (Eat2) at 0.04 and 0.1鈥塵g/mouse, respectively. Data are representative of at least three independent experiments. (a) Colitis scores were evaluated daily (line graph). Bar graph shows the average colitis score on day 7. Statistical significance was determined using Wilcoxon鈥檚 test (*p鈥?lt;鈥?.05, hamster IgG vs Eat2 (0.04); ##p鈥?lt;鈥?.01, hamster IgG vs Eat2 (0.1)). (b) Colons were removed and their lengths measured on day 7. Statistical significance was determined using the Student鈥檚 t-test (*p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01, hamster IgG vs. anti-CD81 antibody).Full size imageAnti-CD81 antibody inhibits migration of T cells induced by human stromal cell-derived factor 1 (SDF-1)Next, we investigated the mechanism of the effect of the anti-mouse CD81 antibody on TNBS-induced colitis, focusing on T cell functions using the clone Eat2. Colitic mice were administered Eat2 and changes in the frequency of T cells among colon lamina propria mononuclear cells (LPMCs) were determined at 2 days after administration. Although the frequencies of CD4+ T and CXCR4+ cells among LPMCs were decreased by Eat2, Eat2 did not affect the frequency of Treg (CD4+FoxP3+) cells (Fig.聽5A). Furthermore, the therapeutic efficacy of Eat2 was associated with decreases in IL-1伪 and IL-6 levels detected in colonic homogenates from mice with TNBS colitis (Supplementary Fig.聽6). Because CD81 was degraded by digestive enzymes in the preparation of colonic cells, the action of the anti-mouse CD81 antibody was determined on splenocytes and not colonic cells in mice with TNBS-induced colitis. However, CD69+ T cells from untreated mice with established TNBS-induced colitis were enriched for CD81+ T cells by 2.1鈥?.2-fold versus CD81鈭?/sup> T cells with or without staphylococcal enterotoxin B (SEB) stimulation (Supplementary Figs.聽7鈥?a data-track=\"click\" data-track-label=\"link\" data-track-action=\"supplementary material anchor\" href=\"/articles/s41598-020-64012-5#MOESM1\">10). The anti-mouse CD81 antibody had no effect on the proliferation of splenocytes from mice with TNBS-induced colitis, which were stimulated with SEB or an immobilised anti-mouse CD3蔚 antibody (Fig.聽5B,聽C). The anti-mouse CD81 antibody also had no influence on the cytokine production of splenocytes from mice with TNBS-induced colitis, which were stimulated with the immobilised anti-mouse CD3蔚 antibody or SEB (Fig.聽5D,聽E). CXCR4+ T cells in mice with established TNBS-induced colitis were enriched among CD81+ T cells by 2.6- or 3.9-fold versus CD81鈭?/sup> T cells with or without SEB stimulation, respectively (Supplementary Figs.聽7鈥?a data-track=\"click\" data-track-label=\"link\" data-track-action=\"supplementary material anchor\" href=\"/articles/s41598-020-64012-5#MOESM1\">10). The anti-CD81 antibody inhibited the migration of splenocytes from mice with TNBS-induced colitis, which was induced by SDF-1, one of the ligands for chemokine receptor CXCR4 and a chemokine involved in the pathogenesis of IBD (Fig.聽5F)1,8. The anti-CD81 antibody also inhibited SDF-1-induced migration of mouse T cell line EL4.IL-2 (Supplementary Fig.聽11)9. Thus, the anti-CD81 antibody reduced migration of activated T cells, which attenuated intestinal inflammation in the TNBS-induced model of colitis.Figure 5Anti-CD81 antibody reduces the frequency CD4+ T cells and inhibits migration induced by SDF-1. (a) Mice with established TNBS-colitis mice were divided into two groups and then intraperitoneally injected with hamster IgG or the anti-CD81 antibody Eat2 (0.1鈥塵g/mouse, n鈥?鈥? per group). After 2 days, lamina propria mononuclear cells (LPMCs) were prepared and stained with anti-CD3蔚, anti-CD4, anti-CXCR4, and anti-FoxP3 antibodies for flow cytometric analysis. Data are representative of three independent experiments. Statistical significance was determined using the Student鈥檚 t-test (*p鈥?lt;鈥?.05). (b,聽c) Splenocytes from mice with established TNBS colitis were cultured with hamster IgG, anti-CD81 (2F7 and Eat2) antibodies at 10鈥壜礸/mL, and CsA at 100鈥塶M under stimulation by (b) 1鈥壜礸/mL SEB or (c) 5鈥壜礸/ml anti-CD3蔚 antibody. After 2 days, BrdU was added to the cultures and detected by an ELISA (n鈥?鈥? per group). Data are representative of at least three independent experiments. Statistical significance was determined using the Student鈥檚 t-test (**p鈥?lt;鈥?.01, hamster IgG vs anti-CD81 antibody, CsA) (d,聽e) Splenocytes from mice with established TNBS-induced colitis mice were prepared and cultured in 96-well plates precoated with 1鈥壜礸/ml anti-CD3蔚 antibody. Hamster IgG, anti-CD81 antibodies (2F7 and Eat2), and an anti-CD28 antibody at 5鈥壜礸/mL were added to the cultures, followed by 3 days of incubation. IFN纬 (d) and TNF伪 (e) in culture supernatants were measured by ELISAs (n鈥?鈥? per group). Data are representative of at least three independent experiments. (f) Splenocytes from mice with established TNBS-induced colitis were prepared and cultured with hamster IgG or anti-CD81 antibodies (2F7 and Eat2) at 10鈥壜礸/mL for 2鈥塰. Cells were seeded in the upper chamber of a 96-well plate containing 5-碌m pore transwells with or without 10鈥塶g/mL SDF-1 in the lower chamber for migration assays. The number of cells was counted by ATPlite (n鈥?鈥? per group). Data are representative of at least three independent experiments. Statistical significance was determined using the Student鈥檚 t-test (**p鈥?lt;鈥?.01, hamster IgG vs. anti-CD81 antibody).Full size imageAnti-CD81 antibody is also effective against sodium dextran sulphate salt (DSS)-induced colitis in miceFinally, we assessed the therapeutic potential of the anti-CD81 antibody in another colitis model, DSS-induced colitis. Mice with DSS-induced colitis were administered the anti-mouse CD81 antibody (clone 2F7) on days 0 and 14. Treatment with the anti-CD81 antibody at 0.2鈥塵g/mouse significantly reduced the colitis score not only on days 4 and 5, but also from day 18 to 20 compared with the vehicle group (Fig.聽6A). In addition, mice with DSS-induced colitis and treated with the anti-CD81 antibody at 0.2鈥塵g/mouse had improved body weights on days 18 and 19 compared with vehicle-treated mice (Fig.聽6B). The length of the colons in mice with DSS-induced colitis was shortened compared with those in untreated mice, and treatment with the anti-CD81 antibody at 0.2鈥塵g/mouse significantly improved the colon length (Fig.聽6C). Histological analysis of DSS-induced colitis mice showed features of colitis, such as depletion of mucus and cell infiltration in the mucosa, and the anti-CD81 antibody treatment improved these pathological changes (Fig.聽6D). Thus, the anti-CD81 antibody had therapeutic effects on DSS-induced acute intestinal inflammation.Figure 6Anti-CD81 antibody (2F7) ameliorates DSS-induced colitis. Mice were administered 5% DSS in drinking water from day 鈭? to 0. Then, the mice were divided into three groups on day 0 (N鈥?鈥? per group). Colitic mice were injected intraperitoneally with hamster IgG or the anti-CD81 antibody on days 0 and 14. Open circle, untreated; closed circle, DSS-induced colitic mice treated with hamster IgG at 0.2鈥塵g/mouse; closed square, DSS-induced colitic mice treated with the anti-CD81 antibody at 0.04鈥塵g/mouse; closed triangle, DSS-induced colitic mice treated with the anti-CD81 antibody at 0.2鈥塵g/mouse. Data are representative of at least three independent experiments. (a) Colitis scores were evaluated daily. Statistical significance was determined using Wilcoxon鈥檚 test (*p鈥?lt;鈥?.05, hamster IgG vs anti-CD81 antibody). (b) Body weights were measured daily. (c) Colons were removed and their lengths measured on day 20. Statistical significances of (b,聽c) were determined using the Student鈥檚 t-test (#p鈥?lt;鈥?.05, untreated vs hamster IgG; *p鈥?lt;鈥?.05, hamster IgG vs anti-CD81 antibody). (d) Representative immunohistochemical staining of colons from treated and untreated mice with DSS-induced colitis (脳40). Hamster IgG or the anti-CD81 antibody was intraperitoneally injected into DSS-induced colitic mice on days 0 and 14. Colons were removed on day 20, and sections were prepared for haematoxylin-eosin staining.Full size imageDiscussionIn this study, we examined the contribution of CD81 to IBD pathophysiology. CD81 has been reported to be expressed on multiple kinds of cells at various expression level and have roles in proliferation, differentiation, cytokine production, migration, and other biological actions10. We found increased CD81 expression on T cells in peripheral blood and Peyer鈥檚 patches of mice with TNBS-induced colitis. An endogenous ligand for CD81 has not been identified, but its signaling is involved in forming complexes consisting of polarised patches of increased CD81 molecules and other associated molecules3. For example, Mittelbrunn et al. reported that immune synapse formation induces CD81 polarisation on T cells and APCs6. A prominent increase in CD81 expression on activated T cells suggests that CD81 contributes to the functions of T cells. We showed that the anti-mouse CD81 antibody improved colitic symptoms, including shortened inflamed colons and reduced body weight, and attenuated histological changes in colons and inflammatory markers in the blood of mice with acute TNBS-induced colitis. Moreover, the anti-CD81 antibody improved colitic symptoms and body weight change of mice with a flare-up of TNBS-induced colitis after stopping anti-CD81 antibody treatment. Interestingly, it was previously shown that CD81 induces quiescence of hematopoietic stem cells11. This function of CD81 may be involved in the reset of inflammatory lymphocytes in colitis and contribute to the long-lasting effect of anti-CD81 antibodies. Because IBD is a chronic remitting/relapsing disease, the long-term effects of the anti-CD81 antibody would be particularly useful for IBD therapy.Dijkstra and colleagues demonstrated that the anti-mouse CD81 antibody clone 2F7 is not effective against experimental autoimmune encephalomyelitis, although another clone, Eat2, is therapeutic12. Different clones of an anti-CD81 antibody exert different effects on biological functions10. We showed that two different anti-mouse CD81 antibody clones, 2F7 and Eat2, were both effective against colitis, although Eat2 was more effective than 2F7. In addition, as another clone of anti-CD81 antibody, Eat1 was also effective against TNBS-induced colitis. Therefore, we hypothesised that CD81 regulated TNBS-induced colitis. We examined the mechanism underlying the therapeutic effect of the anti-CD81 antibody and found that CD4+ T cells were decreased in colons of mice with colitis treated with the anti-CD81 antibody. Although anti-CD81 antibodies had no influence on the proliferation or cytokine release of stimulated lymphocytes from mice with colitis, the anti-CD81 antibody reduced migration of lymphocytes from both colitic mice and that of a mouse T cell line towards SDF-1. IFN纬+ T cells in established TNBS-colitis mice were rich in CD81+ T cells at 3.7-fold (12.8% versus 3.5%) or 4.9-fold (6.4% versus 1.3%) compared with CD81鈭?/sup> T cells with or without SEB stimulation, respectively (Supplementary Figs.聽7 and 9). IL-17+ T cells from mice with established TNBS-induced colitis were rich in CD81+ T cells at 3.7-fold (11.9% versus 3.2%) and 19-fold (3.8% versus 0.2%) compared with CD81鈭?/sup> T cells with or without SEB stimulation, respectively (Supplementary Figs.聽7 and 9). These results indicated that CD81+ T cells were mostly effector T cells in TNBS-induced colitis13. Therefore, it was likely that the anti-CD81 antibody reduced migration of effector T cells in mice with colitis and improved colonic inflammation. However, because this inhibitory effect was not complete, other mechanisms may contribute to the therapeutic effect of the anti-CD81 antibody on murine colitis. Recently, it was reported that CD81 knockout mice have impaired functions of regulatory T cells in a tumour transplantation model14.Finally, we confirmed the therapeutic potential of the anti-CD81 antibody in another model, DSS induced colitis. The anti-CD81 antibody ameliorated the colitis score and improved colon lengths in mice with DSS-induced colitis. Although there are potential limitations in IBD animal models, such as species differences and incomplete disease manifestations, we have shown that CD81 plays a critical role in IBD pathophysiology. Because CD81 is a critical or modified molecule for various biological functions, such as cell migration, resetting the cell cycle, and Treg functions, complete understanding of the mechanism of the therapeutic effect of anti-CD81 antibodies on IBD is necessary prior to implementation of CD81-targeted therapy in the clinic.Materials and methodsMiceFour to 6-week-old male SJL/J mice (Charles River Japan, Japan) and 6-week-old male BALB/cAnNCrlCrlj (Charles River Japan, Japan) were purchased and housed under specific pathogen-free conditions. Mice were quarantined and acclimatised for 1 week before use and then housed in a controlled environment (23鈥壜扁€?鈥壜癈; 55鈥壜扁€?0% humidity) with a 12-hour light/dark cycle (lights on at 8:00 AM) and allowed free access to food (CE-2; Clea Japan, Inc., Japan) and filtered water. All study protocols were approved by the Animal Care and Use Committee of Sumitomo Dainippon Pharma Co., Ltd., and undertaken in accordance with the regulations for animal experiments in this division.AntibodiesA hamster anti-mouse CD81 antibody (2F7) was purchased from Southern Biotech (UK). Anti-mouse CD81 antibody (Eat1) was purchased from Santa Cruz (US). Hamster IgG (HTK888), anti-mouse CD81 (Eat2), biotin-anti-mouse CD81 (Eat2), FITC-anti-hamster IgG (poly4055), FITC-anti-mouse CD69 (H1.2F3), isotype-matched FITC-hamster IgG (HTK888), PE-anti-mouse CD81 (Eat2), isotype-matched PE-hamster IgG (HTK888), and PerCP-streptavidin were obtained from Biolegend (USA). APC-anti-mouse CD4 antibodies (GK1.5), isotype matched APC-rat IgG2b (RTK2071), PE-anti-mouse FoxP3(FJK-16s), an APC-anti-mouse CD3蔚 (145鈥?C11) antibody, and APC-hamster IgG (eBio299Arm) were purchased from eBioscience (USA). FITC-anti-mouse CD3蔚 (145鈥?C11), anti-mouse CD3蔚 (145鈥?C11), anti-mouse CD28 (37.51), PE-anti-mouse CD69 (H1.2F3), PE-anti-mouse CXCR4 (2B11), PE-anti-mouse IFN纬 (XMG1.2), PE-anti-mouse IL-17 (TC11鈥?8H10), and anti-mouse CD16/32 antibodies (Fc block, 24鈥塆.2) were purchased from BD Bioscience (USA).T cell culture and activationPBMCs were prepared using Lympholyte-Mammals (CEDARLANE, Canada). PBMCs (2.5 脳 106/well in 6-well plates) were cultured with PHA (5鈥壜礸/ml; WAKO) and IL-2 (50鈥塶g/ml; R D Systems) in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin at 37鈥壜癈 in a humidified atmosphere containing 5% CO2 for 0, 24, 48, and 72鈥塰.Flow cytometry analysisPBMCs were prepared using Lympholyte-Mammals (CEDARLANE, Canada). Colon lamina propria mononuclear cells (LPMCs) were prepared according to previously described methods15,16. Briefly, colons were cut into pieces, digested with 1鈥塵g/mL collagenase A (Roche, Switzerland) and 0.01% DNase (Sigma-Aldrich) in 1.5% FBS in Hank鈥檚 balanced salt solution (Invitrogen, USA) and then purified from the interface between a 75% and 40% Percoll gradient. Peyer鈥檚 patches, mesenteric lymph nodes, and spleens were ground with white edge glasses (Matsunami Glass, Japan). Splenocytes were treated with ACL lysis buffer (Gibco, USA) to remove red blood cells. The cells were filtered through a 70-碌m pore size mesh to remove tissue debris. PBMCs were cultured in the absence or presence of 5鈥壜礸/ml PHA and 50鈥塶g/ml recombinant human IL-2 (R D Systems) in RPMI-1640 medium (Gibco) containing 10% FBS and penicillin-streptomycin. For intracellular staining, cells were cultured with or without 1鈥壜礸/mL SEB (sigma) for 50鈥塰. The cells were treated with Golgi Stop (BD Bioscience) for 14鈥塰. The cells were then incubated with an anti-mouse CD16/32 antibody for 20鈥塵in to block IgG Fc receptor binding and then stained with fluorescently labelled antibodies against cell surface markers (CD3, CD69, CD81, CXCR4, IFN纬, and IL-17). Stained cells were fixed with 4% paraformaldehyde and analysed on a FACSCalibur or FACSCanto II (BD Bioscience). Dead cells were excluded by forward light scatter and 7-aminoactinomycin D staining. In the lymphoid population, 2 脳 104 cells/sample were analysed. The threshold of positivity was defined as beyond the nonspecific binding observed in the presence of a relevant isotype control antibody.Induction of TNBS colitisSJL/J mice were subcutaneously injected with an emulsion of 2,4,6-trinitrobenzene sulfonic acid (TNBS; Nacalai Tesque Inc., Japan) bound to ovalbumin (OVA; Sigma, USA) in complete Freund鈥檚 adjuvant (CFA; Difco Laboratories, USA). One week later, the mice were anesthetised and infused with 2鈥塵g TNBS in 0.2鈥塵l of 50% ethanol at 3鈥塩m from the anus. To ensure TNBS distribution within the entire colon, the mice were held in a vertical position for 3鈥塵inutes after the infusion. Colitis scores were estimated by stool conditions: score 0, normal stool that was dry or wet, but maintained shape after prodding; score 1, loose stool that was wet and soft, and its shape was easily broken by prodding; score 2, diarrhoea. From 5 to 7 days after rectal infusion of TNBS, mice with colitis score 1 or 2 were determined as having established colitis. Colitic mice were divided into three or four groups by their body weight and stool score on day 0 using the 鈥淏lock randomization with multiple variables鈥?program in Stat Preclinica. Mice were sacrificed on day 7 for the short-term analysis or day 28 for the long-term analysis after the first anti-CD81 antibody administration, and then spleens, colons, and blood were collected for analyses. Serum from mice with colitis was analysed for C-reactive protein, lymphotactin, monocyte chemotactic protein-5 (MCP-5), and macrophage inflammatory protein-2 (MIP-2) using rodents multi-analyte profiles (Charles River Laboratories, Japan). Colons were removed, and sections were prepared for haematoxylin-eosin staining. Analysis of the levels of cytokines IL-1伪 and IL-6 in colon homogenates was performed by ELISAs (Invitrogen, USA).ImmunohistochemistryColons were removed from mice, opened longitudinally, and fixed in 10% formaldehyde neutral buffer solution (Nacalai Tesque Inc.). Samples were embedded in paraffin and cut into 5 碌m-thick sections. The sections were de-paraffinized, blocked, and incubated with a horseradish peroxidase-conjugated anti-mouse CD81 antibody (clone Eat2). Antibody binding was detected with DAB substrate using the Rabbit ImmunoCruz Staining System (Santa Cruz Biotechnology, USA) and counterstained with haematoxylin (Wako). All sections were observed by light microscopy.Anti-mouse CD81 Abs, hamster IgG, and SSZ treatment of TNBS-induced colitic mice for short-and long-term analysesTo examine the effect of anti-CD81 antibodies on colitis, mice with established colitis were randomised by colitis scores and body weights, grouped, and intraperitoneally injected with an anti-CD81 antibody (clone 2F7 or Eat2) or control hamster IgG. In the short-term effect analysis, mice received the antibody once a day on days 0, 2, and 4 (Fig.聽2A鈥揅), days 0 and 2 (Fig.聽2D and Supplementary Fig.聽2), or day 0 (Table聽1, Figs.聽4 and 5, Supplementary Figs.聽3, 5, 6, and 12). In the long-term effect analysis, mice received the antibody once a day from day 0 to 5 (Fig.聽3). SSZ (Sigma-Aldrich) was administered each day orally for the long-term analysis. Then, colons were collected for analyses.Proliferation and cytokine production of lymphoid cells from TNBS-induced colitic miceSpleens were removed from mice with established TNBS-induced colitis and ground with white edge glasses. Splenocytes were treated with ACK lysing buffer to lyse red blood cells. Remaining cells were filtered through a 70-碌m pore size mesh to remove debris. The prepared cells were seeded at 1 脳 106 cells/well in 96-well plates precoated with 5鈥壜礸/ml anti-mouse CD3蔚 antibody or 1鈥壜礸/ml SEB. After culture with hamster IgG, anti-CD81 antibodies (2F7 and Eat2), and an anti-CD28 antibody at 5鈥壜礸/mL each, and cyclosporine A (CsA, Sigma) at 100鈥塶M for 3 days, cell proliferation was measured using the Biotrack ELISA system, version 2, containing BrdU and an anti-BrdU antibody (Amersham, UK) at an absorbance of 450鈥塶m. Culture supernatants were also collected, and IFN纬 and TNF伪 were measured by ELISAs (R D Systems). Spleen cells from mice with TNBS-induced colitis were stimulated with or without 1鈥壜礸/mL SEB for 50鈥塰. Cultured cells were treated with Golgi Stop for 14鈥塰, and then cell surface molecules (CD69 and CXCR4) and intracellular cytokines (IFN纬 and IL-17) were stained and analysed by the FACSCanto II.Migration assaySplenocytes from mice with established TNBS-induced colitis or mouse T cell line EL4.IL-2 (ATCC, TIB-181) were prepared and incubated with hamster IgG and the anti-mouse CD81 antibody. A 75鈥壜祃 cell suspension of 2 脳 107 cells/ml was added to the upper well of an HTS Transwell 96 well permeable support, 5鈥壜祄 pore (Corning, USA) with or without 235鈥壜祃 of 10鈥塶g/ml recombinant human SDF-1 (PEPROTECH, UK) in the lower well. After 2鈥塰 of incubation at 37鈥壜癈 with 5% CO2, 50鈥壜祃/well of the cell suspension in the lower well was transferred to an EIA/RIA 96 well plate (Corning) and 50鈥壜祃/well ATPlite (PerkinElmer, USA) was added to each well. Luminescence was measured using an Envision 2102 multilabel reader (PerkinElmer, USA).Induction of DSS colitisBALB/cAnNCrlCrlj mice were administered 5% sodium dextran sulfate salt (molecular weight: 1,000鈥?,000; Wako Pure Chemicals, Japan) in drinking water from day 鈭? to 0. Colitis scores were estimated by stool conditions: score 0, normal stool that was dry or wet, but maintained shape after prodding; score 1, loose stool without bloody stool or a bloody bowel discharge or bloody stool without loose stool or diarrhoea; score 2, diarrhoea without bloody stool or a bloody bowel discharge or bloody bowel discharge without loose stool or diarrhoea; score 3, loose stool and a bloody bowel discharge or diarrhoea and bloody stool; score 4, diarrhoea and a bloody bowel discharge. On day 0, mice with a body weight decrease at day 鈭? of more than 20% compared with day 鈭? and mice with a colitis score of 鈥?鈥?were excluded from the study. The remaining mice were divided to three groups by their body weight and colitis score on day 0 using the 鈥淏lock randomization with multiple variables鈥?program in Stat Preclinica. Then, mice were administered distilled water from day 0 to 9, followed by 5 days of 5% DSS in their drinking water and 5 days of distilled water administration. Control animals were administered distilled water only. The mice were monitored daily for survival, body weight, and stool consistency. Then, colons were collected on day 20 for analyses.Statistical analysisData are expressed as means 卤 standard error (X鈥壜扁€塖E). The statistical difference of colitis scores between groups was calculated by Wilcoxon鈥檚 test. The unpaired Student鈥檚 t-test was used to calculate the statistical significance of differences in colon length, body weight change, serum markers, migration assay, and frequency of colonic cell populations between two groups. Statistical analysis was performed using Stat Preclinica version 1.0.3295 (Takumi Information Technology Inc., Japan). All data generated or analysed during this study are included in this published article (and its Supplementary Information files). 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Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nat. Protoc. 2, 2307鈥?311 (2007).CAS聽 Article聽Google Scholar聽 Download referencesAcknowledgementsThe authors thank Mr. Takamasa Watanabe for contributing to experiments and helpful discussions. We thank Mitchell Arico from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript and helping to draft the abstract. This study was supported by Sumitomo Dainippon Pharma. Co. Ltd. The author would like to thank the referee for useful suggestions that helped her to improve the original manuscript.Author informationAffiliationsExternal Innovation, Sumitomo Dainippon Pharma Co., Ltd, Osaka, 554-0022, JapanTakuya HasezakiApplied Bioscience Group, Bioscience Research Laboratory, Sumitomo Chemical Co., Ltd, Osaka, 554-0022, JapanTadahiko YoshimaGroup 2, Platform Technology Research Unit, Sumitomo Dainippon Pharma Co., Ltd, Osaka, 554-0022, JapanYukiko MineAuthorsTakuya HasezakiView author publicationsYou can also search for this author in PubMed聽Google ScholarTadahiko YoshimaView author publicationsYou can also search for this author in PubMed聽Google ScholarYukiko MineView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsT.H., Y.M., and T.Y. study concept and design; T.H. and T.Y. acquisition of data; T.H. and T.Y. analysis and interpretation of data; T.H. drafting of the manuscript; Y.M. and T.Y. critical revision of the manuscript; T.H. statistical analysis; Y.M. study supervision.Corresponding authorCorrespondence to Takuya Hasezaki.Ethics declarations Competing interests The authors are current or former employees of Sumitomo Dainippon Pharma Co., Ltd. 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To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and PermissionsAbout this articleCite this articleHasezaki, T., Yoshima, T. Mine, Y. Anti-CD81 antibodies reduce migration of activated T lymphocytes and attenuate mouse experimental colitis. Sci Rep 10, 6969 (2020). https://doi.org/10.1038/s41598-020-64012-5Download citationReceived: 29 August 2019Accepted: 06 April 2020Published: 24 April 2020DOI: https://doi.org/10.1038/s41598-020-64012-5 CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.