Oral Anti-CD3 Antibody Treatment Induces Regulatory T Cells and Inhibits the Development of Atherosclerosis in Mice

Atherosclerosis is an inflammatory condition of the arterial wall involving cells of innate and adaptive immunity.^1,2 It leads to severe clinical events caused by the rupture of plaques and thrombotic occlusion of the artery and is the main cause of acute coronary syndrome and stroke, which account for approximately half of the deaths in Western countries. It is now widely recognized that chronic inflammation via T-cell–mediated pathogenic immune responses plays an important role in atherogenesis.² Accumulating evidence suggests that several subsets of regulatory T cells (Tregs), which have been shown to maintain immunologic unresponsiveness to self-antigens,³ inhibit atherosclerosis development through the downregulation of activated T-cell responses.^4–6 These studies imply that promotion of an endogenous regulatory immune response has a therapeutic potential to suppress atherosclerotic diseases.

Anti-CD3–specific antibodies strongly suppress immune responses by antigenic modulation of the T-cell receptor/CD3 complex.⁷ In 1985, parenteral anti-CD3 antibody was available for use in acute transplant rejection in humans. However, its long-term use is limited because of severe side effects such as mitogenicity and antiglobulin responses.⁷ The mitogenicity of anti-CD3 antibody with Fc portion is caused by interacting with Fc receptors (FcRs) on monocytes or macrophages. Therefore, humanized Fc-mutated antibodies were designed and demonstrated to be effective in human autoimmune diabetes for a long period, although they still induce low levels of cytokine release because of some degree of T-cell activation.⁸ This long-term beneficial effect of FcR-nonbinding anti-CD3 antibody is partly explained by the recent study showing that FcR-nonbinding anti-CD3 antibody induces transforming growth factor (TGF)-β–producing CD4⁺CD25⁺ Tregs.⁹ Notably, it has been shown that intravenously administered FcR-nonbinding CD3-specific antibody induces a regulatory immune response and inhibits atherosclerosis development and progression.¹⁰ Recent studies have revealed that oral or nasal anti-CD3 antibody with or without Fc portion is biologically active and induces CD4⁺LAP⁺ (latency-associated peptide) Tregs that suppress experimental autoimmune encephalitis, autoimmune diabetes mellitus, and lupus in a TGF-β–dependent fashion and that this therapy would not be expected to have side effects even though FcR-binding CD3-specific antibody is used.^11–13

Mucosal tolerance induction has been shown to inhibit various autoimmune diseases in mice¹⁴ and, importantly, atherosclerotic diseases in mice^15,16 and therefore has been gaining much interest clinically. High doses of oral antigen lead to anergy or depletion of antigen-specific T cells. On the other hand, low doses of oral antigen induce many types of antigen-specific Tregs, including T-helper type 3 (Th3) cells, regulatory T-cell type 1 (Tr1) cells, naturally occurring CD4⁺CD25⁺Foxp3⁺ Tregs, and CD4⁺LAP⁺ Tregs,¹⁴ indicating that mucosal tolerance induction seems to be an effective way to treat atherosclerosis. As a possible explanation for the effects of oral anti-CD3 antibody treatment, a mechanism similar to that described in mucosal tolerance is supposed because autoimmune diseases are shown to be suppressed by low doses of oral anti-CD3 antibody but not by high doses.¹¹ However, there is no evidence of antigen specificity with oral anti-CD3 antibody.

Given these backgrounds, we hypothesized that induction of CD4⁺LAP⁺ Tregs by oral anti-CD3 antibody treatment would inhibit atherosclerosis in apolipoprotein E–deficient (ApoE^−/−) mice. Our findings provide for the first time evidence that oral anti-CD3 antibody treatment induces CD4⁺LAP⁺ Tregs and CD4⁺CD25⁺Foxp3⁺ Tregs, which suppress pathogenic immune processes pivotal for atherogenesis through a TGF-β–dependent mechanism and consequently inhibit atherosclerotic plaque formation.

Methods

For expanded Methods, please see the online-only Data Supplement.

Experimental Design

We fed 6-week-old or 16-week-old male ApoE^−/− mice 5 μg hamster CD3-specific antibody (clone 145 to 2C11; R&D Systems, Minneapolis, Minn) or 5 μg hamster immunoglobulin G (IgG; Jackson Immuno Research Laboratories Inc, West Grove, Pa) dissolved in 0.2 mL phosphate-buffered saline by gastric intubation with a plastic tube once a day for 5 consecutive days. Mice were killed at 16 weeks of age, and atherosclerotic lesions were assessed. In some experiments, mice were fed control IgG or anti-CD3 antibody daily for 5 days and were injected with 100 μg of neutralizing anti-TGF-β antibody (clone 1D11; Bio Express Inc, West Lebanon, NH) or control rat IgG (Bio Express Inc). Injections were repeated once a week from age 7 weeks to age 16 weeks.

Atherosclerotic Lesion Assessment

For aortic root lesion analysis, 5 consecutive sections (10-μm thickness), spanning 550 μm of the aortic sinus, were collected from each mouse, stained with Oil Red O (Sigma, St Louis, Mo), and quantified with the use of the Image J (National Institutes of Health) as described previously.¹⁷ Immunohistochemistry was performed with the use of antibodies to identify macrophages (MOMA-2, 1:400; BMA Biomedicals, Augst, Switzerland), T cells (CD4, 1:100; BD Biosciences, San Jose, Calif), smooth muscle cells (α-smooth muscle actin, 1:400; Sigma), and natural Tregs (Foxp3, 1:100; eBioscience, San Diego, Calif). For en face lesion analysis, the thoracic aorta was opened longitudinally, stained with Oil Red O, and analyzed as described previously.¹⁷

Flow Cytometry Analysis

Mesenteric lymph node (MLN) cells and splenocytes were isolated 2 days or 10 weeks after the final oral anti-CD3 antibody administration. Fluorescent-activated cell sorter analysis was performed by FACSCalibur with the use of CellQuest Pro software (BD Biosciences).

Cell Proliferation, Purification, and Cytokine Assays

Lymphocytes from MLNs or spleen were prepared and stimulated with concanavalin A (Sigma) in vitro. Cytokine levels in supernatants were examined by ELISA or a Mouse Cytokine Array kit (R&D Systems). CD4⁺CD25⁻LAP⁺ Tregs, CD4⁺CD25⁻LAP⁻ cells, CD4⁺CD25⁺ Tregs, and CD4⁺CD25⁻ cells were purified from spleens or MLNs with the use of a MoFlo cell sorter (DakoCytomation, Denmark). Cell proliferation assay was performed by assessing [³H]thymidine incorporation.

Real-Time Reverse Transcription Polymerase Chain Reaction Analysis

Total RNA was extracted from MLN cells harvested after 2 days of oral antibody treatment or from the aortas after perfusion with RNA later (Ambion, Austin, Tex) with the use of TRIzol reagent (Invitrogen, Carlsbad, Calif). Quantitative polymerase chain reaction (PCR) was performed as described previously.¹⁸

Results

Effects of Oral Anti-CD3 Antibody on CD4⁺LAP⁺ Tregs or CD4⁺CD25⁺Foxp3⁺ Tregs in MLNs and Spleens

We fed 6-week-old male ApoE^−/− mice 5 μg hamster anti-CD3–specific antibody or 5 μg hamster IgG by gastric intubation once a day for 5 consecutive days. Previous studies suggested a fundamental immunologic difference between intravenous and oral anti-CD3 antibody administration.^11,12 In fact, oral anti-CD3 antibody treatment did not cause T-cell receptor downmodulation or depletion of T cells (Figure I in the online-only Data Supplement). To investigate the effects of oral anti-CD3 antibody treatment on CD4⁺LAP⁺ Treg levels in ApoE^−/− mice, we performed flow cytometry studies. Similar to the previous report,¹¹ 2 days after the last feeding of anti-CD3 antibody, we found a significant increase in LAP⁺ Tregs in the CD4⁺ T-cell population in MLNs and spleens of anti-CD3–treated mice (P<0.05; Figure 1A and 1B). Notably, we observed a significant increase in LAP⁺ Tregs in the CD4⁺CD25⁻ T-cell population in spleens (P<0.05) but not in the CD4⁺CD25⁺ T-cell population (Figure 1A and 1C), suggesting that oral anti-CD3 antibody treatment results in an increase in CD4⁺CD25⁻LAP⁺ Tregs, which are distinct from naturally occurring Tregs expressing CD25⁺ on their surface. We also examined the effect of oral anti-CD3 treatment on natural CD4⁺CD25⁺Foxp3⁺ Tregs by fluorescence-activated cell sorter analyses. Interestingly, we found a modest but significant increase in CD4⁺CD25⁺Foxp3⁺ Tregs in MLNs of anti-CD3–treated mice compared with control mice (P<0.05) but not in spleens (Figure 1D and 1E).

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To determine whether an increased number of Tregs by oral anti-CD3 contributes to the more suppressive potential of lymphoid cells, we performed in vitro proliferation assays of MLN cells or splenocytes in response to anti-CD3 antibody. As shown in Figure 1F and 1G, stimulation with anti-CD3 antibody resulted in the proliferation of MLN cells or splenocytes in control IgG-treated mice in a dose-dependent manner. In contrast, we observed a significant reduction in MLN or spleen cell proliferation of anti-CD3–treated mice (P<0.05), indicating the suppressed immune responses of lymphoid cells in anti-CD3–treated mice on polyclonal stimulation (Figure 1F and 1G). We additionally performed in vitro suppression assays to reveal whether oral anti-CD3 antibody treatment affects the suppressive function of Tregs. These experiments demonstrated that the suppressive function of CD4⁺CD25⁻LAP⁺ Tregs or CD4⁺CD25⁺ Tregs was not altered by oral anti-CD3 treatment (Figure IIA and IIB in the online-only Data Supplement).

Oral Anti-CD3 Antibody Treatment Inhibits Atherosclerotic Plaque Formation and Reduces Inflammatory Cell Recruitment Into Plaques in ApoE^−/− Mice

To determine the effect of oral anti-CD3 treatment on the development of atherosclerosis, we orally treated 6-week-old male ApoE^−/− mice with a 5-μg dose of anti-CD3 antibody or control IgG daily for 5 consecutive days. During the experiments, no adverse effect was observed in either group. No statistical differences in body weight or plasma lipid profiles were detected between the 2 groups (Table). At 16 weeks of age, the mice were euthanized, and cryosections of the aortic root of control and anti-CD3–treated mice were stained with Oil Red O and analyzed quantitatively. Surprisingly, anti-CD3–treated mice showed ≈50% reduction in atherosclerotic lesion formation in the aortic root compared with control IgG-treated mice (aortic sinus mean plaque area of 11 3854±20 329 μm² versus 54 565±5937 μm² in control IgG-treated and anti-CD3–treated mice, respectively; P=0.007; Figure 2A). In parallel with the cross-sectional studies, en face analysis of thoracic aortas was performed. En face analysis of thoracic aortas revealed a significant 33% reduction in aortic plaque burden at 16 weeks in anti-CD3–treated mice (2.31±0.33%; P<0.05 versus controls) compared with control mice (3.44±0.33%) (Figure 2B). To further evaluate the effect of anti-CD3 antibody treatment on the progression of atherosclerosis, we treated 16-week-old male ApoE^−/− mice, euthanized them at 24 weeks, and evaluated the atherosclerotic lesion formation. The results indicated that oral anti-CD3 antibody treatment did not prevent atherosclerosis progression (Figure III in the online-only Data Supplement).

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To determine the effects of oral anti-CD3 antibody treatment on plaque component, we next performed immunohistochemical studies of atherosclerotic lesions in the aortic sinus. Interestingly, the atherosclerotic lesions of anti-CD3–treated mice showed a marked 38% reduction in the accumulation of macrophages (P<0.01) and also a 30% decrease in CD4⁺ T-cell infiltration (P<0.01) compared with control mice (Figure 3A and 3B). In addition, immunohistochemical analysis of the plaques showed that relative smooth muscle cell and collagen contents in the aortic sinus plaques tended to be increased in anti-CD3–treated mice, but this did not reach statistical significance (Figure 3A and 3B). These results suggest that oral anti-CD3 antibody treatment reduces plaque inflammation, which may lead to decreased lesion development.

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Expression of Inflammatory Markers and Regulatory T-Cell Markers in Atherosclerotic Plaques

To reveal the mechanisms of reduced plaque development and inflammatory cell recruitment, we examined mRNA expressions of the proinflammatory molecules in atherosclerotic aortas by quantitative reverse transcription (RT)-PCR. We found that proinflammatory cytokines such as interferon (IFN)-γ and interleukin (IL)-6, adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, chemokine monocyte chemoattractant protein (MCP)-1, and macrophages detected by CD68 were markedly reduced in anti-CD3–treated mice compared with control mice (P<0.05; Figure 4A and data not shown).

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Recent studies suggest that antigen presentation may occur within the atherosclerotic plaque in addition to the lymphoid organs and that Tregs migrate to the atherosclerotic lesions to suppress local immune responses,^19,20 although actual roles of intraplaque Tregs in atherogenesis have not yet been clarified. To determine whether increased accumulation of Tregs into the plaques is involved in the reduction of atherosclerotic lesion formation, we analyzed the expression of Treg-associated markers in the lesions by quantitative reverse transcription PCR (RT-PCR). We found no significant differences in the relative mRNA expressions of CD4⁺LAP⁺ Treg-derived cytokine TGF-β or natural Treg-specific markers Foxp3 and CD25 in the plaques among the 2 groups (Figure 4A). Furthermore, immunohistochemical studies of atherosclerotic lesions with the use of anti-Foxp3 antibody demonstrated an increase in the number of natural Tregs within the plaque of anti-CD3–treated mice (Figure 4B). When it is taken into consideration that anti-CD3–treated mice have less plaque in the aorta than control mice, this result is consistent with the quantitative RT-PCR analysis of Treg-specific markers. These data indicate that oral anti-CD3 antibody treatment could inhibit the migration of effector T cells into the lesion selectively and relatively increase the proportion of natural Tregs in atherosclerotic plaque that might suppress pathogenic T-cell immune responses or macrophage activation as well as in the lymphoid organs.

Effects of Oral Anti-CD3 Antibody on Cytokine Production in Lymphocytes From Spleen and MLNs

Next, to determine whether induction of various Tregs by oral anti-CD3 antibody changed the cytokine production profile from lymphocytes, we examined cytokine secretion from spleen or MLN lymphocytes in response to concanavalin A using cytokine arrays and ELISA. On day 7 after the treatment of oral anti-CD3 antibody, we tried to assess the changes in cytokine production from MLN cells; however, the cytokine levels (IFN-γ, IL-4, and IL-10) were below the detectable levels. Therefore, we next investigated cytokine mRNA expression of unstimulated MLN cells by using quantitative RT-PCR. Interestingly, anti-CD3 antibody treatment resulted in a significant decrease in mRNA expressions of T-helper type 1 (Th1) cytokine (IFN-γ) and T-helper type 2 (Th2) cytokines (IL-4 and IL-10) in MLN cells (P<0.05; Figure 5A). Consistent with previous studies, significantly increased production of antiinflammatory cytokine TGF-β, which was the only detectable cytokine in our experiments, was observed in MLN cells of anti-CD3–treated mice (P<0.05; Figure 5B). CD4⁺LAP⁺ Tregs induced by oral anti-CD3 antibody reach mesenteric lymph nodes and other lymphoid organs such as spleens through the bloodstream, where they may induce deactivation of pathogenic T cells and macrophages. Therefore, we semiquantitatively analyzed various cytokine or chemokine productions in spleen lymphocytes on day 7 after the treatment. Lymphocytes from anti-CD3–treated mice secreted fewer Th1 cytokines (IFN-γ and IL-2), Th2 cytokines (IL-4, IL-10, and IL-13), and inflammation-related cytokines or chemokines (IL-17, MIP-1α, MIP-2, RANTES, and TNF-α) under stimulation with concanavalin A than those from control mice (Figure 5C), indicating suppressed Th1 and Th2 immune responses in anti-CD3–treated mice. ELISA data confirmed that TGF-β production was markedly increased and cytokine levels such as IFN-γ, IL-10, and IL-17 were significantly decreased in spleen lymphocyte supernatants from anti-CD3–treated mice compared with controls (P<0.05; Figure 5D). Although we could not detect IL-6 expression in the cytokine array analysis, ELISA data for lymphocyte IL-6 production revealed lower production in spleen lymphocytes of anti-CD3–treated mice than controls (P<0.05; Figure 5D). We also assessed cytokine production from lymphocytes 10 weeks after the treatment (at 16 weeks). Surprisingly, we found markedly increased TGF-β secretion in spleen lymphocytes of anti-CD3–treated mice (P<0.05), with no differences in IFN-γ or IL-10 levels between the groups (Figure 5E and data not shown), suggesting the long-term protective effect of TGF-β. In contrast to the increased TGF-β production at 16 weeks, we detected no significant difference in the number of CD4⁺LAP⁺ Tregs among the 2 groups, although CD25⁻LAP⁺ Tregs in the CD4⁺ T cell population tended to be slightly increased in spleens of anti-CD3–treated mice (1.78±0.14% versus 2.11±0.15% in control IgG-treated and anti-CD3–treated mice, respectively; P=0.068; data not shown). Previous studies have shown that ani-CD3 antibody treatment not only increases the number of CD4⁺LAP⁺ Tregs but also enhances the regulatory function of these cells.^11,12 Although we did not observe the enhanced suppressive function of Tregs in our in vitro experiments (Figure IIA in the online-only Data Supplement), our data imply that the prolonged TGF-β secretion in lymphocytes of anti-CD3–treated mice may reflect the altered function of these Tregs, which might be maintained for a long time, and suggest an important prolonged contribution of this antiinflammatory cytokine to a reduction in atherosclerosis development.

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Suppressive Function of Oral Anti-CD3 Antibody Is Mediated by TGF-β

Previous data suggest that TGF-β plays a key role in the suppressive function of both natural Tregs²¹ and CD4⁺LAP⁺ Tregs.²² In addition, our ELISA data for cytokine production in lymphocytes show that TGF-β is the only cytokine with an antiinflammatory property that is enhanced by anti-CD3 antibody treatment. To determine whether TGF-β is directly involved in preventing plaque formation after anti-CD3 treatment, we performed an in vivo TGF-β neutralization study. Anti-CD3–treated mice that received anti-TGF-β neutralizing antibody have significantly increased atherosclerotic lesion formation and accumulations of macrophages and CD4⁺ T cells in the plaques compared with anti-CD3–treated mice receiving isotype-matched antibody (Figure 6A and 6B). In addition, when mice were injected with neutralizing antibody, there were no significant differences in aortic root plaque size and inflammatory cell infiltration between control and anti-CD3–treated mice (Figure 6A and 6B). Together, these findings suggest that the suppressive function of oral anti-CD3 antibody in vivo is mediated by TGF-β, at least in part.

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Discussion

Recent work has clearly demonstrated that natural Tregs, which show a high expression of CD25 on their surface²³ and also express the transcription factor forkhead box P3 (Foxp3),²⁴ play a protective role in atherogenesis in mice.⁴ Besides naturally occurring Tregs, previous studies have implicated the protective role of other types of Tregs including Tr1 or Th3 cells in the development of atherosclerosis in mice through the production of antiinflammatory cytokines such as IL-10 or TGF-β, respectively.^16,25 Recently, CD4⁺LAP⁺ Treg has been identified as a new subset of Tregs that suppresses autoimmune diseases in mice.^11–13,22 In the present study, we show that oral anti-CD3 antibody treatment attenuates atherosclerotic lesion formation and that this reduction is associated with an increase in the number of Tregs, including CD4⁺LAP⁺ Tregs and CD4⁺CD25⁺Foxp3⁺ Tregs, which are possibly induced in the gut and migrate into other lymphoid organs, subsequently leading to suppression of Th1 and Th2 immune responses through the production of TGF-β. Our data are the first report demonstrating a possible role of CD4⁺LAP⁺ Tregs in atherogenesis, although further studies are required to provide more direct evidence for the relationship between an enhancement in CD4⁺LAP⁺ Treg number or function and a reduction of atherosclerosis.

Importantly, unlike the case of intravenous FcR-binding anti-CD3 antibody treatment, we observed no evidence of adverse responses of oral FcR-binding anti-CD3 antibody during the experiment, which may be related to the fact that orally administered anti-CD3 antibody is very small in amount (10 times less than that of the usual intravenous administration) and for the most part does not seem to enter the bloodstream.¹¹ It has been reported that immunosuppressive effects by intravenous FcR-binding anti-CD3 antibody may involve mechanisms such as depletion of pathogenic T cells from the bloodstream or lymphoid organs and modulation of the T-cell receptor on T cells.⁷ In contrast to intravenous FcR-binding anti-CD3 antibody administration, we never observed the T-cell receptor downmodulation or depletion of T cells (Figure I in the online-only Data Supplement), which is consistent with the previous report.¹¹ Ochi et al¹¹ speculated that low doses of oral anti-CD3 antibody administration may result in the induction of Tregs by delivering a weak signal to T cells, which is observed similarly in mucosal tolerance induction. They demonstrated that experimental autoimmune encephalitis was suppressed in mice fed 5 μg anti-CD3 antibody with or without Fc portion but not in mice fed 50 μg or 500 μg antibody.¹¹ Consistent with its effect on suppression of experimental autoimmune encephalitis, it was also shown that suppression of proliferation in spleens and MLN cells was observed with 5 μg anti-CD3 antibody but not with 50 μg or 500 μg. In consideration of this, we decided to use a 5-μg dose of FcR-binding anti-CD3 antibody for the treatment of ApoE^−/− mice and found that this treatment results in a dramatic reduction in atherosclerotic lesion formation, associated with a significant increase in CD4⁺LAP⁺ Tregs. Intriguingly, we also detected an increased proportion of CD4⁺CD25⁺Foxp3⁺ Tregs in MLNs of anti-CD3–treated mice.

Administration of FcR-nonbinding anti-CD3 monoclonal antibody has been shown to induce long-lasting immune tolerance and to be an effective treatment for autoimmune diabetes in mice and in humans,^8,9 acute transplant rejection in humans,⁷ and, notably, atherosclerosis in mice.¹⁰ These studies have indicated that the long-term beneficial effect of FcR-nonbinding anti-CD3 antibody may possibly be caused by increasing TGF-β–producing Tregs. When parenteral FcR-nonbinding anti-CD3 antibody is compared with oral FcR-binding anti-CD3 antibody, there may be some common mechanisms underlying suppression of autoimmunity because both appear to work mainly by inducing TGF-β–producing Tregs rather than eliminating pathogenic T cells. In consideration of this, it is possible that after oral administration, FcR-binding anti-CD3 antibody might lose its Fc portion in the gut and consequently produce F(ab′)₂ (FcR-nonbinding anti-CD3 antibody), a small amount of which might enter the bloodstream, leading to induction of Tregs.

Several in vitro and in vivo studies demonstrated that naive T cells in the periphery can differentiate into CD4⁺Foxp3⁺ Tregs in the presence of TGF-β.^26,27 Recent studies also suggest that differentiation of CD4⁺Foxp3⁺ Tregs may be facilitated by dendritic cells in the gut-associated lymphoid tissue in the presence of TGF-β and that retinoic acid produced by dendritic cells plays an important role in their differentiation.²⁸ In this study, we found a significant increase in CD4⁺ LAP⁺ Tregs in MLNs and spleens of anti-CD3–treated mice, in association with markedly increased TGF-β secretion by CD4⁺LAP⁺ Tregs, which have been reported to secrete much more TGF-β than CD4⁺LAP⁻ T cells.¹¹ Therefore, it is highly possible that upregulated TGF-β secretion in the gut from anti-CD3–induced CD4⁺LAP⁺ Tregs could lead to induction of CD4⁺CD25⁺Foxp3⁺ Tregs.

Because previous studies have reported that oral anti-CD3 treatment results in conversion of Th1 immune responses into Th2 or Th3 immune responses in the periphery associated with decreased proliferation in spleen cells and MLN cells,^11,12 it seems possible that similar changes in the cytokine production profile from lymphocytes could contribute to the observed antiatherogenic effect of oral anti-CD3 antibody in our study. Unlike previous studies, however, our ELISA and cytokine array analyses clearly demonstrated significant suppression in both Th1 and Th2 immune responses, as well as markedly increased TGF-β production from lymphocytes in oral anti-CD3–treated mice. In this experiment, we used atherosclerosis-prone ApoE^−/− mice on C57BL/6 background, which are known to show Th1-prone phenotype. Therefore, the difference in mouse genetic background used might be a possible explanation for the distinct immune responses by oral anti-CD3 antibody between our study and others. Accumulating evidence suggests that T-helper 17 (Th17) cells, which produce the inflammatory cytokine IL-17, play an important role in promotion of many autoimmune diseases.²⁹ In addition, it has been reported that TGF-β stimulation leads to differentiation of naive T cells to Th17 cells or CD4⁺Foxp3⁺ Tregs in mice in the presence or absence of IL-6, respectively.³⁰ Our data indicate a shift from Th17 cells to CD4⁺Foxp3⁺ Tregs in oral anti-CD3–treated mice, which might partially affect the atheroprotective effects of oral anti-CD3, although whether Th17 cells accelerate atherosclerosis as well as other autoimmune diseases thus far remains unclear.

TGF-β has been attracting much attention as a potent antiatherosclerotic cytokine.³¹ TGF-β, which is produced by many types of cells in atherosclerotic plaques, suppresses pathogenic immune responses such as recruitment of inflammatory cells into the plaques or foam cell formation and increases collagen biosynthesis.^32,33 In particular, TGF-β signaling in T cells appears to be indispensable for the regulation of atherosclerotic disease progression because ApoE^−/− mice with specific deletion of TGF-β signaling in T cells show remarkably accelerated plaque formation and inflammatory cell infiltration, associated with enhancement of both Th1 and Th2 immune responses.³⁴ In consideration of this, our data suggest that increased TGF-β production from T cells may be a key mechanism for the preventive effect of oral anti-CD3 antibody on atherosclerosis through the downregulation in both Th1 and Th2 immune responses.

We are somewhat surprised because only 5-day oral treatment with a small amount of anti-CD3 antibody has a prolonged effect on the immune system and results in a dramatic atheroprotective phenotype. This long-term protective effect of oral anti-CD3 antibody was observed similarly in other autoimmune diseases such as experimental autoimmune encephalitis and autoimmune diabetes.^11,12 TGF-β appears to play a crucial role in the prolonged antiatherogenic effect because we detected a significant increase in TGF-β production in spleen lymphocytes of oral anti-CD3–treated mice at 10 weeks after the antibody administration. It is reported that CD4⁺LAP⁺ Tregs secrete TGF-β and are also activated by TGF-β produced by themselves,²² indicating that they may act in autocrine and paracrine manners. We speculate that a transient increase in CD4⁺LAP⁺ Tregs may lead to a positive-feedback loop of activation of CD4⁺LAP⁺ Tregs through the production of TGF-β. On the basis of our observed findings, this novel method, which directly governs the inflammatory immune responses in atherogenesis, could be a hopeful strategy to prevent cardiovascular diseases.

In summary, we have demonstrated that oral anti-CD3 antibody treatment inhibits atherosclerosis development via inducing various Tregs and that TGF-β, which may be derived mainly from CD4⁺LAP⁺ Tregs, possibly plays an important role in this suppressive mechanism. Although the mechanism by which oral anti-CD3 antibody increases Tregs remains to be clarified, our data indicate that induction of Tregs in the intestine by oral anti-CD3 antibody could be used as a promising therapeutic approach for atherosclerotic vascular disorders. Recent studies have demonstrated no toxicity of orally administered anti-CD3 antibody not only in mice but also in humans.³⁵ Clinical studies in human are required to identify the efficacy of oral anti-CD3 antibody in the prevention of atherosclerotic diseases.

Footnotes