Artemisinin Analogue SM934 Ameliorates Murine Experimental Autoimmune Encephalomyelitis through Enhancing the Expansion and Functions of Regulatory T Cell

Background

Artemisinin analogue SM934 was previously reported to possess immunosuppressive properties. The aim of this study was to determine the effects and the underlying mechanisms of SM934 in murine experimental autoimmune encephalomyelitis (EAE).

Methods

Female C57BL/6 mice immunized with MOG_35–55 were treated with or without SM934, then the clinical scores and other relevant parameters were assessed. Th1, Th17 and regulatory T (Treg) cell profiles were determined through ELISA, qRT-PCR, flow cytometry and BrdU incorporation assay. The effects of SM934 on Th1, Th17 and Treg cells differentiation were explored through intracellular staining and flow cytometry examination.

Results

In vivo, administration of SM934 significantly inhibited the development of EAE and suppressed the elevation of serum IL-17. Ex vivo, upon antigen-recall stimulation, IL-2, IFN-γ, IL-17 and IL-6 production were decreased, whereas IL-10 and TGF-β production were increased from the splenocytes isolated from SM934-treated mice. Consistently, both flow cytometry and qRT-PCR results showed that SM934 treatment significantly increased the Treg, while strongly suppressed the Th17 and Th1, responses in the peripheral. Furthermore, in the spinal lesion, SM934 treatment dramatically decreased the infiltration of CD4⁺ T cells, within which the Treg cells percentage was enlarged, whereas the Th17, but not Th1 percentage, was significantly decreased comparing with the vehicle-treated groups. Finally, both BrdU incorporation and in vitro Treg differentiation assays revealed that SM934 treatment could directly promote the expansion of Treg cells in vivo and in vitro.

Conclusion

Taken together, this study demonstrated that SM934 treatment could ameliorate the murine EAE disease, which might be mediated by inducing Treg differentiation and expansion.     

iNKT Cells Suppress the CD8(+) T Cell Response to a Murine Burkitt's-Like B Cell Lymphoma

The T cell response to B cell lymphomas differs from the majority of solid tumors in that the malignant cells themselves are derived from B lymphocytes, key players in immune response. B cell lymphomas are therefore well situated to manipulate their surrounding microenvironment to enhance tumor growth and minimize anti-tumor T cell responses. We analyzed the effect of T cells on the growth of a transplantable B cell lymphoma and found that iNKT cells suppressed the anti-tumor CD8(+) T cell response. Lymphoma cells transplanted into syngeneic wild type (WT) mice or Jalpha18(-/-) mice that specifically lack iNKT cells grew initially at the same rate, but only the mice lacking iNKT cells were able to reject the lymphoma. This effect was due to the enhanced activity of tumor-specific CD8(+) T cells in the absence of iNKT cells, and could be partially reversed by reconstitution of iNKT cells in Jalpha 18(-/-) mice. Treatment of tumor-bearing WT mice with alpha -galactosyl ceramide, an activating ligand for iNKT cells, reduced the number of tumor-specific CD8(+) T cells. In contrast, lymphoma growth in CD1d1(-/-) mice that lack both iNKT and type II NKT cells was similar to that in WT mice, suggesting that type II NKT cells are required for full activation of the anti-tumor immune response. This study reveals a tumor-promoting role for iNKT cells and suggests their capacity to inhibit the CD8(+) T cell response to B cell lymphoma by opposing the effects of type II NKT cells.     

Cathepsin L Inhibition Prevents Murine Autoimmune Diabetes via Suppression of CD8+ T Cell Activity

Background

Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet β cells. To determine whether specific lysosomal proteases might influence the outcome of a T cell–mediated autoimmune response, we examined the functional significance of cathepsin inhibition on autoimmune T1D-prone non-obese diabetic (NOD) mice.

Methods and Findings

Here it was found that specific inhibition of cathepsin L affords strong protection from cyclophosphamide (CY)-induced insulitis and diabetes of NOD mice at the advanced stage of CD8⁺ T cell infiltration via inhibiting granzyme activity. It was discovered that cathepsin L inhibition prevents cytotoxic activity of CD8⁺ T cells in the pancreatic islets through controlling dipeptidyl peptidase I activity. Moreover, the gene targeting for cathepsin L with application of in vivo siRNA administration successfully prevented CY-induced diabetes of NOD mice. Finally, cathepsin L mRNA expression of peripheral CD8⁺ T cells from NOD mice developing spontaneous T1D was significantly increased compared with that from control mice.

Conclusions

Our results identified a novel function of cathepsin L as an enzyme whose activity is essential for the progression of CD8⁺ T cell-mediated autoimmune diabetes, and inhibition of cathepsin L as a powerful therapeutic strategy for autoimmune diabetes.     

调节性T细胞与自身免疫性疾病

自身免疫性疾病是由于机体正常免疫耐受功能受损导致免疫系统对自身组织结构和功能的破坏,并出现一定临床表现的一类疾病.调节性T细胞作为一类具有负向免疫调节功能的淋巴细胞亚群在免疫自稳和免疫耐受中起关键作用,既能抑制不恰当的免疫反应,又能限制免疫应答的范围、程度及作用时间,对效应性T细胞的增殖及免疫活性的发挥产生抑制,因此在许多自身免疫性疾病的发病中扮演重要角色.近年来的研究表明调节性T细胞可以通过细胞接触、分泌细胞因子、基因调控等多种途径发挥作用,在不同的疾病,不同的内环境因素作用下可以表现出不同的特点,转录因子Foxp3作为调节性T细胞的特异性标志是其分化成熟及功能维持的根本.     

Galectin-9 Ameliorates Con A-Induced Hepatitis by Inducing CD4+CD25low/int Effector T-Cell Apoptosis and Increasing Regulatory T Cell Number

Background

T cell-mediated liver damage is a key event in the pathogenesis of many chronic human liver diseases, such as liver transplant rejection, primary biliary cirrhosis, and sclerosing cholangitis. We and other groups have previously reported that galectin-9, one of the β-galactoside binding animal lectins, might be potentially useful in the treatment of T cell-mediated diseases. To evaluate the direct effect of galectin-9 on hepatitis induced by concanavalin A (Con A) administration in mice and to clarify the mechanisms involved, we administered galectin-9 into mice, and evaluated its therapeutic effect on Con A-induced hepatitis.

Methodology/Principal Findings

Galectin-9 was administrated i.v. to Balb/c mice 30 min before Con A injection. Compared with no treatment, galectin-9 pretreatment significantly reduced serum ALT and AST levels and improved liver histopathology, suggesting an ameliorated hepatitis. This therapeutic effect was not only attributable to a blunted Th1 immune response, but also to an increased number in regulatory T cells, as reflected in a significantly increased apoptosis of CD4⁺CD25^low/int effector T cells and in reduced proinflammatory cytokine levels.

Conclusion/Significance

Our findings constitute the first preclinical data indicating that interfering with TIM-3/galectin-9 signaling in vivo could ameliorate Con A-induced hepatitis. This strategy may represent a new therapeutic approach in treating human diseases involving T cell activation.     

Regulatory T Cell Suppression of Gag-Specific CD8+ T Cell Polyfunctional Response After Therapeutic Vaccination of HIV-1-Infected Patients on ART

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4⁺ T cells (Treg), thereby masking enhancement of HIV-1-specific CD8⁺ T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4⁺CD25^hiFOXP3⁺ Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8⁺ T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine–pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8⁺ T cell vaccine response by enzyme linked immunosorbent assay for interferon γ production. Although there was no significant change in CD8⁺ T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8⁺ T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection.     

Emergent Group Dynamics Governed by Regulatory Cells Produce a Robust Primary T Cell Response

The currently accepted paradigm for the primary T cell response is that effector T cells commit to autonomous developmental programs. This concept is based on several experiments that have demonstrated that the dynamics of a T cell response is largely determined shortly after antigen exposure and that T cell dynamics do not depend on the level and duration of antigen stimulation. Another experimental study has also shown that T cell responses are robust to variations in antigen-specific precursor frequency.     

调节性T细胞的分化及其影响因素

调节性T细胞是近年发现的一种具有免疫抑制活性的CD4+T细胞亚群,它们可以在胸腺中被选择分化,也可以在外周淋巴组织内由转化生长因子-β等细胞因子诱导分化。本文就调节性T细胞分化过程的关键信号通路及影响因素进行综述。调控调节性T细胞的分化过程能够影响其在体内的数目,可通过对其数量的干预,为自身免疫性疾病、免疫监视、排斥反应及变态反应等相关疾病提供可能的治疗靶点。     

Self-tolerance and Autoimmunity in a Regulatory T Cell Model

The class of immunosuppressive lymphocytes known as regulatory T cells (Tregs) has been identified as a key component in preventing autoimmune diseases. Although Tregs have been incorporated previously in mathematical models of autoimmunity, we take a novel approach which emphasizes the importance of professional antigen presenting cells (pAPCs). We examine three possible mechanisms of Treg action (each in isolation) through ordinary differential equation (ODE) models. The immune response against a particular autoantigen is suppressed both by Tregs specific for that antigen and by Tregs of arbitrary specificities, through their action on either maturing or already mature pAPCs or on autoreactive effector T cells. In this deterministic approach, we find that qualitative long-term behaviour is predicted by the basic reproductive ratio R ₀ for each system. When R ₀<1, only the trivial equilibrium exists and is stable; when R ₀>1, this equilibrium loses its stability and a stable non-trivial equilibrium appears. We interpret the absence of self-damaging populations at the trivial equilibrium to imply a state of self-tolerance, and their presence at the non-trivial equilibrium to imply a state of chronic autoimmunity. Irrespective of mechanism, our model predicts that Tregs specific for the autoantigen in question play no role in the system’s qualitative long-term behaviour, but have quantitative effects that could potentially reduce an autoimmune response to sub-clinical levels. Our results also suggest an important role for Tregs of arbitrary specificities in modulating the qualitative outcome. A stochastic treatment of the same model demonstrates that the probability of developing a chronic autoimmune response increases with the initial exposure to self antigen or autoreactive effector T cells. The three different mechanisms we consider, while leading to a number of similar predictions, also exhibit key differences in both transient dynamics (ODE approach) and the probability of chronic autoimmunity (stochastic approach).     

Dendritic Cells in Chronic Mycobacterial Granulomas Restrict Local Anti-Bacterial T Cell Response in a Murine Model

Background

Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood.

Methodology/Principal Findings

We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c⁺ cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c⁺ cells from acute granulomas. As a consequence of their phenotype, CD11c⁺ cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85B-specific CD4⁺IFNγ⁺ T cells or induce an IFNγ response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c⁺ cells from chronic lesions to stimulate a protective IFNγ T cell response.

Conclusions/Significance

Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explain why mycobacteria are adapted for long-term survival in granulomatous lesions.     

HDAC1 Controls CD8+ T Cell Homeostasis and Antiviral Response

Reversible lysine acetylation plays an important role in the regulation of T cell responses. HDAC1 has been shown to control peripheral T helper cells, however the role of HDAC1 in CD8⁺ T cell function remains elusive. By using conditional gene targeting approaches, we show that LckCre-mediated deletion of HDAC1 led to reduced numbers of thymocytes as well as peripheral T cells, and to an increased fraction of CD8⁺CD4^– cells within the CD3/TCRβ^lo population, indicating that HDAC1 is essential for the efficient progression of immature CD8⁺CD4^– cells to the DP stage. Moreover, CD44^hi effector CD8⁺ T cells were enhanced in mice with a T cell-specific deletion of HDAC1 under homeostatic conditions and HDAC1-deficient CD44^hi CD8⁺ T cells produced more IFNγ upon ex vivo PMA/ionomycin stimulation in comparison to wild-type cells. Naïve (CD44^l°CD62L⁺) HDAC1-null CD8⁺ T cells displayed a normal proliferative response, produced similar amounts of IL-2 and TNFα, slightly enhanced amounts of IFNγ, and their in vivo cytotoxicity was normal in the absence of HDAC1. However, T cell-specific loss of HDAC1 led to a reduced anti-viral CD8⁺ T cell response upon LCMV infection and impaired expansion of virus-specific CD8⁺ T cells. Taken together, our data indicate that HDAC1 is required for the efficient generation of thymocytes and peripheral T cells, for proper CD8⁺ T cell homeostasis and for an efficient in vivo expansion and activation of CD8⁺ T cells in response to LCMV infection.     

Selected MicroRNAs Define Cell Fate Determination of Murine Central Memory CD8 T Cells

During an immune response T cells enter memory fate determination, a program that divides them into two main populations: effector memory and central memory T cells. Since in many systems protection appears to be preferentially mediated by T cells of the central memory it is important to understand when and how fate determination takes place. To date, cell intrinsic molecular events that determine their differentiation remains unclear. MicroRNAs are a class of small, evolutionarily conserved RNA molecules that negatively regulate gene expression, causing translational repression and/or messenger RNA degradation. Here, using an in vitro system where activated CD8 T cells driven by IL-2 or IL-15 become either effector memory or central memory cells, we assessed the role of microRNAs in memory T cell fate determination. We found that fate determination to central memory T cells is under the balancing effects of a discrete number of microRNAs including miR-150, miR-155 and the let-7 family. Based on miR-150 a new target, KChIP.1 (K ⁺ channel interacting protein 1), was uncovered, which is specifically upregulated in developing central memory CD8 T cells. Our studies indicate that cell fate determination such as surface phenotype and self-renewal may be decided at the pre-effector stage on the basis of the balancing effects of a discrete number of microRNAs. These results may have implications for the development of T cell vaccines and T cell-based adoptive therapies.     

Negative Selection by an Endogenous Retrovirus Promotes a Higher-Avidity CD4+ T Cell Response to Retroviral Infection

Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4⁺ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4⁺ T cells and diminished the response to FV infection. Surprisingly, however, CD4⁺ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4⁺ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4⁺ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4⁺ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity.     

B7-H3 Promotes Pathogenesis of Autoimmune Disease and Inflammation by Regulating the Activity of Different T Cell Subsets

B7-H3 is a cell surface molecule in the immunoglobulin superfamily that is frequently upregulated in response to autoantigens and pathogens during host T cell immune responses. However, B7-H3''s role in the differential regulation of T cell subsets remains largely unknown. Therefore, we constructed a new B7-H3 deficient mouse strain (B7-H3 KO) and evaluated the functions of B7-H3 in the regulation of Th1, Th2, and Th17 subsets in experimental autoimmune encephalomyelitis (EAE), experimental asthma, and collagen-induced arthritis (CIA); these mouse models were used to predict human immune responses in multiple sclerosis, asthma, and rheumatoid arthritis, respectively. Here, we demonstrate that B7-H3 KO mice have significantly less inflammation, decreased pathogenesis, and limited disease progression in both EAE and CIA mouse models when compared with littermates; these results were accompanied by a decrease in IFN-γ and IL-17 production. In sharp contrast, B7-H3 KO mice developed severe ovalbumin (OVA)-induced asthma with characteristic infiltrations of eosinophils in the lung, increased IL-5 and IL-13 in lavage fluid, and elevated IgE anti-OVA antibodies in the blood. Our results suggest B7-H3 has a costimulatory function on Th1/Th17 but a coinhibitory function on Th2 responses. Our studies reveal that B7-H3 could affect different T cell subsets which have important implications for regulating pathogenesis and disease progression in human autoimmune disease.     

Reduced Epithelial Na+/H+ Exchange Drives Gut Microbial Dysbiosis and Promotes Inflammatory Response in T Cell-Mediated Murine Colitis

Inflammatory bowel diseases (IBD) are associated with functional inhibition of epithelial Na⁺/H⁺ exchange. In mice, a selective disruption of NHE3 (Slc9a3), a major apical Na⁺/H⁺ exchanger, also promotes IBD-like symptoms and gut microbial dysbiosis. We hypothesized that disruption of Na⁺/H⁺ exchange is necessary for the development of dysbiosis, which promotes an exacerbated mucosal inflammatory response. Therefore, we performed a temporal analysis of gut microbiota composition, and mucosal immune response to adoptive T cell transfer was evaluated in Rag2^-/- and NHE3^-/-/Rag2^-/- (DKO) mice with and without broad-spectrum antibiotics. Microbiome (16S profiling), colonic histology, T cell and neutrophil infiltration, mucosal inflammatory tone, and epithelial permeability were analyzed. In adoptive T cell transfer colitis model, Slc9a3 status was the most significant determinant of gut microbial community. In DKO mice, NHE3-deficiency and dysbiosis were associated with dramatically accelerated and exacerbated disease, with rapid body weight loss, increased mucosal T cell and neutrophil influx, increased mucosal cytokine expression, increased permeability, and expansion of CD25^-FoxP3⁺ Tregs; this enhanced susceptibility was alleviated by oral broad-spectrum antibiotics. Based on these results and our previous work, we postulate that epithelial electrolyte homeostasis is an important modulator in the progression of colitis, acting through remodeling of the gut microbial community.     

Late Engagement of CD86 after Influenza Virus Clearance Promotes Recovery in a FoxP3+ Regulatory T Cell Dependent Manner

Influenza A virus (IAV) infection in the respiratory tract triggers robust innate and adaptive immune responses, resulting in both virus clearance and lung inflammation and injury. After virus clearance, resolution of ongoing inflammation and tissue repair occur during a distinct recovery period. B7 family co-stimulatory molecules such as CD80 and CD86 have important roles in modulating T cell activity during the initiation and effector stages of the host response to IAV infection, but their potential role during recovery and resolution of inflammation is unknown. We found that antibody-mediated CD86 blockade in vivo after virus clearance led to a delay in recovery, characterized by increased numbers of lung neutrophils and inflammatory cytokines in airways and lung interstitium, but no change in conventional IAV-specific T cell responses. However, CD86 blockade led to decreased numbers of FoxP3⁺ regulatory T cells (Tregs), and adoptive transfer of Tregs into αCD86 treated mice rescued the effect of the blockade, supporting a role for Tregs in promoting recovery after virus clearance. Specific depletion of Tregs late after infection mimicked the CD86 blockade phenotype, confirming a role for Tregs during recovery after virus clearance. Furthermore, we identified neutrophils as a target of Treg suppression since neutrophil depletion in Treg-depleted mice reduced excess inflammatory cytokines in the airways. These results demonstrate that Tregs, in a CD86 dependent mechanism, contribute to the resolution of disease after IAV infection, in part by suppressing neutrophil-driven cytokine release into the airways.     

Induction of a Cross-Reactive CD8+ T Cell Response following Foot-and-Mouth Disease Virus Vaccination

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. Current inactivated FMDV vaccines generate short-term, serotype-specific protection, mainly through neutralizing antibody. An improved understanding of the mechanisms of protective immunity would aid design of more effective vaccines. We have previously reported the presence of virus-specific CD8⁺ T cells in FMDV-vaccinated and -infected cattle. In the current study, we aimed to identify CD8⁺ T cell epitopes in FMDV recognized by cattle vaccinated with inactivated FMDV serotype O. Analysis of gamma interferon (IFN-γ)-producing CD8⁺ T cells responding to stimulation with FMDV-derived peptides revealed one putative CD8⁺ T cell epitope present within the structural protein P1D, comprising residues 795 to 803 of FMDV serotype O UKG/2001. The restricting major histocompatibility complex (MHC) class I allele was N*02201, expressed by the A31 haplotype. This epitope induced IFN-γ release, proliferation, and target cell killing by αβ CD8⁺ T cells, but not CD4⁺ T cells. A protein alignment of representative samples from each of the 7 FMDV serotypes showed that the putative epitope is highly conserved. CD8⁺ T cells from FMDV serotype O-vaccinated A31⁺ cattle recognized antigen-presenting cells (APCs) loaded with peptides derived from all 7 FMDV serotypes, suggesting that CD8⁺ T cells recognizing the defined epitope are cross-reactive to equivalent peptides derived from all of the other FMDV serotypes.Foot-and-mouth disease virus (FMDV) is a member of the family Picornaviridae, genus Aphthovirus. The FMDV particle consists of a positive-strand RNA molecule of approximately 8,500 nucleotides, enclosed within an icosahedral capsid. The genome encodes a unique polyprotein from which four structural proteins (P1A, P1B, P1C, and P1D; also referred to as VP4, VP2, VP3, and VP1, respectively) and nine nonstructural proteins are cleaved by viral proteases (48). FMDV shows a high genetic and antigenic variability, which is reflected in the seven serotypes and multiple subtypes reported so far (13). The virus causes a highly contagious infection in cloven-hoofed animals which is characterized by the formation of vesicles on the mouth, tongue, nose, and feet. In addition, most infected animals develop viremia.The virus elicits a rapid humoral response in both infected and vaccinated animals (26). Virus-specific antibodies protect animals in a serotype-specific manner against reinfection or against infection in the case of vaccination, and protection is generally correlated with high levels of neutralizing antibodies (38). Control of the disease is achieved by vaccination with a chemically inactivated whole-virus vaccine emulsified with adjuvant; however, this provides only short-term, serotype-specific protection (2). The introduction of this vaccine has been very successful in areas of the world where the disease is enzootic. However, one of the major difficulties in implementing vaccination is the inability to distinguish vaccinated animals from infected/recovered animals, which may still be shedding virus. Currently, a number of assays specifically developed for this purpose are being validated (29, 41), and the success of these assays is dependent on the use of purified vaccine antigen. A strategy using replication-deficient adenovirus 5 expressing FMDV antigens has been shown to provide early protection against homologous challenge (39).The identification and characterization of T cell epitopes are important for understanding protective immunity mediated by CD8⁺ and CD4⁺ T lymphocytes. Such T cell responses are pathogen specific and are restricted by major histocompatibility complex (MHC) class I and class II molecules, which present foreign peptides to the immune system (55, 56). The role of cellular immunity in the protection of animals from FMDV is still a matter of some controversy. Specific T cell-mediated antiviral responses have been observed in cattle and swine following either infection or vaccination (3, 7, 24). CD4⁺ T cell responses are suggested to play an important role in protection against FMDV, and published studies demonstrate the presence of FMDV-specific MHC class II-restricted responses in cattle and pigs (22, 24). CD4⁺ epitopes within both P1A and P1D proteins have recently been identified in cattle (23). We have recently reported the presence of FMDV-specific, MHC class I-restricted CD8⁺ T cell responses in cattle following infection or vaccination. Despite these observations, the significance of cell-mediated immune responses in protective immunity to FMDV remains unclear.Cattle MHC (bovine leukocyte antigen [BoLA]) is relatively complex, with variable haplotypes expressing one, two, or three of the six classical class I genes (6, 15). At present, about 60 full-length validated cattle MHC class I cDNA sequences have been identified (www.ebi.ac.uk/ipd/mhc/bola), and the haplotypes commonly found in the Holstein breed are well characterized. We have previously identified amino acid motifs present in peptides binding to BoLA class I alleles N*02101, N*02201, and N*01301 (20). More recently, a number of Theileria parva CD8⁺ T cell epitopes presented through these and additional class I alleles have been described (25). Identification of such epitopes allows detailed analysis of cellular immune responses to vaccination and infection.In the present study, we aimed to identify MHC class I-restricted CD8⁺ T cell epitopes within the FMDV capsid protein. Using a panel of overlapping peptides, we have identified a BoLA A31-restricted epitope that is similar in all FMDV serotypes.