Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells

The cannabinoid system is immunomodulatory and has been targeted as a treatment for the central nervous system (CNS) autoimmune disease multiple sclerosis. Using an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we investigated the role of the CB(1) and CB(2) cannabinoid receptors in regulating CNS autoimmunity. We found that CB(1) receptor expression by neurons, but not T cells, was required for cannabinoid-mediated EAE suppression. In contrast, CB(2) receptor expression by encephalitogenic T cells was critical for controlling inflammation associated with EAE. CB(2)-deficient T cells in the CNS during EAE exhibited reduced levels of apoptosis, a higher rate of proliferation and increased production of inflammatory cytokines, resulting in severe clinical disease. Together, our results demonstrate that the cannabinoid system within the CNS plays a critical role in regulating autoimmune inflammation, with the CNS directly suppressing T-cell effector function via the CB(2) receptor.     

Delta-like ligand 4 regulates central nervous system T cell accumulation during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated inflammatory demyelinating disease of the CNS that serves as a model for multiple sclerosis. Notch receptor signaling in T lymphocytes has been shown to regulate thymic selection and peripheral differentiation. In the current study, we hypothesized that Notch ligand-receptor interaction affects EAE development by regulating encephalitogenic T cell trafficking. We demonstrate that CNS-infiltrating myeloid dendritic cells, macrophages, and resident microglia expressed Delta-like ligand 4 (DLL4) after EAE induction. Treatment of mice with a DLL4-specific blocking Ab significantly inhibited the development of clinical disease induced by active priming. Furthermore, the treatment resulted in decreased CNS accumulation of mononuclear cells in the CNS. Anti-DLL4 treatment did not significantly alter development of effector cytokine expression by Ag-specific T cells. In contrast, anti-DLL4 treatment reduced T cell mRNA and functional cell surface expression of the chemokine receptors CCR2 and CCR6. Adoptive transfer of Ag-specific T cells to mice treated with anti-DLL4 resulted in decreased clinical severity and diminished Ag-specific CD4(+) T cell accumulation in the CNS. These results suggest a role for DLL4 regulation of EAE pathogenesis through modulation of T cell chemokine receptor expression and migration to the CNS.     

Proinflammatory bacterial peptidoglycan as a cofactor for the development of central nervous system autoimmune disease

Upon stimulation by microbial products through TLR, dendritic cells (DC) acquire the capacity to prime naive T cells and to initiate a proinflammatory immune response. Recently, we have shown that APC within the CNS of multiple sclerosis (MS) patients contain peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria, which signals through TLR and NOD. In this study, we report that Staphylococcus aureus PGN as a single component can support the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model for MS. Mice immunized with an encephalitogenic myelin oligodendrocyte glycoprotein peptide in IFA did not develop EAE. In contrast, addition of PGN to the emulsion was sufficient for priming of autoreactive Th1 cells and development of EAE. In vitro studies demonstrate that PGN stimulates DC-mediated processes, reflected by increased Ag uptake, DC maturation, Th1 cell expansion, activation, and proinflammatory cytokine production. These data indicate that PGN-mediated interactions result in proinflammatory stimulation of Ag-specific effector functions, which are important in the development of EAE. These PGN-mediated processes may occur both within the peripheral lymph nodes as well as in the CNS and likely involve recognition by TLR on DC. Thus, PGN may provide a physiological trigger of DC maturation, and in this way disrupt the normal tolerance to self Ag. As such, PGN signaling pathways may serve as novel targets for the treatment of MS.     

The role of antibodies in multiple sclerosis

B cells, plasma cells, and antibodies are commonly found in active central nervous system (CNS) lesions in patients with multiple sclerosis (MS). B cells isolated from CNS lesions as well as from the cerebrospinal fluid (CSF) show signs of clonal expansion and hypermutation, suggesting their local activation. Plasma blasts and plasma cells maturating from these B cells were recently identified to contribute to the development of oligoclonal antibodies produced within the CSF, which remain a diagnostic hallmark finding in MS. Within the CNS, antibody deposition is associated with complement activation and demyelination, indicating antigen recognition-associated effector function. While some studies indeed implied a disease-intrinsic and possibly pathogenic role of antibodies directed against components of the myelin sheath, no unequivocal results on a decisive target antigen within the CNS persisted to date. The notion of a pathogenic role for antibodies in MS is nevertheless empirically supported by the clinical benefit of plasma exchange in patients with histologic signs of antibody deposition within the CNS. Further, such evidence derives from the animal model of MS, experimental autoimmune encephalomyelitis (EAE). In transgenic mice endogenously producing myelin-specific antibodies, EAE severity was substantially increased accompanied by enhanced CNS demyelination. Further, genetic engineering in mice adding T cells that recognize the same myelin antigen resulted in spontaneous EAE development, indicating that the coexistence of myelin-specific B cells, T cells, and antibodies was sufficient to trigger CNS autoimmune disease. In conclusion, various pathological, clinical, immunological, and experimental findings collectively indicate a pathogenic role of antibodies in MS, whereas several conceptual challenges, above all uncovering potential target antigens of the antibody response within the CNS, remain to be overcome.     

Ninjurin1 is expressed in myeloid cells and mediates endothelium adhesion in the brains of EAE rats

Ninjurin1 (nerve injury-induced protein, Ninj1) is an adhesion molecule that is essential for cell-to-cell interactions. However, little is known about the function of Ninj1 in the central nervous system (CNS). To address its role in the CNS, we analyzed the expression pattern of Ninj1 in normal rats and in an experimental autoimmune encephalomyelitis (EAE) model. Ninj1 was expressed in three major compartments of brains, meninges, the choroid plexus, and parenchymal perivascular spaces. In the EAE brains, Ninj1 was strongly expressed in myeloid cells (macrophages/monocytes and neutrophils) and partially expressed in endothelial cells (ECs). Furthermore, Ninj1 enhanced adhesion between BV2 cells (murine monocyte lineage microglia) and HBMECs (human brain microvascular endothelial cells). Collectively, our findings suggest that Ninj1 may mediate the entry of myeloid cells into the CNS in normal and EAE brains, and it is a potential therapeutic target for regulating myeloid cell trafficking across the blood-brain barrier (BBB) in CNS immune processes.     

Inflammation-mediated memory dysfunction and effects of a ketogenic diet in a murine model of multiple sclerosis

A prominent clinical symptom in multiple sclerosis (MS), a progressive disorder of the central nervous system (CNS) due to heightened neuro-inflammation, is learning and memory dysfunction. Here, we investigated the effects of a ketogenic diet (KD) on memory impairment and CNS-inflammation in a murine model of experimental autoimmune encephalomyelitis (EAE), using electrophysiological, behavioral, biochemical and in vivo imaging approaches. Behavioral spatial learning deficits were associated with motor disability in EAE mice, and were observed concurrently with brain inflammation. The KD improved motor disability in the EAE model, as well as CA1 hippocampal synaptic plasticity (long-term potentiation) and spatial learning and memory (assessed with the Morris Water Maze). Moreover, hippocampal atrophy and periventricular lesions in EAE mice were reversed in KD-treated EAE mice. Finally, we found that the increased expression of inflammatory cytokines and chemokines, as well as the production of reactive oxygen species (ROS), in our EAE model were both suppressed by the KD. Collectively, our findings indicate that brain inflammation in EAE mice is associated with impaired spatial learning and memory function, and that KD treatment can exert protective effects, likely via attenuation of the robust immune response and increased oxidative stress seen in these animals.     

Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence). Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs. The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.     

Tissue concentration changes of amino acids and biogenic amines in the central nervous system of mice with experimental autoimmune encephalomyelitis (EAE)

We have characterized the changes in tissue concentrations of amino acids and biogenic amines in the central nervous system (CNS) of mice with MOG_35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model commonly used to study multiple sclerosis (MS). High performance liquid chromatography was used to analyse tissue samples from five regions of the CNS at the onset, peak and chronic phase of MOG_35-55 EAE. Our analysis includes the evaluation of several newly examined amino acids including d-serine, and the inter-relations between the intraspinal concentration changes of different amino acids and biogenic amines during EAE. Our results confirm many of the findings from similar studies using different variants of the EAE model as well as those examining changes in amino acid and biogenic amine levels in the cerebrospinal fluid (CSF) of MS patients. However, several notable differences were observed between mice with MOG_35-55-induced EAE with findings from human studies and other EAE models. In addition, our analysis has identified strong correlations between different amino acids and biogenic amines that appear to change in two distinct groups during EAE. Group I analyte concentrations are increased at EAE onset and peak but then decrease in the chronic phase with a large degree of variability. Group II is composed of amino acids and biogenic amines that change in a progressive manner during EAE. The altered levels of these amino acids and biogenic amines in the disease may represent a critical pathway leading to neurodegenerative processes that are now recognized to occur in EAE and MS.     

IFN-gamma determines distinct clinical outcomes in autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS initiated by autoreactive CD4(+) T cells. EAE classically presents with a progressive ascending paralysis and is a model of multiple sclerosis that recapitulates some aspects of the disease. In this report we describe a mouse strain that spontaneously develops a severe, nonclassical form of EAE with 100% incidence. The distinct clinical phenotype is marked initially by a slight head tilt, progressing to a severe head tilt, spinning, or a rotatory motion. Classical EAE spontaneously occurs in myelin basic protein (MBP)-specific TCR transgenic RAG-1(-/-) mice (referred to as T/R(-)), whereas nonclassical EAE spontaneously occurs in T/R(-) IFN-gamma(-/-) mice (T/R(-)gamma(-)). Thus, the TCR recognizes the same Ag (MBP) and uses identical TCR in both cases. The cellular infiltrate in nonclassical EAE is predominantly found in the brainstem and cerebellum, with very little inflammation in the spinal cord, which is primarily affected in classical disease. Importantly, depending on the genetic makeup and priming conditions of the MBP-specific T cells, nonclassical disease can occur in the presence of an inflammatory infiltrate with eosinophilic, neutrophilic, or monocytic characteristics. Finally, we believe that nonclassical spontaneous EAE could be a useful model for the study of some characteristics of multiple sclerosis not observed in classical EAE, such as the inflammatory responses in the brainstem and cerebellum that can cause vertigo.     

Detailed analysis of early immunopathologic events during lesion formation in acute experimental autoimmune encephalomyelitis

To investigate early immunopathologic events, SJL/J mice were challenged for acute experimental autoimmune encephalomyelitis (EAE) and sampled between 12 hr and 14 days postinoculation (PI). Complete Freund's adjuvant (CFA)-inoculated mice served as controls. T cells, T cell subsets, Class II major histocompatibility (MHC) antigen (Ia)-positive and immunoglobulin (Ig)-positive cells, albumin and Ig deposits, and myelin antigens were localized in frozen sections of central nervous system (CNS) and non-CNS tissue (heart, liver, kidney) by immunocytochemical techniques. In both experimental groups, a few Ia-positive endothelial cells and low-grade diffuse infiltration by T cells, T cell subsets, and Ia+ and Ig+ cells were seen from 12 hr PI onward in CNS and non-CNS tissue. Only in acute EAE but not in CFA-challenged mice were these early changes followed at 10 days PI by extensive inflammation which was restricted to the CNS and was accompanied by Ia-positive astrocytes. Thus, in acute EAE, immunopathologic changes appear to develop in two stages. During the early low-grade generalized phase, recirculation of lymphocytes is moderately enhanced while during the late phase, extensive immunopathology is focused upon the target organ, the CNS.     

CD24 on the resident cells of the central nervous system enhances experimental autoimmune encephalomyelitis

CD24 is a cell surface glycoprotein that is expressed on both immune cells and cells of the CNS. We have previously shown that CD24 is required for the induction of experimental autoimmune encephalomyelitis (EAE), an experimental model for the human disease multiple sclerosis (MS). The development of EAE requires CD24 expression on both T cells and non-T host cells in the CNS. To understand the role of CD24 on the resident cells in the CNS during EAE development, we created CD24 bone marrow chimeras and transgenic mice in which CD24 expression was under the control of a glial fibrillary acidic protein promotor (AstroCD24TG mice). We showed that mice lacking CD24 expression on the CNS resident cells developed a mild form of EAE; in contrast, mice with overexpression of CD24 in the CNS developed severe EAE. Compared with nontransgenic mice, the CNS of AstroCD24TG mice had higher expression of cytokine genes such as IL-17 and demyelination-associated marker P8; the CNS of AstroCD24TG mice accumulated higher numbers of Th17 and total CD4+ T cells, whereas CD4+ T cells underwent more proliferation during EAE development. Expression of CD24 in CD24-deficient astrocytes also enhanced their costimulatory activity to myelin oligodendrocyte glycoprotein-specific, TCR-transgenic 2D2 T cells. Thus, CD24 on the resident cells in the CNS enhances EAE development via costimulation of encephalitogenic T cells. Because CD24 is increased drastically on resident cells in the CNS during EAE, our data have important implications for CD24-targeted therapy of MS.     

Relapsing murine experimental allergic encephalomyelitis induced by myelin basic protein

Experimental allergic encephalomyelitis (EAE), an experimental autoimmune disease of the central nervous system (CNS), is readily induced in many mammalian species by immunization with CNS tissue or myelin basic protein (MBP) purified from the CNS. EAE has been frequently used as a model for multiple sclerosis (MS). However, EAE generally presents as an acute monophasic disease in the adult animal after immunization with MBP. After recovery, the animal is resistant to rechallenge with encephalitogen (1). Two exceptions to these observations have been reported. McFarlin et al. (2) reported that a variable number of Lewis rats showed signs of a single, mild relapse about a week after recovery from MBP-induced acute EAE. Panitch and Ciccone (3) have reported induction of recurrent EAE in rats immunized with human MBP. Chronic, relapsing EAE has been induced in the mouse; however, an apparent requirement for CNS tissue had been noted (4, 5). Recently, during the course of a series of experiments on the induction of EAE in SJL/J, PL/J, and (SJL/J X PL/J)F1 (SPL F1) mice, it was observed that the F1 mice frequently had paralytic relapses after recovery from MBP-induced symptoms. Experiments were initiated to examine this phenomenon, and the findings are presented below.     

Time-Dependent Progression of Demyelination and Axonal Pathology in MP4-Induced Experimental Autoimmune Encephalomyelitis

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal pathology. Myelin basic protein/proteolipid protein (MBP-PLP) fusion protein MP4 is capable of inducing chronic experimental autoimmune encephalomyelitis (EAE) in susceptible mouse strains mirroring diverse histopathological and immunological hallmarks of MS. Limited availability of human tissue underscores the importance of animal models to study the pathology of MS.MethodsTwenty-two female C57BL/6 (B6) mice were immunized with MP4 and the clinical development of experimental autoimmune encephalomyelitis (EAE) was observed. Methylene blue-stained semi-thin and ultra-thin sections of the lumbar spinal cord were assessed at the peak of acute EAE, three months (chronic EAE) and six months after onset of EAE (long-term EAE). The extent of lesional area and inflammation were analyzed in semi-thin sections on a light microscopic level. The magnitude of demyelination and axonal damage were determined using electron microscopy. Emphasis was put on the ventrolateral tract (VLT) of the spinal cord.ResultsB6 mice demonstrated increasing demyelination and severe axonal pathology in the course of MP4-induced EAE. In addition, mitochondrial swelling and a decrease in the nearest neighbor neurofilament distance (NNND) as early signs of axonal damage were evident with the onset of EAE. In semi-thin sections we observed the maximum of lesional area in the chronic state of EAE while inflammation was found to a similar extent in acute and chronic EAE. In contrast to the well-established myelin oligodendrocyte glycoprotein (MOG) model, disease stages of MP4-induced EAE could not be distinguished by assessing the extent of parenchymal edema or the grade of inflammation.ConclusionsOur results complement our previous ultrastructural studies of B6 EAE models and suggest that B6 mice immunized with different antigens constitute useful instruments to study the diverse histopathological aspects of MS.     

Passive induction of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a widely used animal model of the human demyelinating disease multiple sclerosis. EAE is initiated by immunization with myelin antigens in adjuvant or by adoptive transfer of myelin-specific T cells, resulting in inflammatory infiltrates and demyelination in the central nervous system. Induction of EAE in rodents typically results in ascending flaccid paralysis with inflammation primarily targeting the spinal cord. This protocol describes passive induction of EAE by adoptive transfer of T cells isolated from mice primed with myelin antigens into na?ve mice. The advantages of using this method versus active induction of EAE are discussed.     

Suppression of experimental autoimmune encephalomyelitis by selective blockade of encephalitogenic T-cell infiltration of the central nervous system

Multiple sclerosis (MS) is a devastating neuroinflammatory disorder of the central nervous system (CNS) in which T cells that are reactive with major components of myelin sheaths have a central role. The receptor for advanced glycation end products (RAGE) is present on T cells, mononuclear phagocytes and endothelium. Its pro-inflammatory ligands, S100-calgranulins, are upregulated in MS and in the related rodent model, experimental autoimmune encephalomyelitis (EAE). Blockade of RAGE suppressed EAE when disease was induced by myelin basic protein (MBP) peptide or encephalitogenic T cells, or when EAE occurred spontaneously in T-cell receptor (TCR)-transgenic mice devoid of endogenous TCR-alpha and TCR-beta chains. Inhibition of RAGE markedly decreased infiltration of the CNS by immune and inflammatory cells. Transgenic mice with targeted overexpression of dominant-negative RAGE in CD4+ T cells were resistant to MBP-induced EAE. These data reinforce the importance of RAGE-ligand interactions in modulating properties of CD4+ T cells that infiltrate the CNS.     

Cytosolic phospholipase A2 plays a key role in the pathogenesis of multiple sclerosis-like disease

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that results in motor and sensory deficits. Although MS and its animal model, experimental autoimmune encephalomyelitis (EAE), are thought to be T cell-mediated diseases, the mechanisms underlying the lesions in the CNS are not fully understood. We propose that a strong candidate as a central mediator in evoking the complex pathological changes seen in MS and EAE is the enzyme cytosolic phospholipase A2 (cPLA2). One of the metabolic products of this enzyme is pro-inflammatory, while the other induces myelin breakdown, demyelination, and chemokine/cytokine expression. We provide evidence that cPLA2 is highly expressed in EAE lesions and show that blocking this enzyme leads to a remarkable reduction in the onset and progression of EAE.     

Estrogen receptor α signaling in T lymphocytes is required for estradiol-mediated inhibition of Th1 and Th17 cell differentiation and protection against experimental autoimmune encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.     

De novo central nervous system processing of myelin antigen is required for the initiation of experimental autoimmune encephalomyelitis

We demonstrate the absolute requirement for a functioning class II-restricted Ag processing pathway in the CNS for the initiation of experimental autoimmune encephalomyelitis (EAE). C57BL/6 (B6) mice deficient for the class II transactivator, which have defects in MHC class II, invariant chain (Ii), and H-2M (DM) expression, are resistant to initiation of myelin oligodendrocyte protein (MOG) peptide, MOG(35-55)-specific EAE by both priming and adoptive transfer of encephalitogenic T cells. However, class II transactivator-deficient mice can prime a suboptimal myelin-specific CD4(+) Th1 response. Further, B6 mice individually deficient for Ii and DM are also resistant to initiation of both active and adoptive EAE. Although both Ii-deficient and DM-deficient APCs can present MOG peptide to CD4(+) T cells, neither is capable of processing and presenting the encephalitogenic peptide of intact MOG protein. This phenotype is not Ag-specific, as DM- and Ii-deficient mice are also resistant to initiation of EAE by proteolipid protein peptide PLP(178-191). Remarkably, DM-deficient mice can prime a potent peripheral Th1 response to MOG(35-55), comparable to the response seen in wild-type mice, yet maintain resistance to EAE initiation. Most striking is the demonstration that T cells from MOG(35-55)-primed DM knockout mice can adoptively transfer EAE to wild-type, but not DM-deficient, mice. Together, these data demonstrate that the inability to process antigenic peptide from intact myelin protein results in resistance to EAE and that de novo processing and presentation of myelin Ags in the CNS is absolutely required for the initiation of autoimmune demyelinating disease.     

Severe disease, unaltered leukocyte migration, and reduced IFN-gamma production in CXCR3-/- mice with experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) Th1 T cell-mediated disease of the CNS, used to study certain aspects of multiple sclerosis. CXCR3, the receptor for CXCL10, CXCL9, and CXCL11, is preferentially expressed on activated Th1 T cells and has been proposed to govern the migration of lymphocytes into the inflamed CNS during multiple sclerosis and EAE. Unexpectedly, CXCL10-deficient mice were susceptible to EAE, leaving uncertain what the role of CXCR3 and its ligands might play in this disease model. In this study, we report that CXCR3(-/-) mice exhibit exaggerated severity of EAE compared with wild-type (CXCR3(+/+)) littermate mice. Surprisingly, there were neither quantitative nor qualitative differences in CNS-infiltrating leukocytes between CXCR3(+/+) and CXCR3(-/-) mice with EAE. Despite these equivalent inflammatory infiltrates, CNS tissues from CXCR3(-/-) mice with EAE showed worsened blood-brain barrier disruption and more von Willebrand factor-immunoreactive vessels within inflamed spinal cords, as compared with CXCR3(+/+) mice. Spinal cords of CXCR3(-/-) mice with EAE demonstrated decreased levels of IFN-gamma, associated with reduced inducible NO synthase immunoreactivity, and lymph node T cells from CXCR3(-/-) mice primed with MOG(35-55) secreted less IFN-gamma in Ag-driven recall responses than cells from CXCR3(+/+) animals. CXCR3(-/-) lymph node T cells also showed enhanced Ag-driven proliferation, which was reduced by addition of IFN-gamma. Taken with prior findings, our data show that CXCL10 is the most relevant ligand for CXCR3 in EAE. CXCR3 does not govern leukocyte trafficking in EAE but modulates T cell IFN-gamma production and downstream events that affect disease severity.