CD8+ T cells in inflammatory demyelinating disease

We review the contribution made by CD8+ T cells to inflammation in the central nervous system (CNS) in Multiple Sclerosis (MS), and discuss their role in the animal model Experimental Autoimmune Encephalomyelitis (EAE). We show that the inflammatory cytokines interferon-gamma and interleukin-17 are differentially regulated in CNS-infiltrating CD4+ and CD8+ T cells in EAE, and that CD8+ T cells regulate disease. In MS, CD8+ T cells appear to play a role in promotion of disease, so cytokine regulation is likely different in CD8+ T cells in MS and EAE.     

The dual role of CD8+ T lymphocytes in the development of stress-induced herpes simplex encephalitis

Despite the generally restrictive nature of the blood–brain barrier (BBB), circulating lymphocytes can infiltrate into the central nervous system (CNS) during a variety of disease states. Although the contributions of these lymphocytes to CNS-associated disease have been identified in some viral models, the factors which govern this infiltration following herpes simplex virus (HSV) infection remain to be elucidated. We have developed a murine model of HSV encephalitis (HSE) to define the relationship among psychological stress, the recruitment of HSV-specific T cells into the CNS, and the development of HSE. Naive mice, as well as mice that had been vaccinated with a recombinant vaccinia virus (rVVESgB498–505) that elicits the generation of HSV-1 gB498–505-specific CD8⁺ T cells, were infected intranasally (i.n.) with HSV-1 McIntyre. Beginning one day prior to HSV-1 infection and continuing for a total of 9 days, naive and vaccinated mice were exposed to a well-established stressor, restraint stress. Naive, stressed mice exhibited increased symptoms of HSE and HSE-associated mortality as compared to non-stressed controls. A concomitant increase in CD4⁺ and CD8⁺ T cells in the brain was observed throughout the infection, with CD8⁺ T cells outnumbering CD4⁺ T cells. The development of HSE in these naive, stressed mice was accompanied by a delayed infiltration of gB498–505-specific CD8⁺ T cells after HSV spread into the brain. In contrast, both stressed and non-stressed rVVESgB498–505-vaccinated mice possessed gB498–505-specific CD8⁺ T cells prior to HSV challenge and were protected against HSE despite having detectable HSV-1 DNA in the brain. Together, these findings suggest that a delayed infiltration of CD8⁺ T cells into the brain may promote HSE in naive mice, while the presence of HSV-specific CD8⁺ T cells in the brain prior to HSV challenge is protective, possibly by limiting HSV replication and spread within the CNS.     

Anti-IL-16 therapy reduces CD4+ T-cell infiltration and improves paralysis and histopathology of relapsing EAE

Infiltration of the central nervous system (CNS) by CD4+ Th1 cells precedes onset and relapses of experimental autoimmune encephalomyelitis (EAE). We reported that (B6xSJL) F1 (H-2b/s) mice with severe relapsing-remitting disease had extensive infiltration by CD4+ T cells compared to that in C57BL/6 (B6) (H-2b) mice, which developed mild low-relapsing disease in response to myelin oligodendrocyte peptide 35-55 (MOG(35-55)). This observation led us to search for mechanisms that specifically regulate trafficking of CD4+ cells in relapsing H-2b/s mice. We show that the CD4+ cell chemoattractant cytokine interleukin (IL)-16 has an important role in regulation of relapsing EAE induced by MOG(35-55) in the (B6xSJL) F1 (H-2b/s) mice. We found production of IL-16 in the CNS of mice with EAE. IL-16 levels in the CNS correlated well with the extent of CD4+ T-cell and B-cell infiltration during acute and relapsing disease. Infiltrating CD4+ T cells, B cells, and to a lesser extent CD8+ T cells all contained IL-16 immunoreactivity. Treatment with neutralizing anti-IL-16 antibody successfully reversed paralysis and ameliorated relapsing disease. In treated mice, diminished infiltration by CD4+ T cells, less demyelination, and more sparing of axons was observed. Taken together, our results show an important role for IL-16 in regulation of relapsing EAE. We describe a novel therapeutic approach to specifically impede CD4+ T cell chemoattraction in EAE based on IL-16 neutralization. Our findings have high relevance for the development of new therapies for relapsing EAE and potentially MS.     

Preferential increase of IL-2R+ CD4+ T cells and CD45RB- CD4+ T cells in the central nervous system in experimental allergic encephalomyelitis.

We examined lymphocytes isolated from the spinal cord (SC), peripheral blood (PB) and lymph nodes (LN) draining the immunization site of Lewis rats with acute experimental allergic encephalomyelitis (EAE). Cells were analysed for T cell subset markers CD4 (mAb W3/25) and CD8 (mAb OX8), for IL-2R (mAb OX39), and for high molecular mass leukocyte common antigen (LCA, CD45RB) expression (mAb OX22). T cells expressing high (CD45RB+) or low (CD45RB-) molecular mass LCA are of different maturational stages and/or separate lineages. CD4+ T cells were more predominant in SC than in PB and LN; CD8+ T cells were scarce in SC but common in PB and LN. Activated CD4+ T cells (IL-2R+) were common in the SC and LN but infrequent in blood. CD4+ T cells that were CD45RB+ were scarce in the SC. In contrast, the majority of CD4+ T cells in the PB and LN were CD45RB+. The preferential accumulation of IL-2R+ CD4+ T cells and of CD45RB- CD4+ T cells in the central nervous system (CNS) indicates that a selective mechanism directs cell egress into CNS lesions in EAE.     

Deficient Fas expression by CD4+ CCR5+ T cells in multiple sclerosis

OBJECTIVE: To evaluate whether T cells expressing CCR5 and CXCR3 from multiple sclerosis (MS) patients are more resistant to apoptosis. METHODS: Expression of CD69, TNF-R1, Fas, FasL, bcl-2, and bax was investigated in 41 MS patients and 12 healthy controls by flow cytometry in CD4+ and CD8+ T cells expressing CCR5 and CXCR3. RESULTS: In MS patients, the percentage of CD69 was increased and Fas expression decreased in CD4+ CCR5+ T cells. INTERPRETATION: The lower Fas expression in activated CD4+ CCR5+ T cells might contribute to disease pathogenesis by prolonging cell survival and favoring their migration into the CNS.     

Increase in peripheral CD4 bright+ CD8 dull+ T cells in Parkinson disease

BACKGROUND: Immune abnormalities are known to be involved in the pathogenesis of sporadic Parkinson disease. OBJECTIVE: To examine whether abnormalities in peripheral lymphocytes exist in Parkinson disease. METHODS: Immune mediators, including CD1a, CD3, CD4, CD8, CD45RO, and Fas (CD95), were examined in peripheral lymphocytes of patients by 3-color flow cytometry. RESULTS: Patients with Parkinson disease displayed a significantly greater population of circulating CD3+ CD4 bright+ CD8 dull+ lymphocytes than age-matched control subjects (P =.005) and patients with cerebrovascular disease (P =.002). The increase in these cells appeared to continue for at least 17 months. These T cells also expressed CD45RO and Fas, markers for activated T cells, while CD1a, a marker for thymic T cells, was negative, suggesting that these cells are mature T cells with immune activities. CONCLUSIONS: As CD4+ CD8+ T cells are known to increase after some specific viral infections, the continuous increase in CD4 bright+ CD8 dull+ T cells shown here may indicate postinfectious immune abnormalities that are possibly associated with the pathogenesis of this slowly progressive, multifactorial neurodegenerative disease.     

Identification of T lymphocytes in simian immunodeficiency virus encephalitis: Distribution of CD8+ T cells in association with central nervous system vessels and virus

T lymphocytes are found within brains infected with human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) where they are a minor, but consistently identified, population. However, little analysis of their phenotypes has been done, and questions concerning whether or not they are viral antigen specific has not been thoroughly examined. We investigated the central nervous system (CNS) of SIV-infected rhesus macaques to identify T-lymphocyte subsets in relation to virus-infected cells and brain microvessels. We have found that a sensitive antigen-retrieval technique greatly enhanced immunohistochemical detection of CD4+ and CD8+ T lymphocytes in control studies. In encephalitic brains of SIV-infected monkeys with acquired immunodeficiency syndrome (AIDS), we found a significant accumulation of CD8+ T lymphocytes but little-to-no accumulation of CD4+ T lymphocytes. CD4+ cells, when detected, were mostly monocyte/macrophages closely associated with CNS vessels. Using a combination of in situ hybridization for SIV RNA, and immunohistochemistry for CD8+ T lymphocytes and/or Glut-1 for endothelial cells on brain microvessels, we found CD8+ T lymphocytes with an angiocentric distribution often adjacent to virus-infected cells. In the CNS of animals with SIV encephalitis, there was a trend of CD8+ T lymphocytes that were not directly juxtaposed with CNS vessels. These data suggest that in brains of SIV-infected monkeys and HIV-infected humans, CD8+ T lymphocytes traffic to and are retained in the CNS in an angiocentric and possibly antigen-specific manner.     

CD4+T-bet+, CD4+pSTAT3+ and CD8+T-bet+ T cells accumulate in peripheral blood during NZB treatment

Circulating T cells and monocytes expressing T-bet, pSTAT1 and pSTAT3 increase in relapsing-remitting multiple sclerosis (RRMS) during relapse. Natalizumab (NZB) is an effective drug in RRMS, but exacerbation of the disease after its discontinuation has been described in some patients. The aim of this research was to study the effect of NZB treatment on circulating lymphomonocyte subpopulations expressing T-bet, pSTAT1, pSTAT3 and CD4+CD25+Foxp3+ regulatory T cells. Flow cytometry was used to evaluate the percentages of circulating CD4+ and CD8+ T cells, CD14+ monocytes and B cells expressing T-bet, pSTAT1, and pSTAT3, and CD4+CD25+Foxp3+ regulatory T cells from RRMS patients before and after 6-12 NZB infusions. In NZB-treated RRMS patients, the percentages of CD4+pSTAT1+ and CD8+pSTAT1+ T cells, CD14+pSTAT1+ monocytes, CD4+T-bet+, CD8+T-bet+ and CD4+pSTAT3+ T cells and CD14+pSTAT3+ monocytes increased after 12 drug infusions and were similar to those observed in untreated relapsing RRMS patients. Otherwise in vitro NZB exposure of peripheral blood mononuclear cells from untreated RRMS patients and controls had no effect. It was concluded that NZB treatment determines an accumulation of CD4+pSTAT1+, CD8+pSTAT1+, CD4+T-bet+, CD8+T-bet+ and CD4+STAT3+ T cells in peripheral blood that may account for the exacerbation of the disease observed in some patients after the discontinuation of the drug.     

Suppressor role of rat CD8+CD45RClow T cells in experimental autoimmune uveitis (EAU)

To determine whether decreased regulatory T cell activity contributes to the pathogenesis of recurrent experimental autoimmune uveitis (EAU), we compared the immunoregulatory activity of CD8+CD45RClow T cells isolated from rats that had recovered from acute EAU with those from rats with the progressive, recurrent disease. Our results showed that CD8+CD45RClow T cells isolated from the recovered rats showed suppressive activity in vitro, whereas those from rats with progressive, recurrent EAU do not. Depletion of CD8+CD45RClow T cells from T cells used for adoptive transfer of EAU increased the pathogenic activity of the T cells. Co-transfer of CD8+CD45RClow T cells with uveitogenic T cells prevented the relapse of disease in the recipient rats. The suppressive CD8+CD45RClow T cells expressed increased levels of Foxp3 after stimulation in vitro with the autoantigen, and inhibited the production of IFN-gamma by autoreactive T cells. Our data indicate that the decreased suppressive activity of CD8+CD45RClow T cells is correlated with disease development in this autoimmune disease. Further studies on the biology of this T cell population should provide much needed insights into disease pathogenesis.     

Neuroprotection by IL-10-producing MOG CD4+ T cells following ischemic stroke

Mucosal tolerance has been used successfully to treat animal models of autoimmune diseases and is being tested in human diseases. In this work we demonstrate the reduction of infarct size following mucosal tolerance by myelin oligodendrocyte glycoprotein (MOG) (35-55) peptide in mouse stroke model. Nasal MOG was most efficacious and reduced ischemic infarct size by 70% at 24 h as well as improving behavior score. Using immunohistological methods and IL-10 -/- mice, we demonstrate the importance of IL-10-producing CD4+ T cells in the reduction of the ischemic infarct volume following middle cerebral artery occlusion (MCAO). Furthermore, adoptive transfer of CD4+ T cells from nasally tolerized mice to untreated mice prior to MCAO surgery significantly decreased stroke size (p<0.001 vs. control), whereas CD4+ T cells from nasally tolerized IL-10-deficient mice had no significant effect. Based on these results, modulation of cerebral inflammation by mucosal tolerance to myelin antigens may have applicability both as prophylactic therapy and treatment following ischemia attacks.     

Isolation and characterization of CD8+ regulatory T cells in multiple sclerosis

To investigate CD8+ regulatory T cell influence on multiple sclerosis development, peripheral blood and cerebrospinal fluid (CSF) CD8+ T cell clones (TCCs) recognizing MBP_83–102 and MOG_63–87-specific CD4+ T cells were isolated from 20 patients during acute exacerbations, 15 in remission and 15 controls. Blood and CSF CD8+ regulatory TCC cloning frequency decreased more during exacerbations than remissions or controls. Target cell pre-activation significantly enhanced CD8+ T granule-mediated cell killing of CD4+ targets, and was restricted by HLA-E. During exacerbations, killer-inhibitory receptor CD94/NKG2A expression was significantly higher in CD8+ TCCs, limiting their cytotoxic activity. Moreover, IL-15 and IFN-γ significantly increased CD94 and NKG2A expression. These data provide evidence that CD94/NKG2A receptors play an important role in regulating T cell activity during the course of MS.     

CNS-derived CCL21 is both sufficient to drive homeostatic CD4+ T cell proliferation and necessary for efficient CD4+ T cell migration into the CNS parenchyma following Toxoplasma gondii infection

Injury, infection and autoimmune triggers increase CNS expression of the chemokine CCL21. Outside the CNS, CCL21 contributes to chronic inflammatory disease and autoimmunity by three mechanisms: recruitment of lymphocytes into injured or infected tissues, organization of inflammatory infiltrates into lymphoid-like structures and promotion of homeostatic CD4+ T-cell proliferation. To test if CCL21 plays the same role in CNS inflammation, we generated transgenic mice with astrocyte-driven expression of CCL21 (GFAP-CCL21 mice). Astrocyte-produced CCL21 was bioavailable and sufficient to support homeostatic CD4+ T-cell proliferation in cervical lymph nodes even in the absence of endogenous CCL19/CCL21. However, lymphocytes and glial-activation were not detected in the brains of uninfected GFAP-CCL21 mice, although CCL21 levels in GFAP-CCL21 brains were higher than levels expressed in inflamed Toxoplasma-infected non-transgenic brains. Following Toxoplasma infection, T-cell extravasation into submeningeal, perivascular and ventricular sites of infected CNS was not CCL21-dependent, occurring even in CCL19/CCL21-deficient mice. However, migration of extravasated CD4+, but not CD8+ T cells from extra-parenchymal CNS sites into the CNS parenchyma was CCL21-dependent. CD4+ T cells preferentially accumulated at perivascular, submeningeal and ventricular spaces in infected CCL21/CCL19-deficient mice. By contrast, greater numbers of CD4+ T cells infiltrated the parenchyma of infected GFAP-CCL21 mice than in wild-type or CCL19/CCL21-deficient mice. Together these data indicate that CCL21 expression within the CNS has the potential to contribute to T cell-mediated CNS pathology via: (a) homeostatic priming of CD4+ T-lymphocytes outside the CNS and (b) by facilitating CD4+ T-cell migration into parenchymal sites following pathogenic insults to the CNS.     

CD45 isoform alteration in CD4+ T cells as a potential diagnostic marker of Alzheimer's disease

Aging represents the greatest risk for development of Alzheimer's disease (AD), and changes in peripheral immune cell phenotypes have been found to be associated with aging. Using flow cytometry, we measured the relative expression levels of CD45 isoforms, a marker of nai;ve versus memory CD4+ T cell status, on isolated CD4+ T lymphocytes from patients with a clinical diagnosis of probable Alzheimer's disease, normal elderly, cognitively abnormal elderly, and patients with clinically diagnosed other forms of dementia. Data show significantly lower levels of CD45RA, and an increase in the CD45RO/CD45RA ratio, on CD4+ T cells in patients diagnosed with probable Alzheimer's disease (n=46) and in cognitively abnormal individuals (n=37) compared to age-matched normal participants (n=90). Patients diagnosed with other forms of dementia (n=19) did not significantly differ from normal individuals. Both CD45RA and the CD45RO/CD45RA ratio had higher positive and negative predictive values and were more sensitive biomarkers of probable AD than the apolipoprotein E epsilon 4 allele, and had greater predictive ability for probable AD by regression analyses. Additionally, a testing strategy employing apolipoprotein E genotyping and CD45RA or the CD45RO/CD45RA ratio revealed increased sensitivity, positive and negative predictive values, and predictive ability over the apolipoprotein E epsilon 4 allele. These data show altered peripheral immunity in AD patients, and raise the possibility that a testing strategy using CD45 isoform alteration on CD4+ T cells and apolipoprotein E genotype may be clinically valuable for diagnosing probable AD.     

多发性硬化患者CD8+记忆性T细胞亚群穿孔素和颗粒酶-B表达的研究

目的 测定多发性硬化(MS)患者外周血中CD8+记忆性T细胞亚群效应细胞因子的表达,并将其与MS病情严重程度进行相关分析.方法 利用四色-流式细胞术检测未经治疗的MS患者、其他神经系统疾病(OND)患者和正常对照(NC)成员组外周血表达穿孔素和颗粒酶-B的CD8+记忆性T细胞亚群数量,并利用扩展的残疾状况量表(EDSS)对MS患者病情严重程度做评分.结果 与NC组比较,MS患者外周血表达颗粒酶-B的CD8+效应记忆性T细胞(TEM)和终末效应记忆性T细胞(TerTEM)均明显减少,比较差异有统计学意义(P＜0.05);表达穿孔素和颗粒酶-B的TEM数量与EDSS呈负相关(r=-0.493,P=0.027;r=-0.594,P=0.009).结论 CD8+TEM参与MS相关的CNS内炎性免疫应答,外周血穿孔素和颗粒酶-B表达阳性的CD8+TEM数量可在一定程度上反映MS患者CNS的病损程度.     

Loss rather than downregulation of CD4 T cells as a mechanism for remission from experimental allergic encephalomyelitis

SJL/J mice recover from clinical signs of experimental allergic encephalomyelitis (EAE) 2 to 3 days following the onset of the initial attack. The immunoregulatory events that induce clinical recovery are not well understood. In this paper we have compared the activation state of the T cells infiltrating the central nervous system (CNS) in symptomatic and remitted mice. We isolated mononuclear cells from the CNS at various time points during the course of EAE and used flow cytometry to describe the kinetics of CNS infiltration by CD45⁺, CD2⁺, CD3⁺, TCRαβ⁺, CD4⁺ cells. There was a 30-fold reduction in the number of CNS CD4⁺ T cells in remitted mice 10 days following the initial attack. More than 60% of CNS CD4⁺ cells were of a CD44^high, CD45RB^low memory/effector phenotype both in active EAE, peak EAE and in remission, contrast to lymph nodes where this phenotype never constituted more than 17%. The proportion of CD8⁺ T cells was not increased in remitted mice, and we detected no TCRγδ⁺ cells within the CNS. Our findings demonstrate an overt loss of CD4⁺ T cells from the CNS and the maintenance of an activated state by T cells within the CNS and during remission from EAE. This argues against downregulation of T cell function as a mechanism for remission.     

Myelin oligodendrocyte glycoprotein peptide-induced experimental allergic encephalomyelitis and T cell responses are unaffected by immunoproteasome deficiency

The inoculation of MOG peptides into C57BL/6 mice induces CD4(+) and CD8(+) T cells, and recent work has shown that adoptive transfer of the latter population, after extensive in vitro stimulation, can cause EAE in na?ve recipient mice. Herein, we have evaluated the incidence and severity of EAE, and the induction of CD4(+) and CD8(+) T cells, following MOG peptide inoculation of wt mice and of LMP-2KO mice that lack an intact immunoproteasome, a cytoplasmic organelle that is induced by chronic inflammation and that may be important for the presentation of MHC class I epitopes to CD8(+) T cells. We report that EAE, evaluated by both clinical and histological criteria, is similar in LMP-2KO mice and wildtype C57B/6 mice (wt) in response to immunization with MOG peptides MOG(35-55) and MOG(40-54), suggesting that the immunoproteasome does not play a key role in the development of demyelinating disease. Furthermore, and consistent with previous reports, peptide-specific CD8(+) T cells were barely detectable in the CNS of peptide-immunized mice, although peptide-specific CD4(+) T cells were abundant. Therefore, we used a new technique to look for autoreactive CD8(+) T cells in MOG peptide-immunized mice, and we report the identification of CD4(+) and CD8(+) T cells that, as late as 19 days after peptide injection, are actively producing IFNgamma in vivo, in response to in vivo antigen contact.     

Comparing the pathogenesis of experimental autoimmune encephalomyelitis in CD4-/- and CD8-/- DBA/1 mice defines qualitative roles of different T cell subsets

Experimental autoimmune encephalomyelitis (EAE) was induced with myelin oligodendrocyte glycoprotein (MOG(1-125)) in CD4(-/-) and CD8(-/-) DBA/1 mice. Both gene-deleted mice developed clinical signs of EAE, albeit milder than in wild-type mice, suggesting that both CD4(+) and CD8(+) cells participate in disease development. Demyelination and inflammation in the central nervous system was reduced in the absence of CD8(+) T cells. Antibody depletion of CD4(+) cells completely protected CD8(-/-) mice from MOG-induced EAE while depletion of CD8(+) cells in CD4(-/-) mice resulted in fewer EAE incidence compared to that in control antibody-treated mice. Antibody depletion of CD4(+) cells in wild-type mice protected from EAE, but not depletion of CD8(+) cells, although demyelination was reduced on removal of CD8(+) T cells. Immunization with immunodominant MOG(79-96) peptide led to EAE only in the presence of pertussis toxin (PT) in the inoculum. PT also triggered an earlier onset and more severe EAE in CD8(-/-) mice. We interpret our findings such that in an ontogenic lack of CD4(+) T cells, EAE is mediated by CD8(+) and elevated levels of alphabetaCD4(-)CD8(-) cells, and that CNS damage is partly enacted by the activity of CD8(+) T cells.     

颅内动脉瘤破裂与CD8~+CD28~-调节性T细胞的关系

目的探讨CD8+CD28-调节性T细胞在颅内动脉瘤发生破裂中所起的作用。方法收集确诊未破裂动脉瘤患者20例,破裂者17例,采用流式细胞术检测其外周血CD8+CD28-调节性T细胞的百分含量;并选择16例正常人及12例脑胶质瘤患者作为对照。对未破裂的患者进行为期2年随访,观察CD8+CD28-T细胞的变化情况。结果健康对照组的CD8+CD28-T细胞含量为27.82%±3.59%,胶质瘤患者的含量为23.71%±8.21%,未破裂动脉瘤患者为13.87%±4.17%,而破裂者为6.93%±2.08%。破裂患者的CD8+CD28-T细胞水平显著低于未破裂者(t=-1.865,P=0.041),且两组动脉瘤患者均低于正常人(均为P<0.01),但胶质瘤患者与正常人与无统计学差异(P=0.117);经过2年随访,共有5例患者失访,在余下的15例患者当中有3例发生破裂(分别在第16、18及22个月发生破裂),此3例患者第1年末的CD8+CD28-T细胞含量已明显低于随访前(P<0.05),且发生破裂后该细胞的含量显著低于第一年末(P<0.05)。结论 CD8+CD28-调节性T细胞减少可能与动脉瘤发生破裂相关。     

CD8+ T cell activation correlates with disease activity in clinically isolated syndromes and is regulated by interferon-beta treatment

An increased percentage of blood CD8+ T cells from patients with clinically isolated syndromes (CIS) suggestive of multiple sclerosis (MS) was found to express CD26 and CD69. The percentage of CD26 or CD69 positive CD8+ T cells was higher in patients with MRI evidence of disease dissemination in space or with active MRI lesions than in the remaining patients. Treatment of MS with interferon (IFN)-beta resulted in a decrease in the percentage of CD26 and CD71 positive CD8+ T cells and an increase in the percentage of CD8+ T cells that expressed interleukin (IL)-10 and IL-13. CD8+ T cell activation in MS may be linked to disease activity already at disease onset, and is regulated by treatment with IFN-beta.     

Direct demonstration of the infiltration of murine central nervous system by Pgp-1/CD44high CD45RB(low) CD4+ T cells that induce experimental allergic encephalomyelitis.

In experimental allergic encephalomyelitis (EAE), autoimmune T cells infiltrate the central nervous system (CNS) and initiate demyelinating pathology. We have used flow cytometry to directly analyse the migration to the CNS of MBP-reactive CD4+ T cells labelled with a lipophilic fluorescent dye (PKH2), in SJL/J mice with passively transferred EAE. Labelled cells constituted about 45% of the CNS CD4+ population at the time of EAE onset. Almost all (greater than 90%) of the PKH2-labelled CD4+ T cells from EAE CNS were blasts and were alpha/beta T cell receptor (TCR)+, CD44(Pgp-1)high, and the majority were CD45RB(low). By contrast, most PKH2-labelled CD4+ T cells in lymph nodes, although CD44high, were CD45RBhigh cells. The cells that were transferred to induce EAE were essentially similar to antigen-primed lymph node cell populations, containing less than 15% CD44high cells, and most of them were CD45RBhigh. The CD44high CD45RB(low) phenotype is characteristic of memory/effector T cells that have been activated by antigen recognition. The difference in CD45RB expression between CNS and LN could therefore reflect differential exposure and/or response to antigen. Consistent with this, PKH2-labelled CD4+ cells isolated from the CNS were responsive to MBP in vitro, whereas PKH2+ CD4+ cells from lymph nodes showed almost undetectable responses. In control experiments in which ovalbumin (OVA)-reactive T cells were transferred, a small number of fluorescent-labelled CD4+ T cells were also detected in CNS, but there were very few blasts, and these remained CD45RBhigh. These results argue for induction of the memory/effector phenotype of CD4+ T cells, and their selective retention in the CNS, as a consequence of antigen recognition.