Systemic T-cell activation in acute clinically isolated optic neuritis

Journal of Neuroimmunology 162 (2005) 165 – 172 www.elsevier.com/locate/jneuroim

Systemic T-cell activation in acute clinically isolated optic neuritis Hanne Roeda,T, Jette Frederiksena, Annika Langkildeb, Torben Lykke Sbrensena, Martin Lauritzenc, Finn Sellebjerga b

a The MS Clinic, Department of Neurology, University of Copenhagen, Glostrup Hospital, DK-2600 Glostrup, Denmark Danish Research Centre for Magnetic Resonance, University of Copenhagen, Hvidovre Hospital, DK-2630 Hvidovre, Denmark c Department of Clinical Neurophysiology, University of Copenhagen, Glostrup Hospital, DK-2600 Glostrup, Denmark

Received 8 October 2004; received in revised form 6 January 2005; accepted 3 February 2005

Abstract We examined untreated 60 patients with acute monosymptomatic optic neuritis (ON). Patients examined early after onset showed increased expression of HLA-DR and CD45R0 on CD4 and CD8 T cells. Expression of HLA-DR on CD4 T cells was higher in patients without IgG oligoclonal bands. Expression of HLA-DR on CD4 and CD8 T cells correlated negatively with measures of disease activity and positively with measures of good visual function, and expression of CD45R0 on CD4 T cells correlated negatively with measures of disease activity. We hypothesize that HLA-DR expression may characterize a protective T-cell subset in ON. D 2005 Published by Elsevier B.V. Keywords: Optic neuritis; T-cell immunology; Flow cytometry; HLA-DR; CD45R0; MRI

1. Introduction Optic neuritis (ON) is an acute demyelinating disease of the optic nerve, and may occur in patients with confirmed multiple sclerosis (MS) or as a clinically isolated syndrome (CIS). However, many patients presenting with ON will later develop MS, and ON is a common onset symptom of MS (Sorensen et al., 1999a). The risk of developing MS is increased in CIS patients with clinically silent white matter lesions on MRI of the brain and in patients with oligoclonal IgG bands (OB) in the cerebrospinal fluid (CSF), two findings which are strongly associated (Brex et al., 2002; Sellebjerg et al., 2000a; Soderstrom et al., 1998). ON is a well-characterized model of an MS relapse, and the clinical severity is readily assessed by psycho physiological tests of visual function and by studies of visual evoked potentials (VEP) (Frederiksen, 1999). Interest in immune activation in CIS

T Corresponding author. Tel.: +45 43 23 30 55; fax: +45 43 23 39 26. E-mail address: [email protected] (H. Roed). 0165-5728/$ - see front matter D 2005 Published by Elsevier B.V. doi:10.1016/j.jneuroim.2005.02.002

is increasing because recent studies have shown that irreversible axonal damage occurs earlier than previously thought (Filippi et al., 2003), and that treatment with interferons can postpone the development of MS (Comi et al., 2001; Jacobs et al., 2000). The hallmark of MS pathology is demyelinating lesions characterized by the presence of T cells, B cells and phagocytic macrophages (Lucchinetti et al., 2000). Disease activity is probably initiated by systemic activation of myelin-reactive CD4 T cells, followed by T-cell migration into the CNS and the sequential recruitment of additional inflammatory cells to the CNS. Cells primarily accumulate in the perivascular space and subsequently migrate into the CNS parenchyma. The recruitment and migration of leukocytes into the CNS are orchestrated by chemokines, and activated blood cells that express specific combinations of adhesion molecules and chemokine receptors are preferentially attracted to CNS by chemokines produced within the target tissue (Hemmer et al., 2002; Sellebjerg and Sorensen, 2003). Hence, in MS patients the expression of adhesion molecules and chemokines on the surface of activated cells govern the recruitment of immune cells to the CNS,

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and the expression of other activation and differentiation molecules reflects the function of the recruited cells (Sellebjerg and Sorensen, 2003). However, the association between disease activity and expression of activation markers is still controversial. In some previous studies, T cells expressing CD26 were increased during relapses and correlated with MRI disease activity (Jensen et al., 2004; Khoury et al., 2000), but this finding was not confirmed in other studies. The preferential recruitment of cells expressing CXCR3 and CCR5 to active lesions is well established (Balashov et al., 1999; Sorensen et al., 1999b). Although an increased percentage of blood T cells expressing CCR5 and CXCR3 was reported in active MS and ON (Misu et al., 2001; Strunk et al., 2000; Teleshova et al., 2002), these findings are also inconsistent. CD45R0, along with several other molecules, is preferentially expressed by memory T cells (De Rosa et al., 2001). CSF T cells have a memory cell phenotype, but previously reported changes in the expression of isoforms of the CD45 molecule in patients with MS are difficult to interpret, since the memory T-cell compartment consists of several subsets including resting memory cells, activated memory effector cells, anergic cells, and regulatory T cells (Engelhardt et al., 1998; Svenningsson et al., 1993). The expression of CD25, the alfa-chain of the interleukin-2 receptor complex has been used as an activation marker in MS, but no consistent changes in the expression of CD25 on T cells have been observed (Khoury et al., 2000; Wu et al., 2000). However, in CIS patients, we found a negative relationship between the percentage of CD25+ CD4 T cells in CSF and measures of demyelination and IgG OB in CSF (Jensen et al., 2004). This may reflect that CD25+ CD4 T cells are also functionally heterogeneous. Some are activated effector T cells, others express high levels of CD25 and have a regulatory phenotype, and the functional activity of these cells is impaired in MS (Viglietta et al., 2004). The expression of HLA-DR following T-cell activation is a late phenomenon, and is temporally associated with decreased T-cell proliferation (Ko et al., 1979; Yachie et al., 1983). Furthermore, in patients with MS, MRI disease activity is preceded by a decrease in the percentage of T cells expressing HLA-DR (Khoury et al., 2000) and, indeed, regulatory CD25+ CD4 T cells have been reported to express high levels of HLA-DR (BaecherAllan et al., 2001). The remarkable differences in results obtained in different studies may at least partly reflect the different selection of patients for study (i.e., relapse versus remission) and different definitions of disease activity (i.e., clinical versus MRI-based). In order to more accurately assess the relationship between T-cell activation and disease activity in MS, we chose to study this in patients presenting with a bout of clinically isolated ON. We used psycho-physiological, neurophysiologic, and MRI methods to measure disease activity, and we studied the relationship between disease activity, systemic and intra-

thecal T-cell activation in patients with active disease based on several different definitions.

2. Materials and methods 2.1. Patients Sixty untreated consecutive patients with acute monosymptomatic ON and onset within 30 days prior to examination were included in the study. Patients were referred to our department from ophthalmologists and neurologists based on Sealand and Funen, an area with approximately 2.9 million inhabitants. Median age was 36 years (range 17–60 years) and 38 (63%) of patients were female. Median delay from symptom onset to phlebotomy and lumbar puncture was 16 days (range 5–30 days). Twenty-five patients had onset within 14 days prior to phlebotomy. All 60 patients underwent MRI studies, 57 with gadolinium-DTPA (Gd). MRI was performed within the same week as phlebotomy. No patients had received glucocorticoid treatment within four weeks prior to study entry, and no patients received treatment with interferons or other immunomodulating agents prior to study entry. For comparison of blood analyses, 26 age and sex-matched healthy volunteers were included as a control group. The study was conducted in accordance with the Helsinki declarations, and was approved by the regional scientific ethics committee. All participants gave written informed consent before study participation. 2.2. Routine examinations Before inclusion, patients were examined by an ophthalmologist to ensure a correct diagnosis, and a thorough medical history was obtained to ensure that patients had no previous neurological symptoms or any other potential cause of visual loss than ON. To further exclude other potential causes of visual loss, a battery of blood analyses were performed including: red and white blood cell counts, sedimentation rate, autoantibody and syphilis screening, and vitamin B12 levels. Visual acuity was assessed by the Snellen chart, using optimal correction. When visual acuity was below 0.05, arbitrary scores were assigned for patients’ ability: to count fingers (0.02) or to register movement or presence of light (0.01). Patients with no light perception on the affected eye were assigned scores of 0. Contrast sensitivity was assessed by Arden gratings (Arden and Gucukoglu, 1978). Contrast sensitivity scores ranged from 0 to 150, with a score of 150 reflecting severely impaired contrast sensitivity. Colour vision was assessed by the Lanthony desaturated 15 hue test (Geller and Hudnell, 1997). Colour vision scores ranged from 0 to 105, with a score of 105 reflecting severely impaired colour vision. VEP latency was recorded using the pattern reversal technique (Frederiksen et al., 1996). When

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no reproducible response was recorded, an arbitrary value of 250 ms was assigned. VEP amplitudes were recorded for N75-P100 and P100-N145, and the mean of the two recordings was calculated and used for further analysis. MRI was performed using a 1.5-T Siemens Vision scanner (Erlangen, Germany). Twenty-five 5-mm-thick axial slices were positioned according to the pituitary— fastigium line, and the following sequences were measured: Double spin echo, TR/TE=2875/20, 80 ms; T1 weighted images, TR/TE=750/14 ms. T1 weighted images were repeated after injection of double-dose (0.2 mmol/kg) gadolinium (Gd)-DTPA. A sagittal turbo T2 with TR/ TE=4600/99 ms was measured in 17 slices each 5 mm thick. Number of enhancing and T2 lesions were counted by a radiologist, blinded to the results of the visual function, VEP and blood studies, and patients were classified according to Paty’s criteria (Paty et al., 1988). IgG oligoclonal bands were detected by isoelectric focusing and immunoblotting (Sellebjerg and Christiansen, 1996). 2.3. Preparation of blood and CSF and flow cytometry All samples were collected between 8 a.m. and 12 a.m. Within 5 min after phlebotomy, 50 Al of whole blood was incubated with antibodies for 30 min, lysed, and washed twice in ice cold FACS PBS with 1% human serum albumin and fixed with 1% paraformaldehyde. CSF was obtained directly in tubes immersed in ice water, and centrifuged within 5 min after lumbar puncture. Cells were resuspended in ice cold FACS PBS, incubated on ice with antibodies for 30 min, washed twice in ice cold FACS PBS with 1% human serum albumin and fixated by 1% paraformaldehyde. The low number of CSF cells from many study participants precluded the analysis of all molecules in each patient. Blood and CSF samples were stored on ice until analysis the same day on a 4-colour FACSCalibur flow cytometer (BD Biosciences, San Jose, CA), using CellQuest software (BD Biosciences). For analysis, cells were gated according to forward and side scatter profiles, and the expression of CD4, CD8 or CD3. Isotype levels were determined as immunoflourescence level of 1% by CD3 positive T cells. Data are given as percentage of CD4, CD8 or CD3 cells with expression above isotype level. The following antibodies were purchased from BD Biosciences: FITC and PE-conjugated IgG1 (clone X40), and IgG2 (clone X39); PerCP-conjugated anti-CD4 (clone SK3) and anti-CD3 (clone SK7); APC-conjugated anti-CD8 (clone SK1); FITC-conjugated anti-CD26 (clone L272 and clone PHP/BA5), anti-CCR5 (clone 2D7), anti-CD95 (clone DX2), anti-CD25 (clone 2A3), and anti-CD154 (clone TRAP1); PE-conjugated anti-HLA-DR (clone L243), antiCD122 (clone TU27), anti-CD45R0 (clone UCHL-1), antiCD69(clone L78) and anti- CXCR3 (clone 1C6). The following antibodies were purchased from R&D systems: PE-conjugated anti-CCR2 (clone 48607) and anti-CCR1 (clone 53504).

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2.4. Statistics As the data did not follow the Normal distribution, the Mann–Whitney test for unpaired data was used for comparison of groups. The Spearman rank test was used for correlation analysis. A 5% significance level was applied to all tests.

3. Results 3.1. Visual function, VEP, MRI, and CSF studies Table 1 lists the results of the visual function tests, VEP, routine laboratory studies, and MRI studies in the entire patient material and in the subgroup of patients studied within 2 weeks from symptom onset. All patients with ON had impaired contrast sensitivity, and the majority of patients also had impaired visual acuity, abnormal colour vision tests, and abnormal VEP results. The median Kurtzke EDSS score was 3.5 (range 0–4). T2-weighted MRI abnormalities were observed in 60% of the patients. MRI was highly suggestive of MS in 37% of patients, 23% of patients had lesser abnormalities, and 40% had a normal MRI of the brain. One or more Gd-enhancing lesion was found in 37% of patients. IgG oligoclonal bands were found in 52% of patients, and 48% had a CSF leukocyte count above 4 cells/Al. 3.2. Flow cytometry studies of peripheral blood cells When comparing all patients to healthy controls, no difference in expression of surface markers on T cells was found. However, when the analysis was restricted to patients seen within 2 weeks from onset, we observed a significantly higher percentage of CD4 T cells expressing CD45R0 ( p=0.05) and HLA-DR ( p=0.05) in patients with ON compared to healthy controls (Fig. 1). This subgroup of patients with ON also had a higher percentage of CD8 T cells expressing CD45R0 ( p=0.01) and HLA-DR ( p=0.02) than did controls (Fig. 1). When the analysis Table 1

Age Symptom duration, days Visual acuity Contrast sensitivity Colour perception VEP amplitude, AV VEP latency, ms T2 lesions Gd lesions IgG index CSF leukocyte count

All patients n=60, median (range)

Patients examined V14 days after onset n=25, median (range)

35 16 0.1 150 105 3.5 165 1 0 0.63 4

35 11 0 140 105 2.4 173 2 0 0.56 4

(17–60) (5–30) (0–10) (87–150) (2–105) (0–22) (89–250) (0–30) (0–7) (0.42–2.9) (0–115)

(20–58) (5–14) (0–0.9) (81–150) (6–105) (0–13) (89–250) (0–6) (0–2) (0.42–2.9) (0–26)

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A 100

80

**

60

40

*

20

HLA-DR on CD4

0

HLA-DR on CD8

<14 days

> 14 days

Controls

B 100

80

60

40 §

20

§§ CD45RO on CD4

0

CD45RO on CD8

< 14 days

> 14 days

controls

Fig. 1. Expression in peripheral blood of HLA-DR and CCD45R0 within and after 14 days after onset. Peripheral blood expression of HLA-DR and CCD45R0 in patients examined within 14 days after onset of ON and more than 14 days after onset of ON compared to controls. (A) The expression of HLA-DR on CD4 and CD8 T cells in blood is increased in patients examined within 14 days after symptom onset of ON compared to controls. *p=0.05 and **p=0.02. (B) The expression of CD45R0 on CD4 and CD8 T cells is increased in patients examined within 14 days after symptom onset compared to controls. §p=0.05 and §§ p=0.01.

was restricted to patients seen within 2 weeks from symptom onset, we also observed a trend to a higher percentage of CD4 T cells expressing CCR5 in patients than in controls ( p=0.07). We found no significant differences in the percentage of T cells expressing any of the other molecules under study in this subgroup of patients and controls (data not shown). Neither did the analysis of patients not yet in clinical remission nor patients with Gd-enhancing lesions on MRI discloses other differences between patient and controls (data not shown).

3.3. Peripheral blood T-cell activation and disease activity We went on to study the relationship between clinical and MRI disease activity and the percentage of CD4 and CD8 T cells expressing HLA-DR and CD45R0, respectively. This analysis was carried out in the entire patient material as well as in the subgroup seen within 2 weeks from symptom onset, i.e., the subgroup of patients in which there was evidence of alterations in the T-cell phenotype in blood. In the entire patient material, we

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found a negative correlation between VEP latencies and the percentage of CD4 T cells (Spearman’s rho= 0.36, p=0.004) and CD8 T cells (Spearman’s rho= 0.31, p=0.02) expressing HLA-DR. In addition, the percentage of CD8 T cells expressing HLA-DR correlated positively with VEP amplitudes (Spearman’s rho=0.30, p=0.02). The number of T2 lesions on MRI correlated negatively with the percentage of CD4 T cells expressing HLA-DR (Spearman’s rho= 0.26, p=0.04) and CD45R0 (Spearman’s rho= 0.29, p=0.03) and the percentage of CD8 T cells expressing HLA-DR (Spearman’s rho= 0.27, p=0.04). Finally, the percentage of CD4 T cells expressing HLA-DR was higher in patients without IgG OB (median 9%, range 3–35%) than in patients with IgG OB in CSF (median 6%, range 3–16%, p=0.04). When these analyses were repeated in the patients seen within 2 weeks from symptom onset, the inverse relationship between lower percentages of T cells expressing HLA-DR and CD45R0 and impaired visual function was even more remarkable (Table 2). Furthermore, lower percentages of HLA-DR positive CD4 T cells ( p=0.003) and CD8 T cells ( p=0.04) were observed in patients with IgG oligoclonal bands in CSF than in those without IgG OB (Fig. 2). Twenty three untreated patients were followed for 6 months as the placebo-group of a treatment trial with IVIG. During this follow up, three patients were diagnosed with MS, In two patients the diagnosis was based on additional relapses and in one patient the diagnosis was based on development of new T2 or Gd lesions on MRI according to McDonald et al. (2001), no difference was found in baseline expression of surface markers between patients diagnosed with MS and patients continuously monosymptomatic after 6 months and no correlation was found between expression of surface markers baseline and number of T2 or enhancing lesions after 6 months.

Table 2 Correlation between measures of disease activity and activation markers in peripheral blood in patients examined within 14 days after onset of ON

Contrast sensitivity EDSS VEP amplitude VEP latency Number of T2 lesions Number of Gd lesions T pV0.05. TT pV0.01.

CD4 T cells HLA-DR

CD4 T cells CD45R0

CD8 T cells HLA-DR

0.54TT p=0.005 0.33 p=0.11 0.52TT 0.008 0.36TT p=0.004 0.54TT p=0.005 0.45T p=0.028

0.56TT p=0.007 0.14 p=0.55 0.60TT p=0.003 0.23 p=0.10 0.56TT p=0.007 0.33 p=0.14

0.41T p=0.05 0.41T p=0.045 0.55TT p=0.006 0.51TT p=0.01 0.44T p=0.034 0.40 p=0.059

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3.4. CSF T-cell studies CSF T-cell studies were carried out in 24 patients out of the entire cohort. In eight patients, CSF samples were obtained within 14 days after onset. The percentage of CD8 T cells expressing HLA-DR ( p=0.001) and the percentage of CD3 T cells expressing CD25 ( pb0.001) were augmented in CSF compared to peripheral blood. Additionally the expression of CXCR3 ( pb0.001), CCR5 ( p=0.016) and CD40L ( p=0.001) on CD4 T cells was augmented in CSF compared to peripheral blood, as was the percentage of CD8 T cells expressing CXCR3 ( p=0.015) and CD40L ( p=0.039) and the percentage of CD3 T cells expressing CCR2 ( p=0.013). The expression of CD122 ( p=0.002) on CD3 T cells was lower in CSF compared to peripheral blood. Further analysis of patients examined within 14 days after onset showed an increased expression only of CXCR3 on CD4 T cells ( p=0.012) and of CD25 on CD3 T cells ( p=0.018) in CSF compared to peripheral blood. However, in patients examined more than 14 days after symptom onset, we found increased expression of CD40L ( p=0.001) and CXCR3 ( p=0.001) on CD4 T cells of HLA-DR ( p=0.004) on CD8 T cells and of CCR2 ( p=0.003) and CD25 ( p=0.001) on CD3 T cells in CSF compared to peripheral blood. Finally in patients examined after 14 days after onset, the expression of CD122 ( pb0.001) on CD3 T cells was decreased in CSF compared to perioheral blood. No correlation between the expression of surface markers in CSF and clinical measures of disease activity was found.

4. Discussion In the present study, we investigated visual function, VEP, MRI, CSF and the expression of several inflammatory markers on CD4 and CD8 T-cell subsets in a population of untreated patients with ON and symptom onset within 30 days. In order to investigate the impact of measures of disease activity, we performed subgroup analyses of patients with onset within 14 days prior to inclusion and of patients without clinical remission. Furthermore by comparing patients with and without IgG oligoclonal bands in CSF and patients with and without lesions on MRI, we analysed if the risk of subsequent development of MS influenced the expression of surface markers on T cells in blood and CSF. We found no difference in the expression of activation markers on T cells in the whole patient cohort. The expression of HLA-DR and CD45R0 on peripheral blood T cells was upregulated in patients examined within 14 days after onset of symptoms and in patients without IgG OB in CSF. The expression of HLA-DR in peripheral blood correlated positively with measures of good visual function and negatively with MRI disease activity. Twenty-three patients were followed for 6 months, during follow-up three patients were diagnosed with MS, no difference was found

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100

80

60

**

40

* 20 HLA-DR on CD4

0

HLA-DR on CD8

not present

present

IgG oligoclonal bands Fig. 2. Peripheral blood expression of HLA-DR in patients examined within 14 days after onset. In patients with ON examined within 14 days after onset, the expression of HLA-DR in peripheral blood is increased on CD4 and CD8 T cells in patients without IgG oligoclonal bands in CSF compared to patients with IgG oligoclonal bands in CSF. *p=0.003, **p=0.04.

in baseline expression of surface markers between patients diagnosed with MS during 6 months of follow-up and patients continuously monosymptomatic. The expression of HLA-DR, CD25, CCR5, CXCR3, CCR2 and CD40L was augmented in CSF compared to blood in all patients and in patients examined after 14 days after onset however only expression of CXCR3 and CD25 was augmented in patients examined within 14 days after onset. The expression of surface markers in CSF did not correlated to clinical measures of disease activity. The expression of CD45R0 is associated with a memory phenotype of T cells (Michie et al., 1992) but T cells expressing CD45R0 are heterogeneous in terms of functional properties (Ohara et al., 2002; Sallusto et al., 1999b). The expression of HLA-DR is often considered to simply reflect T-cells activation. However, in patients with MS, MRI disease activity is preceded by a transient decrease in the expression of HLA-DR on T cells in peripheral blood (Khoury et al., 2000). This decrease does not seem to reflect recruitment of cells expressing HLA-DR to CNS, since the percentage of T cells in CSF expressing HLA-DR is lower in patients with active MS (Fredrikson et al., 1987; Oksaranta et al., 1995; Sellebjerg et al., 2000b) and since HLA-DR is not expressed by T cells in MS lesions (Hayashi et al., 1988). Instead, our results suggest that HLA-DR positive T cells could be recruited actively to the CSF during the late stages of a demyelinating attack. The role of expression of HLA-DR on T cells is not clear. HLA-DR expressing T cells may present antigen, but T-cell antigen presentation induces T-cell anergy in responsive T cells (Satyaraj et al., 1995; Taams et al., 1999). In addition, a

subset of human T cells with a memory phenotype and high expression of CD25 coexpress HLA-DR, and possess regulatory capacities (Baecher-Allan et al., 2001). Thus the augmented expression of HLA-DR in CSF in patients examined more than 2 weeks after onset may reflect an increased percentage of regulatory T cells in CSF during remission after monosymptomatic ON. We found comparable blood expression of CD25 in ON patients and controls in the present study. This is consistent with the results of previous studies of CD25 expression on blood T cells in ON and MS (Jensen et al., 2004; Khoury et al., 2000; Wu et al., 2000). We hypothesize that the expression of HLA-DR which is expressed on regulatory CD4 T cells could reflect the regulatory activity associated with a subset of CD25 positive CD4 T cells, but functional studies are required to substantiate this. Data on expression of chemokine receptors in acute relapses of MS and acute ON are not conclusive. Some authors find an increase in the percentage of CD4 T cells in blood expressing CCR5 and CXCR3 during acute relapses of MS and ON, while others have not been able to confirm this (Misu et al., 2001; Sorensen et al., 2002, 2003; Teleshova et al., 2002). We found a trend towards an increased expression of CCR5 on CD4 T cells in patients with clinically isolated ON examined within 14 days after onset. The reason for the ambiguous data on expression of CCR5 and CXCR3 may be the selective recruitment of T cells expressing CCR5 and CXCR3 to the CSF, which may differ in early and later stages of an MS attack (Balashov et al., 1999; Simpson et al., 2000; Sorensen et al., 1999b), also the expression of CCR5 is rapidly down regulated in the

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absence of Il-2 and by stimulation with anti-CD3 and antiCD28 (Sallusto et al., 1998, 1999a). Furthermore chemokine receptors are internalised after ligand binding, resulting in reduced surface expression and finally, the results of chemokine receptor studies may differ to some extent in whole blood-based and isolated mononuclear cells (Kivisakk et al., 2002). In conclusion we found changes in systemic T-cell activation early after onset in clinically isolated ON, and changes in CSF T-cell activation after 2 weeks. Systemic changes were normalized already within 2 to 4 weeks from symptom onset. We found negative correlations between HLA-DR and CD45R0 expression on CD4 and CD8 T cells in peripheral blood and measures of MRI disease activity and visual impairment and VEP latencies. We hypothesize that a subset of T cells co-expressing HLA-DR and CD45R0 is protective in patients with clinically isolated ON. Further studies are needed to characterize these cells regarding their functional activity and co-expression of other molecules.

Acknowledgements The study was supported by the Danish Multiple Sclerosis Society, Johnsen’s Foundation and the Novo Nordisk foundation. The authors wish to thank laboratory technicians Merete Corfixen and Kirsten Junker for their kind assistance.

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