Cerebrospinal fluid B cells from multiple sclerosis patients are subject to normal germinal center selection

Previous findings from our laboratory demonstrated that some clonally expanded cerebrospinal fluid (CSF) B cells from MS patients exhibit diminished mutation targeting patterns in comparison to typical B cells selected in the context of germinal centers (GCs). In order to determine whether the overall CSF B cell repertoires adhered to mutation patterns typical of GC-selected B cells, we analyzed the immunoglobulin repertoires from CSF B cells of 8 MS patients for mutation characteristics typical of GC-derived B cells. Mutation targeting was preserved. Thus, clonal expansion of some CSF B cells may occur independently of GC, but the CSF B cell pool is governed by typical GC selection. Interestingly, the heavy chain CDR3's of CSF B cells from MS patients had a net acidic charge, similar to GC-derived B cells, but a tendency towards longer CDR3's, consistent with autoreactive B cells. How these findings may support current hypotheses regarding the origin of CSF B cells is discussed.     

The effects of natalizumab on the innate and adaptive immune system in the central nervous system

Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the central nervous system (CNS). Natalizumab ((R)Tysabri) is a humanized recombinant monoclonal antibody that binds to the alpha (alpha)(4) chain of the alpha(4) beta (beta)(1) integrin (very late activation antigen 4; VLA-4), and alpha(4)beta(7) integrin. Recently, two patients with MS and one patient with Crohn's disease who were treated with natalizumab in the setting of clinical trials developed progressive multifocal leukoencephalopathy (PML), an opportunistic infection of the brain with the polyoma virus JC. We recently showed that natalizumab decreases the numbers of CD4(+) and CD8(+) T lymphocytes, CD19(+) B cells, and CD138(+) plasma cells in the cerebrospinal fluid (CSF) of patients with MS on natalizumab therapy. In addition, we demonstrated that the cell numbers in CSF remained unchanged even 6 months after cessation of natalizumab treatment.     

The effects of natalizumab on inflammatory mediators in multiple sclerosis: prospects for treatment‐sensitive biomarkers

Background: Natalizumab affects systemic cytokine expressions and clinical course in relapsing–remitting multiple sclerosis (RRMS). We analyzed levels of inflammatory cytokines in cerebrospinal fluid (CSF) cells and peripheral blood mononuclear cells (PBMCs), levels of matrix metalloproteinase (MMP)‐9 and osteopontin (OPN) in CSF, and clinical outcome measures in 22 natalizumab‐treated RRMS patients. Methods: mRNA levels of cytokines in cells were detected with real‐time RT‐PCR. Protein levels of OPN and MMP‐9 were measured by ELISA. Results: Natalizumab reduced CSF cell counts (P < 0.0001). Tumor necrosis factor (TNF) and interferon‐γ (IFN‐γ) mRNAs were significantly increased in PBMCs. In contrast, expressions of IFN‐γ and interleukin (IL)‐23 were decreased but IL‐10 increased in the CSF cells. OPN and MMP‐9 were reduced in the CSF. Patients being in remission at baseline showed the same deviations of mediators as those in relapse after natalizumab treatment. The open label clinical outcome measures were either stable or improved during therapy. Conclusions: Natalizumab attenuates pro‐inflammatory mediators intrathecally and the reduced pro‐inflammatory milieu may allow increased production of the anti‐inflammatory mediator IL‐10. The increased systemic cytokines may impede the improvement of certain clinical measures like fatigue. The affected mediators seem to be sensitive to an immune‐modifying treatment which could be used as biomarkers for this therapy.     

JC polyomavirus expression and bell-shaped regulation of its SF2/ASF suppressor during the follow-up of multiple sclerosis patients treated with natalizumab

Natalizumab is effective against multiple sclerosis (MS), but is associated with progressive multifocal leukoencephalopathy (PML), fatal disease caused by the JCV polyomavirus. The SF2/ASF (splicing factor2/alternative splicing factor) inhibits JCV in glial cells. We wondered about SF2/ASF modulation in the blood of natalizumab-treated patients and if this could influence JCV reactivation. Therefore, we performed a longitudinal study of MS patients under natalizumab, in comparison to patients under fingolimod and to healthy blood donors. Blood samples were collected at time intervals. The expression of SF2/ASF and the presence and expression of JCV in PBMC were analyzed. A bell-shaped regulation of SF2/ASF was observed in patients treated with natalizumab, increased in the first year of therapy, and reduced in the second one, while slightly changed, if any, in patients under fingolimod. Notably, SF2/ASF was up-regulated, during the first year, only in JCV DNA-positive patients, or with high anti-JCV antibody response; the expression of the JCV T-Ag protein in circulating B cells was inversely related to SF2/ASF protein expression. The SF2/ASF reduction, parallel to JCV activation, during the second year of therapy with natalizumab, but not with fingolimod, may help explain the increased risk of PML after the second year of treatment with natalizumab, but not with fingolimod. We propose that SF2/ASF has a protective role against JCV reactivation in MS patients. This study suggests new markers of disease behavior and, possibly, help in re-evaluations of therapy protocols.     

Altered CD4+/CD8+ T-cell ratios in cerebrospinal fluid of natalizumab-treated patients with multiple sclerosis

BACKGROUND: Treatment with natalizumab, a monoclonal antibody against the adhesion molecule very late activation antigen 4, an alpha4beta(1) integrin, was recently associated with the development of progressive multifocal leukoencephalopathy, a demyelinating disorder of the central nervous system caused by JC virus infection. OBJECTIVE: To test the effect of natalizumab treatment on the CD4(+)/CD8(+) T-cell ratios in cerebrospinal fluid (CSF) and peripheral blood. DESIGN: Prospective longitudinal study. SETTING: Academic and private multiple sclerosis centers. PATIENTS: Patients with multiple sclerosis (MS) treated with natalizumab, untreated patients with MS, patients with other neurologic diseases, and human immunodeficiency virus-infected patients. MAIN OUTCOME MEASURES: CD4(+) and CD8(+) T cells were enumerated in CSF and peripheral blood. The mean fluorescence intensity of unbound alpha4 integrin on peripheral blood CD4(+) and CD8(+) T cells was analyzed before and after natalizumab therapy. RESULTS: Natalizumab therapy decreased the CSF CD4(+)/CD8(+) ratio of patients with MS to levels similar to those of human immunodeficiency virus-infected patients. CD4(+)/CD8(+) ratios in peripheral blood in patients with MS progressively decreased with the number of natalizumab doses, but they remained within normal limits. Six months after the cessation of natalizumab therapy, CSF CD4(+)/CD8(+) ratios normalized. The expression of unbound alpha4 integrin on peripheral blood T cells decreases with natalizumab therapy and was significantly lower on CD4(+) vs CD8(+) T cells. CONCLUSIONS: Natalizumab treatment alters the CSF CD4(+)/CD8(+) ratio. Lower expression of unbound alpha4 integrin on CD4(+) T cells is one possible mechanism. These results may have implications for the observation that some natalizumab-treated patients with MS developed progressive multifocal leukoencephalopathy.     

Receptor revision and atypical mutational characteristics in clonally expanded B cells from the cerebrospinal fluid of recently diagnosed multiple sclerosis patients

PURPOSE: To determine whether cerebrospinal fluid (CSF) B cells exhibit clonal expansion in patients recently diagnosed with multiple sclerosis (MS). CSF B cell clonal expansion was detected early in the disease process. Evidence of receptor revision was present in at least one MS patient who had been recently diagnosed with MS. Targeting of mutations to RGYW/WRCY motifs within CDRs was nominally observed in the CSF B cell clones despite the high mutational frequencies (MF). These observations are consistent with the presence of intense specific B cell stimulation and expansion in the CNS of MS patients early in the disease process.     

The same TCR (N)Dbeta(N)Jbeta junctional region is associated with several different vbeta13 subtypes in a multiple sclerosis patient at the onset of the disease

In multiple sclerosis (MS), the T-cell receptors (TCRS) of autoreactive T lymphocytes recognize various myelin components or derivatives including peptides of the myelin basic protein (MBP). Using the exhaustive immunoscope approach we showed that the T-cell repertoires of MS patients differ from those of healthy controls, with expansion of Vβ13 cell clones in cerebrospinal fluid (CSF) and in peripheral blood lymphocytes (PBLs). Sequencing of the β13⁺ chains of T cells recovered from the CSF revealed high interindividual diversity, and no particular Vβ13⁺ rearrangements were shown to be myelin-autoreactive. Within the overall Vβ13 repertoire in the CSF of patient MS3 at the onset of the disease, most of the overrepresented (N)Dβ(N)Jβ junctional regions were found to be associated with two or three different Vβ13 segments. These rearrangements were most common in the PBLs of patient MS3. No such associations were detected in the Vβ5 multigene family that was used as a control. Thus, Vβ13 T cells infiltrating the CSF from patient MS3 may have been selected on the basis of both the Vβ13 segments and the (N)Dβ(N)Jβ junctional CDR3 sequence.     

Immune surveillance in multiple sclerosis patients treated with natalizumab

OBJECTIVE: Our objective was to test whether natalizumab, an antibody against very late activating antigen (VLA)-4, interferes with central nervous system immune surveillance as assessed by leukocyte cell numbers and cellular phenotypes in cerebrospinal fluid (CSF) and peripheral blood. METHODS: Cell numbers and cellular phenotypes in CSF and peripheral blood were analyzed in multiple sclerosis (MS) patients treated with natalizumab, untreated MS patients, and patients with other neurological disease (OND). JC virus DNA in the CSF and peripheral blood was quantified by kinetic polymerase chain reaction. RESULTS: CSF leukocyte counts, CD4(+) and CD8(+) T cells, CD19(+) B cells, and CD138(+) plasma cells were significantly lower in natalizumab-treated MS patients compared with OND patients and untreated MS patients. JC virus DNA was not detected in CSF or peripheral blood from natalizumab-treated patients. Six months after cessation of natalizumab therapy, low lymphocyte counts in the CSF persisted. The patient with the highest total leukocyte and CD4(+) and CD8(+)T-cell counts in the CSF experienced a clinical relapse. INTERPRETATION: These data suggest that natalizumab treatment results in a prolonged decrease of lymphocytes in the CSF and are consistent with the hypothesis that natalizumab impairs immune surveillance of the central nervous system.     

Evidence for a missed signal to the CD8+ cells in CSF of Multiple Sclerosis patients

Peripheral blood (PB) and cerebrospinal fluid (CSF) lymphocyte subpopulations, defined by various T-cell specific monoclonal antibodies and flow cytometry, were analysed in 44 relapsing remitting multiple sclerosis (RRMS) patients (including 21 subjects in the acute phase and 23 in the stable phase), 40 chronic-progressive multiple sclerosis (CPMS) patients, and 24 patients with other neurological diseases (OND), in order to verify the presence of any abnormality in the lymphocyte subset pattern. A significant increase in the total number of T-lymphocytes and the CD4+ subpopulation was found in the PB of the MS patients in comparison with the OND group. Moreover, a not statistically significant increase in CD4⁺ cells was observed in the CSF of MS patients. A statistically significant increase was also found in the CD4⁺ Leu 8⁺ (suppressor inducer) cells in the CSF of all of the MS groups. Finally, the CD8⁺ (suppressor/cytotoxic) cell levels, were significantly lower in the CSF of CPMS and stable RMS patients than in the CSF of the OND patients. As a whole, our data suggest that the immunosuppressive deficit that seems to be a constant finding in MS is not due to a decrease in suppressor inducer cell levels, as previously suggested, but may be caused by a missed or altered signal from the suppressor inducer to CD8⁺ suppressor cells.This Work was partially supported by an IRCCS Current Research Grant 1994.     

Evidence for a missed signal to the CD8+ cells in CSF of Multiple Sclerosis patients

Peripheral blood (PB) and cerebrospinal fluid (CSF) lymphocyte subpopulations, defined by various T-cell specific monoclonal antibodies and flow cytometry, were analysed in 44 relapsing remitting multiple sclerosis (RRMS) patients (including 21 subjects in the acute phase and 23 in the stable phase), 40 chronic-progressive multiple sclerosis (CPMS) patients, and 24 patients with other neurological diseases (OND), in order to verify the presence of any abnormality in the lymphocyte subset pattern. A significant increase in the total number of T-lymphocytes and the CD4+ subpopulation was found in the PB of the MS patients in comparison with the OND group. Moreover, a not statistically significant increase in CD4⁺ cells was observed in the CSF of MS patients. A statistically significant increase was also found in the CD4⁺ Leu 8⁺ (suppressor inducer) cells in the CSF of all of the MS groups. Finally, the CD8⁺ (suppressor/cytotoxic) cell levels, were significantly lower in the CSF of CPMS and stable RMS patients than in the CSF of the OND patients. As a whole, our data suggest that the immunosuppressive deficit that seems to be a constant finding in MS is not due to a decrease in suppressor inducer cell levels, as previously suggested, but may be caused by a missed or altered signal from the suppressor inducer to CD8⁺ suppressor cells.     

Intrathecal immunotherapy in CNS tumors disseminating via CSF: preliminary evaluation using different treatment schedules

Eight patients affected by central nervous system tumours disseminating via cerebrospinal fluid received rIL-2 immunotherapy according to three different protocols involving intrathecal administration followed or not by systemic infusion. Immunological monitoring included serial evaluation of CSF leukocytes, CSF and peripheral blood CD3^– CD56⁺ cells, and NK activity. The most marked rise in CSF leukocyte levels was induced by daily intrathecal rIL2 administration, which also induced increased PB NK activity. Systemic rIL2 infusion following intrathecal treatment maintained a high percentage of CSF CD3^– CD56⁺ cells, but not CSF leukocytes at high levels. Clinical conditions improved after treatment in two patients, worsened in one and remained substantially unchanged in the remaining five. The side effects of intrathecal rIL2 treatment included fever, confusion, and seizures, and there were marked interindividual variations in the immunological response.

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Sommario 8 pazienti con tumore del sistema nervoso centrale con disseminazioni via liquido cefalo-rachidiano, sono stati sottoposti ad immunoterapia con rIL-2 secondo tre diversi schemi di trattamento che contemplavano la somministrazione intratecale seguita o no da somministrazione per via sistemica. Sono stati valutati alcuni parametri immunologici tra cui: citometria liquorale, percentuale di cellule CD3^– CD56⁺ ed attività Natural Killer (NK) sia nel liquor che nel sangue. Nonostante vi fosse una marcata variabilità interindividuale nella risposta immunologica al trattamento, il protocollo che procurava il più marcato aumento della citometria liquorale comprendeva la somministrazione intratecale giornaliera di rIL-2. Il trattamento con rIL-2 per via intratecale — a prescindere dallo schema di somministrazione — induceva un aumento dell'attività NK nel sangue. La somministrazione di rIL-2 per via sistemica, in seguito al trattamento intratecale, era in grado di mantenere una elevata percentuale di cellule CD3^– CD56⁺ nel liquor, ma non di mantenere l'elevata citometria. Le condizioni cliniche erano, alla fine del trattamento, migliorate in due pazienti, peggiorate in uno e sostanzialmente invariate negli altri. Gli effetti collaterali del trattamento erano principalmente febbre, stato confusionale e convulsioni.

     

Psychological disturbance differentially alters CD4 + and CD8 + leukocytes in the blood and intrathecal compartments

To evaluate cellular changes in the intrathecal compartment in response to psychological stress, cerebrospinal fluid (CSF) and peripheral blood (PB) samples were obtained from rhesus monkeys under baseline and challenge conditions. Juvenile monkeys separated from their social companions overnight had elevated cortisol, increased polymorphonuclear (PMN), and fewer CD4⁺ and CD8⁺ leukocytes in PB. In contrast, in CSF there were more CD4⁺ and fewer CD8⁺ leukocytes, raising the CD4/CD8 ratio. Dexamethasone given intramuscularly caused similar hematological changes: i.e. neutrophilia and lymphocytopenia with fewer CD4⁺ and CD8⁺ leukocytes in PB. However, it did not induce similar changes in CSF, indicating that the stress-related shift of CD4⁺ leukocytes in the intrathecal compartment involves physiological processes beyond adrenocortical steroids.     

Effects of Fingolimod and Natalizumab on Brain T1-/T2-Weighted and Magnetization Transfer Ratios: a 2-Year Study

Fingolimod and natalizumab significantly reduce disease activity in relapsing-remitting multiple sclerosis (RRMS) and could promote tissue repair and neuroprotection. The ratio between conventional T1- and T2-weighted sequences (T1w/T2w-ratio) and magnetization transfer ratio (MTR) allow to quantify brain microstructural tissue abnormalities. Here, we compared fingolimod and natalizumab effects on brain T1w/T2w-ratio and MTR in RRMS over 2 years of treatment. RRMS patients starting fingolimod (n = 25) or natalizumab (n = 30) underwent 3T brain MRI scans at baseline (T0), month 6 (M6), month 12 (M12), and month 24 (M24). White matter (WM) lesions, normal-appearing (NA) WM, and gray matter (GM) T1w/T2w-ratio and MTR were estimated and compared between groups using linear mixed models. No baseline demographic, clinical, and MRI difference was found between groups. In natalizumab patients, lesion T1w/T2w-ratio and MTR significantly increased at M6 vs. T0 (p ≤ 0.035) and decreased at subsequent timepoints (p ≤ 0.037). In fingolimod patients, lesion T1w/T2w-ratio increased at M12 vs. T0 (p = 0.010), while MTR gradually increased at subsequent timepoints vs. T0 (p ≤ 0.027). Natalizumab stabilized NAWM and GM T1w/T2w-ratio and MTR. In fingolimod patients, NAWM T1w/T2w-ratio and MTR significantly increased at M24 vs. M12 (p ≤ 0.001). A significant GM T1w/T2w-ratio decrease at M6 vs. T0 (p = 0.014) and increase at M24 vs. M6 (p = 0.008) occurred, whereas GM MTR was significantly higher at M24 vs. previous timepoints (p ≤ 0.017) with significant between-group differences (p ≤ 0.034). Natalizumab may promote an early recovery of lesional damage and prevent microstructural damage accumulation in NAWM and GM during the first 2 years of treatment. Fingolimod enhances tissue damage recovery being visible after 6 months in lesions and after 2 years in NAWM and GM.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00997-1.Key Words: Multiple sclerosis, MRI, disease-modifying drugs, magnetization transfer ratio, T1w/T2w-ratio, neuroprotection, repair     

Surface markers on lymphocytes from human cerebrospinal fluid : Identification by monoclonal antibodies

Lympocyte subpopulations in cerebrospinal fluid (CSF) and peripheral blood (PB) were studied using monoclonal antibodies and the common membrane markers. The results in three groups of patients were compared: 36 subjects with ‘non-immunological disorders’ (NID), 14 subjects with multiple sclerosis (MS) and 6 with subacute sclerosing panencephalitis (SSPE). It was found that, in patients with NID, (1) 90% of cells were T lymphocytes, reactive with OKT3; (2) the helper/suppressor (T4/T8) ratios were the same in the CSF and the PB; (3) the OKIa1 percentage was lower in the CSF than in the PB; and (4) only a few cells were ‘immature’, reacting with OKT10. Using the membrane markers (E rosettes, Fc IgG receptors and surface immunoglobulins), on the other hand, it was noted that the majority of cells in the CSF were identified as suppressor T lymphocytes and surface immunoglobulin-positive B cells were less common than the Ia1 marker suggested.There were no significant differences between the CSF results in patients with NID and MS but the OKT3 lymphocytes were reduced in CSF samples from patients with SSPE.     

JCV detection in multiple sclerosis patients treated with natalizumab

Natalizumab therapy is associated with an increased risk of progressive multifocal leukoencephalopathy (PML). Because the prognosis of established PML is uniformly dismal, identification of highly susceptible patients to the disease may improve outcomes. We wanted to investigate whether serial plasma and cerebrospinal fluid (CSF) screening for polyomavirus would identify patients with laboratory evidence of viral infection prior to the development of clinical PML. Two hundred MS patients had pre-treatment CSF/plasma screening for JC virus (JCV) and BK virus (BKV) DNA, and thereafter every six treatments of natalizumab. In all positive patients treatment is stopped (due to potential risk of PML), they have follow-up clinical examinations and plasma/CSF JCV/BKV tests until all evaluations are normal. No patient developed clinical evidence of PML. Eight of the 200 patients had detectable JCV or BKV DNA. Five patients were positive for BKV DNA in the CSF and three patients were positive for JCV DNA (one in plasma, two in CSF). After cessation of natalizumab treatment, all patients converted to undetectable viral DNA. Screening for JCV in CSF in natalizumab-treated patients could help identify those at heightened risk for developing PML and discontinuing treatment in these patients may abort development of the clinical illness.     

MS and clinically isolated syndromes: Shared specificity but diverging clonal patterns of virus-specific IgG antibodies produced in vivo and by CSF B cells in vitro

Background:  Intrathecal synthesis of oligoclonal IgG antibodies against measles virus (MeV), varicella zoster virus (VZV) and herpes simplex virus type-1 (HSV-1) is a characteristic feature multiple sclerosis (MS).
Methods:  We have used isoelectric focusing-immunoblot to define the clonal patterns of IgG and of IgG antibodies to MeV, VZV and HSV-1 in supernatants of in vitro cultures of peripheral blood lymphocytes (PBL) and cerebrospinal fluid (CSF) cells and in sera and CSF from three patients with MS and three patients with clinically isolated syndromes (CIS) suspective of demyelinating disease.
Results:  In vitro synthesis of IgG by PBL was not detected in any patient. In contrast , in vitro synthesis by CSF cells of oligoclonal IgG and oligoclonal IgG antibodies to one or two of the three viruses tested was observed in all six patients. The clonal patterns of the in vitro synthesized IgG and virus specific IgG differed to varying extent from those synthesized intrathecally in vivo . However, in each patient, the in vitro and in vivo intrathecally produced antibodies displayed specificity for the same viruses. The addition of B cell activating factor (BAFF) had no effect on the amounts or clonal patterns of either total IgG or virus-specific IgG produced by CSF cells in vitro .
Conclusion:  Virus specific B cells capable of spontaneous IgG synthesis are clonally expanded in the CSF of patients with MS. The B-cell repertoire in CSF samples is only partially representative of the intrathecal B-cell repertoire.     

Spontaneous proliferation of cerebrospinal fluid mononuclear cells in multiple sclerosis : A longitudinal study

Cerebrospinal fluid (CSF) and peripheral blood (PB) specimens from 32 MS patients longitudinally followed for up to 24 months and from a group of control patients without intrathecal inflammation were studied for the occurrence of activated lymphocytes with an autoradiography method. MS patients had higher numbers of proliferating mononuclear cells in CSF than did the controls, both during remission and exacerbation phases, whereas this difference was not found in the PB. ACTH treatment decreased the number of proliferating cells in CSF but had no effect on those of the PB of MS patients. A large variation in spontaneous proliferation of CSF cells was evident during the follow-up of individual patients, and there seemed to be no uniform correlation to the clinical fluctuations.     

Lymphocyte phenotype and subset distribution in normal cerebrospinal fluid

The distribution of lymphocyte subpopulations in cerebrospinal fluid (CSF) and their phenotypic characteristics were extensively investigated in a group of 18 healthy individuals using two- and three-color flow cytometry. Generally, CDS⁺ T lymphocytes constituted the vast majority of CSF lymphocytes while the number of B lymphocytes and NK cells were low. Most T lymphocytes exhibited the phenotype of memory /primed cells in both the CD4⁺ and CD8⁺ subpopulations. Two markers for recent activation, HLA-DR and interleukin-2 receptor (CD25) were not upregulated when compared with peripheral blood (PB) in the majority of CSF T lymphocytes. However, a fraction of T lymphocytes co-expressing the NK cell markers CD56 and/or CD16 showed a pronounced upregulation of HLA-DR in CSF as compared with PB. This study documents that the cellular composition of the normal CSF differs profoundly from PB regarding all major lymphocyte subpopulations. This has to be taken into account in studies addressing questions regarding cellular immune reactions in the central nervous system under pathological conditions.     

CD4+ lymphocyte subsets in the cerebrospinal fluid of multiple sclerosis and non-inflammatory neurological diseases

Summary We studied paired cerebrospinal fluid (CSF) and peripheral blood (PB) samples from 18 inactive multiple sclerosis (MS) patients and 10 with non-inflammatory neurological diseases. By means of a dual-colour cytofluorimetric micromethod we were able to count 1500 cells on average in each CSF sample. We found a significant reduction of CD45RA+ and CD4+CD45RA+ cells in the CSF of MS patients. Similarly, CD45RA+ and CD4+CD45RA+ CSF/PB ratios were lower compared with controls. The reduction of suppressor-inducer T-cells did not correlate with CD8+ cell levels in the CSF. The CD4+ subset ratio (CD4+CD45RA–/CD4+CD45RA+) was significantly increased in the CSF of MS patients. Our data suggest that the reduction of CD4+CD45RA+ cells in the PB is not due to a segregation of such cells in the CSF. Conversely, CSF changes reflect changes in the PB similar to these found for other T-cell subsets.