Cytokine Secretion by γδ and αβ T cells in Monophasic Experimental Autoimmune Encephalomyelitis

Article No. jaut.1998.0263, available online at http://www.idealibrary.com on

Journal of Autoimmunity (1999) 12, 73–80

Cytokine Secretion by ãä and áâ T cells in Monophasic Experimental Autoimmune Encephalomyelitis Mark A. Jensen, Amit Dayal and Barry G. W. Arnason Department of Neurology and the Brain Research Institute, University of Chicago, Chicago, IL, USA

Received 7 May 1998 Accepted 30 November 1998 Key words: cytokine, autoimmune encephalomyelitis, áâ lymphocyte, ãä lymphocyte

Mononuclear cells were isolated from the central nervous system (CNS), lymph nodes (LN), spleen and blood, over the course of murine monophasic experimental autoimmune encephalomyelitis (EAE). Individual cytokine secreting T cells were enumerated. IL-2-secreting áâ T cells were numerous at all sites at disease onset. By disease peak their numbers had fallen profoundly; they remained low thereafter. IL-2 secreting ãä T cells were rare throughout. IFN-ã-secreting cells were plentiful at all sites at disease onset. ãä T cells comprised 7% of total and 20% of IFN-ã-secreting CNS-derived cells at disease onset; values at disease peak were 12 and 40% respectively. IL-4-secreting áâ T cells were rare in the CNS and LN throughout and did not increase in the spleen from baseline values. In contrast, splenic IL-4-secreting ãä T cells had increased to four-fold baseline values at disease onset and seven-fold at disease peak. Recovery from EAE is associated with a global inhibition of IL-2-secreting áâ T cells and to a lesser extent with IFN-ã-secreting áâ and ãä T cells, whereas IL-4-secreting ãä T cells increase in the spleen as disease evolves. © 1999 Academic Press

ãä T cells are found in the CNS of patients with multiple sclerosis (MS) and in animals with EAE [17–24]. Although activated ãä T cells secrete a wide array of cytokines and differentiate along Th1- or Th2-type pathways similar to those observed for áâ T cells, their role in EAE remains unclear [25, 26], since administration of anti-ãä TCR antibody in vivo may either lessen or increase EAE severity in rodents [19, 27]. We have characterized the cytokines secreted by áâ and ãä T cells cells over the course of monophasic EAE in an attempt to delineate mechanisms that may contribute to the initiation and ending of the attack and to the post-attack tolerant state. MBP-specific Ig isotype concentrations in serum were characterized over the course of disease as an independent correlate of systemic Th1 and Th2 function in vivo. We find that a Th1-type response predominates for both áâ and ãä T cell subsets, both in the CNS and the periphery at the onset of disease. As disease evolves, a profound decline in Th1-type cytokine secretion occurs both in the CNS and in the peripheral lymphoid organs, and Th2-type ãä T cells accumulate in the spleen.

Introduction Experimental autoimmune encephalomyelitis (EAE) serves as an animal model for multiple sclerosis (MS). EAE may evolve as a monophasic, a relapsing– remitting, or as a chronic paralytic illness. Tissue destruction in EAE is orchestrated by Th1-type áâ T cells. Th2-type T cells can inhibit Th1-type T cells under many circumstances and accordingly a protective role for them in EAE has been proposed by many authors [1–13]. For example, forced deviation of the immune response within the lymphoid organs from a Th1- towards a Th2-type response suppresses EAE induction. On the other hand IL-4 secreting cells within the CNS remain few in number throughout the course of actively induced EAE [1, 2, 14]; so whether Th2-type T cells within the central nervous system (CNS) participate in ending an EAE attack remains uncertain. Th2-type T cells may even contribute to EAE under some circumstances. Purposeful induction of a Th2 response by intraperitoneal injection of myelin oligodendrocyte glycoprotein leads to a hyperacute form of EAE in marmosets [15] and adoptive transfer of neuroantigen reactive Th2-type cells to immune deficient mice induces a paralytic CNS disease with an eosinophilic infiltrate [16].

Materials and Methods Animal model

Correspondence to: Dr Mark A. Jensen, Department of Neurology MC2030, University of Chicago, 5812 S. Ellis Ave., Chicago, IL 60637, USA. Fax: 773–702–9076. E-mail: [email protected]

Six- to eight-week-old female PL/J mice (Jackson Laboratory, Bar Harbor, ME, USA) were housed in 73

0896–8411/99/020073+08 $30.00/0

© 1999 Academic Press

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laminar flow units in a high barrier virus free facility. Mice were anesthetized with 1.5 mg of pentobarbital sodium in saline (Abbott Laboratories, Chicago, IL, USA), and immunized at the tail base with 0.05 ml of adjuvant containing 0.020 mg MBP in 0.025 ml of saline emulsified with 0.020 mg Mycobacterium tuberculosis H37 RA (Difco Laboratories, Detroit, MI, USA) in 0.025 ml IFA. Saline (0.10 ml) containing 40 ng of pertussis toxin (LIST Biologicals, Campbell, CA, USA) was injected into the tail vein of all mice 12 h and 48 h after immunization [28]. Clinical disease was assessed as 0, intact; 1, flaccid tail, clumsiness; 2, hind limb paresis; 3, hind limb paraplegia; 4, moribund. Mice were killed at disease onset (score <2), at disease peak (score ≥2), or during recovery (score ≤2 with clinical improvement ≥1). Myelin basic protein (MBP) was prepared according to Diebler et al. using brains (Hilltop Lab Animals Inc., Scottsdale, PA, USA) from mice maintained in a virus-free colony [29].

Monoclonal antibodies (MAbs) Anti-CD3 mAb 145–2C11 (hereafter referred to as anti-CD3 mAb) was a kind gift of Dr J.A. Bluestone. Anti-IL2 mAb JES6-1A12, anti-IFN-ã mAb R4-6A2, anti-IL4 mAb BVD4-1D11, anti-áâ TCR mAb H57–597 (hereafter referred to as anti-áâ mAb), anti-ãä TCR mAb GL4 (hereafter referred to as anti-ãä mAb), anti-trinitrophenol (TNP) mAb UC8-4B3, anti-CD32/ CD16 mAb 2.4G2, FITC-conjugated anti-CD3 mAb, and biotinylated anti-IL2 mAb JES6-5H4, anti-IFN-ã mAb XMG1.2, and anti-IL4 mAb BVD6-24G2 were all purchased from Pharmingen Corporation (San Diego, CA, USA).

Mononuclear cell (MNC) isolation Mice were anesthetized, exsanguinated from the heart, and then perfused with normal saline. CNS tissue, spleens and inguinal lymph nodes (LN), were disrupted using a tissue homogenizer. Disrupted CNS tissue was diluted in 60 ml of PBS. Six aliquots (each 10 ml) of cell suspension were overlayed onto 10 ml of modified FD (sg =1.064 g/cm3) and spun at 250×g for 30 min. MNC traversed the FD layer to form a pellet. CNS MNC collected from the first FD separation and the spleen and LN cell suspensions (also blood) were overlayed onto 2 ml of ficoll-sodium diatrizoate (FD) (sg =1.089 g/cm3) and spun at 250×g for 15 min. MNC were collected from the interface and washed with PBS.

Blood cell counts White blood cells (WBC) of tail vein blood were counted with a hemacytometer. Differential counts (100 cells/mouse) were carried out on Giemsa-stained smears.

Immunofluorescence CNS (1×105 cells/stain), LN, spleen and blood (3× 105 cells/stain) MNC were incubated with anti-Fc receptor mAb for 15 min to block FcR; stained with FITC-conjugated mAbs to áâ TCR and ãä TCR for 30 min at 4°C; washed extensively with buffer (2% BSA plus 0.05% NaN3 in PBS), fixed with 1% paraformaldehyde, and mounted on slides with Bacto FA mounting fluid (Difco Laboratories). Percent T cells was determined from 100 cells counted using a Zeiss Orthomat fluorescent microscope (Wildleitz USA Inc., Rockleigh, NJ, USA). To determine background fluorescence, cells were stained with FITC-conjugated anti-TNP mAb.

Enzyme-linked immunospot (ELISPOT) plates The method used for detecting cytokine secreting cells has been described in detail elsewhere [30]. Briefly, microwell plates with nitrocellulose bottoms (Millipore Corporation, Bedford, MA, USA) were coated overnight with 50 ìl (6 ìg/ml) of cytokine capture mAb in bicarbonate buffer. Purified rat IgG (Sigma Corporation, St. Louis, MO, USA) was added to control wells to assess the number of non-specific spots (always <5%). Anti-áâ and anti-ãä mAb were also coated onto some wells to specifically activate áâ or ãä T cells. Wells were blocked with 2% BSA in PBS for >5 h.

MNC incubation Varying numbers of MNC plus 2×105 T cell-depleted irradiated splenocytes/well (to ensure comparable cell densities and as a source of APC) were added to ELISPOT wells prepared as described above and incubated for 24 h at 37°C, 7.5% CO2 in a humidified incubator. Preliminary studies showed <5% variation in frequency of cytokine producing cells in wells plated with 5×103 to 5×104 CNS MNC/well or with 5×103 to 1×105 LN, blood, or splenic MNC/well so that for these studies, 5–20×103 CNS MNC and 15–50×103 LN, spleen, or blood MNC were added to each well.

Counting cytokine-specific spots After incubation, plates were washed extensively with PBS and overlaid for 4 h with 50 ìl of anti-cytokine biotinylated mAb, at 3 ìg/ml in 2% BSA/PBS. Plates were again washed extensively with PBS, and overlaid with peroxidase-conjugated goat anti-biotin IgG (Vector Laboratories Inc., Burlingame, CA, USA) diluted 1:200 in 0.2% BSA/PBS for 2 h. Wells were again washed extensively with PBS. One hundred microlitres of 2-amino-9-ethylcarbazole (0.5 mg/ml) plus 0.01% H2O2 in 0.05 M sodium acetate buffer was next added to each well for 30 min to visualize spots marking the sites of individual cytokine-secreting

ãä T cell responses in EAE

cells. Plates were then washed with tap water, and dried. Spots were counted at ×50 magnification using a dissecting microscope. Total cytokine secreting cell number/well was determined by subtracting the number of spots detected in wells coated with irrelevant Ab (always <5% of responding cells) from wells coated with anti-cytokine capture mAb. Cytokine-secreting cells/106 cells were calculated as: (relevant spots per well/number of cells plated)×10. T cell-depleted splenocytes did not secrete cytokines when plated in medium alone or in the presence of any of the stimuli used.

Proliferative responses CNS, LN, spleen and blood-derived MNC were suspended at 5×105 cells/ml in culture medium [30] and tested for proliferative responses to anti-CD3 mAb 145 2C11, or to medium alone. MNC (0.1 ml; 5×104 cells) were added to each well of a 96-well flat bottom microculture plate. Irradiated Thy1-depleted syngeneic splenocytes (0.08 ml; 2.5×105), prepared using rabbit antisera to Thy1, and low-tox M rabbit complement according to the manufacturers directions (Accurate Chemical & Scientific Corp., Westbury, NY, USA), were added to each well as a source of antigen presenting cells (APC). Medium (0.02 ml) containing anti-CD3 mAb (10 ìg/ml), or medium alone was added to each well. Plates were incubated at 37°C in 5% CO2 under humidified conditions for 48 h. Each well was then pulsed with 1 ìCi of 3H-methylthymidine (Amersham Corporation, Arlington Heights, IL, USA) for 15 h. Cells were harvested onto glass fiber filter paper (Cambridge Technology Inc., Watertown, MA, USA) using a PhD cell harvester; radioactivity was determined by liquid scintillation spectroscopy (Model LS 5000TD; Beckman Instruments Inc., Irvine, CA, USA).

ELISA for quantitation of relative concentrations of serum anti-MBP IgG1 and IgG2a To detect serum MBP-specific IgG1 and IgG2a ELISA plates (Gibco BRL) were overlaid with 50 ìl of murine MBP (50 ìg/ml) in carbonate buffer (pH 9.0). Plates were incubated overnight, washed with 0.05% Tween20/DPBS, blocked with 2% BSA/DPBS for 4 h, and then washed with 0.05% Tween 20/DPBS. One hundred microlitre aliquots of mouse serum (diluted 1:5 in 0.2% BSA) were added to quadruplicate wells. Half the wells received PBS containing 0.2% BSA to permit measurement of background signal. As a positive control, rabbit polyclonal Ab against bovine MBP (a generous gift from Dr G. E. Diebler) was added at a dilution of 1:300. Plates were incubated in humid conditions at 4°C overnight and then washed with 0.05% Tween 20/DPBS biotinylated rat anti-mouse IgG1 mAb G1-7.3 (50 ìg/ml) or rat anti-mouse IgG2a mAb R19–15 (6 ìg/ml in 0.2% BSA; Pharmingen, San Diego, CA, USA) was added to each well. Wells containing rabbit anti-MBP, as well as duplicate

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blanks, received 50 ìl or 8 ìg/ml polyclonal peroxidase conjugated goat anti-serum to rabbit IgG (Caltag) diluted in 0.2% BSA in PBS and 0.25% normal goat serum. Plates were incubated at room temperature for 3 h and washed 10 times with PBS. Wells were developed using an avidin-peroxidase kit (Vector Laboratories Inc., Burlingame, CA, USA). Orthophenylenediamine (4 mg/ml) in citrate buffer was added to each well. Plates were read on a ThermoMax Microplate Reader (Molecular Devices Corp., Menlo Park, CA, USA) with a test wavelength of 450 nm and a reference one of 650 nm. Data were analysed using the program Softmax.

Statistical analysis Student’s unpaired two-tailed t-test was used for data analysis.

Results Clinical disease Clinical disease began 13–21 days post-immunization and followed a consistent self-limited course over 5–7 days. MNC were isolated for study at onset of disease, peak disease and during recovery. MNC were also isolated from unimmunized (UI) mice.

Quantitative and phenotypic T cell analysis according to disease phase CNS We have previously shown that the number of T cells recoverable from the CNS increases with disease severity [30]. To evaluate the effect of disease phase on T cell subset representation, the distribution of áâ and ãä T cells was determined over the course of EAE (Figure 1). áâ T cells doubled in number from disease onset to disease peak. Numbers declined three-fold from peak values during recovery. ãä T cells were 13-fold (onset), eight-fold (peak), and nine-fold (recovery) fewer than áâ T cells. The greatest number of ãä T cells was recovered at disease peak.

LN At disease onset áâ T cell numbers were double the values obtained in UI mice (Figure 1). They then fell so as to approximate values in UI mice at disease peak, and fell further during recovery. áâ T cells vastly outnumbered ãä T cells, but even so, ãä T cells were increased three-fold at disease onset compared to UI controls. ãä T cell numbers in LN decreased steadily thereafter.

Spleen áâ T cell numbers were lower at disease onset than in UI mice and their number declined further as disease

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Blood Circulating áâ T cell numbers were two-fold increased at disease onset compared to UI controls, but then fell to values below baseline at peak disease and during recovery (Figure 1). No change was observed in the number of circulating ãä T cells at any time.

Proliferative responses At disease onset CNS-derived T cells proliferated sixto seven-fold less briskly than LN, blood, and spleenderived T cells (Figure 2). The finding suggests that T cells refractory to proliferative signaling enter the CNS selectively or become refractory soon after entering the CNS. CNS-T cells isolated at peak disease were totally unresponsive to proliferative triggering through their TCR. T cells isolated from the LN, blood and spleen proliferated far less in response to antiCD3 mAb at disease peak than T cells isolated from these compartments at disease onset. Thus, a total inhibition in proliferative responsiveness of T cells

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progressed (Figure 1). ãä T cells were 10-fold fewer than áâ T cells at all phases of disease, with a decrease in number as disease evolved which paralleled that observed for áâ T cells.

H-Thymidine incorporation (cpm)

Figure 1. Mean number ± SEM of áâ ( ) and ãä T cells ( ) recovered from CNS, LN, spleen and blood of unimmunized mice (UI), and of mice with EAE at disease onset, peak disease and during recovery. For LN the number of MNC isolated from both inguinal lymph nodes is given. n=6–12 mice for each data point.

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Figure 2. Proliferative responses at disease onset and disease peak to anti-CD3 mAb stimulation in MNC derived from the CNS, LN, spleen and blood. MNC (5×104) were cultured with irradiated (1500 rads) T cell-depleted splenic MNC in medium alone or in the presence of soluble anti-CD3 mAb (1 ìg/ml). Thymidine incorporation was measured during the last 15 h of a 62-h culture (medium alone at onset ( ); at peak ( ); in presence of anti-CD3 mAb at onset ( ; at peak ( )). Results are expressed as mean cpm ±SEM of three different experiments.

isolated from the CNS is observed at peak disease and a profound inhibition in T cells obtained from other sites is also observed at this time.

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Figure 3. Mean number of cells ± SEM/10 cells secreting IL-2 (A–D), IFN-ã (E–H), or IL-4 (I–L). MNC derived from the CNS, LN, spleen, and blood were activated with anti-áâ or anti-ãä TCR mAb. Unimmunized mice (UI) were also studied. The mean number of cells secreting IL-2/106 cells in medium alone was: ≤ 1 for CNS; ≤ 3 for LN, ≤ 33 for blood and ≤ 13 for spleen. The mean number of cells secreting IFN-ã/106 cells in medium alone was: ≤ 240 for CNS; ≤ 183 for LN, ≤ 550 for blood, and ≤ 209 for spleen. The mean number of cells secreting IL-4/106 cells in medium alone was: ≤ 306 for CNS; ≤ 33 for LN, ≤ 1592 for blood, and ≤ 781 for spleen. n=3–7 for each.

Cytokine secretion profiles of áâ and ãä T cells over the course of disease IL-2-secreting cells CNS. Cells secreting IL-2 upon activation with anti-áâ TCR mAb were numerous at disease onset but had fallen four-fold by the time of disease peak and remained low throughout recovery (P<0.01 for onset vs. peak or recovery; Figure 3). IL-2-secreting ãä T cells were scarce in CNS-derived MNC at all phases of disease. Periphery. IL-2 secreting áâ T cells were increased at disease onset in LN, blood, and spleen compared to UI controls, but their numbers had decreased markedly by time of peak disease and remained low throughout

recovery (Figure 3; for onset vs. peak plus recovery P=0.08 for LN; P<0.04 for blood; P<0.002 for spleen). IL-2 secreting ãä T cells were barely detectable in LN, blood and spleen-derived MNC of UI mice but were found in modest numbers in LN, blood and spleen-derived MNC over the course of disease.

IFN-ã-secreting cells CNS. Large numbers of cells that secreted IFN-ã in response to either anti-áâ or anti-ãä TCR mAbs were found at disease onset (Figure 3). IFN-ã-secreting áâ T cell and IFN-ã-secreting ãä T cell numbers both declined as disease evolved (P<0.02 for onset vs. peak and recovery for áâ T cells). Note that the ãä T cell contribution to the total IFN-ã-secreting cell pool

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Periphery. IFN-ã-secreting áâ and ãä T cells were markedly increased in LN, spleen and blood at disease onset over values obtained for UI mice. IFN-ã secreting cell numbers in LN, spleen, and blood declined as disease evolved (Figure 3; for áâ T cells at onset vs. peak plus recovery P=0.3 for LN; P<0.02 for blood; P<0.004 for spleen). Note again the inordinate contribution of ãä T cells to the total IFN-ã-secreting cell pool as compared to numbers of ãä T cells retrievable from the various organs. IL-4-secreting cells CNS. Only low numbers of cells that secreted IL-4 in response to either anti-áâ TCR or anti-ãä TCR mAbs were found at all phases of disease (Figure 3). At disease onset IFN-ã-secreting áâ T cells outnumbered IL-4-secreting áâ T cells by 30:1; at disease peak by 12:1; and during recovery by 8:1 (Figure 3). For ãä T cells the IFN-ã:IL-4-secreting cell ratios were 15:1 at disease onset; 5:1 at disease peak; and 5:1 during recovery. Periphery. IL-4-secreting áâ T cells were present in very low numbers in LN at all phases of disease (Figure 3). Numbers of IL-4-secreting áâ T cells in spleen and blood did not change appreciably over the course of disease. Increases in IL-4-secreting ãä T cells were noted in the spleen and blood at all phases of disease. Again the contribution of ãä T cells to the IL-4-secreting cell pools in the blood and spleen at all post-immunization time points far exceeded their numerical contribution to the total T cell pool. Effect of disease progression on relative titers of IgG1 and IgG2a anti-MBP antibody Immune deviation from a Th1 response to a Th2 response is often accompanied by a change in relative serum concentrations of IgG1 (Th2 cell-driven) and IgG2a (Th1 cell-driven). Sera collected at disease onset and disease peak contained a five-fold shift in favour of anti-MBP IgG2a relative to anti-MBP IgG1 (Figure 4). This anti-MBP IgG2a bias was much reduced during recovery (P<0.05 for recovery vs. onset or peak). Thus a shift from an anti-MBP IgG2a response towards an anti-MBP IgG1 response occurred as disease progressed to recovery.

Discussion We have characterized áâ and ãä T cell cytokine responses in the CNS and the peripheral lymphoid organs of mice with EAE. ãä T cells represent only a small percentage of the total T cell population in all compartments analysed, but they comprise a large percentage of the cytokine-secreting T cell population.

0.7 Ratio MBP reactive IgG1/IgG2a

(between 20 and 40%) was substantially higher than the contribution of ãä T cells to the total T cell pool (7–12%) (Figure 3).

0.6 0.5 0.4 0.3 0.2 0.1 0

Onset

Peak

Recovery

Figure 4. Effect of disease progression on relative titers of anti-MBP IgG1 and IgG2a. Data are expressed as the mean ratio±SEM of the optical density obtained for anti-MBP IgG1 to the optical density obtained for anti-MBP IgG2a. The ratio at disease onset and at peak disease is significantly lower than at recovery (P<0.05). n=5–6 mice for each data point.

In addition, the cytokine-secreting profile of ãä T cells differs from that of áâ T cells, suggesting distinct functions for the two T cell populations. Encephalitogenic antigens are traditionally administered in mycobacterium containing adjuvant since this manœuvre facilitates disease induction. ãä T cells are activated promptly by mycobacterial components and their initial response is strongly skewed towards IFN-ã production [31]. This skew may contribute to disease induction by forcing áâ T cells to differentiate along the Th-1-type T cell path. We show here that IFN-ã-secreting ãä T cells are markedly enriched at disease onset in LN, spleen and blood, far out of proportion to their numerical representation; a finding consistent with this formulation. Also consistent is the observation that depletion of ãä T cells with anti-ãä Ab can reduce both the severity of EAE and the expression of IFN-ã mRNA in the CNS of mice with disease [32]. Recovery from disease in the model studied here is preceded by an abrupt inhibition of Th1-type cytokine secretion. Inhibition of IL-2 secretion is more pronounced than that of IFN-ã, both at disease peak and into recovery. Inhibition is observed at all sites sampled in a concerted fashion, arguing for some global mechanism that specifically downregulates Th1-type cytokine secretion. Proliferative responsiveness also falls. Glucocorticoid production surges at the peak of monophasic EAE and is crucial for recovery from disease [33]. Glucocorticoids suppress IL-2 production and inhibit T cell proliferation and they are probably responsible for the global inhibition of IL-2 secretion and the global loss of proliferative responses that we observed. CNS-restricted inhibition (e.g. by astrocytes) may well be superimposed, however, as loss of proliferative responsiveness in CNS-derived cells antedates that observed at other sites and at later times loss of responsiveness is total within the CNS yet partial at other sites.

ãä T cell responses in EAE

One proposed mechanism for inhibition of Th1-type T cell function within the CNS is entry into the CNS of activated Th2-type T cells. Cytokines released by Th2type T cells, including IL-4 and IL-10, can inhibit Th1-type T cells. Purposeful deviation of the immune response towards a Th2-type during EAE, which can be achieved by administering Th2-type cytokines, suppresses disease activity, though whether the Th2-type cytokines act peripherally, within the CNS, or at both sites, remains unresolved [2]. That some effect could be exerted within the CNS is supported by the observation that neuroantigen reactive Th2-type T cells inhibit EAE [34, 35]. Yet, we find few IL-4-secreting cells within the CNS at any time point, suggesting no obligate role for Th2 cells within the CNS as abrogators of ongoing EAE. It is also known that IL-4 knockout mice recover from EAE with kinetics similar to those of their normal littermates [36]. Furthermore, although IL-4 inhibits IFN-ã synthesis, it is less certain that it directly inhibits IL-2 production, as was observed here. It follows that inhibition of IL-2 and IFN-ã production within the CNS during monophasic EAE involves mechanisms beyond activation of Th2 cells within the CNS. A profound decline in IL-2 secretion was seen surprisingly early in the course of disease. Perhaps failed IL-2 production is required for recovery from EAE. If so, purposeful inhibition of IL-2 might be considered as therapy for EAE, and by extension, for MS. IFN-ã secreting cell number also decreased as disease evolved but IFN-ã-secreting cells remained retrievable from the CNS even during the recovery phase, suggesting that ongoing IFN-ã synthesis within the CNS, including IFN-ã synthesis by ãä T cells, may contribute to recovery. This postulate finds some support in the observation that IFN-ã knockout mice fail to recover from EAE as promptly as their normal littermates [37]. EAE in the B10PL mouse is monophasic and hence akin to EAE in the PL/J mouse studied here. Treatment of B10PL mice with anti-ãä T cell Ab GL3 depletes ãä T cells from the peripheral lymphoid organs for a prolonged period, delays the onset of EAE, and decreases its severity [19]. In contrast, treatment of mice with the anti-ãä T cell Ab UC7-13D5 aggravates disease severity, and EAE relapses, ordinarily rare in this strain, occur [27]. It is likely that Ab UC7-13D5 activates ãä T cells in vivo. Thus, the apparent discrepancy between the results obtained with the two Abs may be more apparent than real but, regardless, it is evident that ãä T cells play a modulating role in EAE and possibly more than one. Absolute numbers of IL-4-secreting áâ T cells change little in the spleen, LN, or blood over the course of disease. Thus, the global ‘off signal’ for both Th1-type áâ, and for Th1-like ãä T cells spares Th2type áâ T cells. Th2-like ãä T cells are similarly spared. Indeed, at disease onset the number of IL-4-secreting ãä T cells within the spleen is increased four-fold over values for spleens from unimmunized controls. This number is increased to seven-fold by disease peak, at which time IL-4-secreting ãä T cells constitute half the IL-4-secreting cell population of the spleen and

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account for the quasi-totality of the increment in IL-4-secreting cells above baseline. We observe that MBP-reactive IgG is skewed towards IgG2a (i.e. is Th1 cytokine-driven) both at disease onset and at disease peak but, as disease wanes, response shifts towards a Th2 cell-driven isotype. IL-4-secreting áâ T cell numbers did not change over the course of disease but IL-4-secreting ãä T cell numbers increased. ãä T cells do not respond to MBP so that, if IL-4 secreting ãä T cells influence anti-MBP IgG isotype, this would, perforce, be a bystander effect.

Acknowledgements This work was supported by PO1-NS24575 and a gift from the Butz Foundation.

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