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Controls Th1 Cells in Experimental Autoimmune Encephalomyelitis1
* Division of Molecular Immunology and
Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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(PKC) is critical for the development of Ag-specific Th1 cells in experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis. PKC-deficient mice immunized with myelin oligodendrocyte glycoprotein failed to develop cell infiltrates and Th1 cytokines in the CNS and were resistant to the development of clinical EAE. CD4 T cells became primed and accumulated in secondary lymphoid organs in the absence of PKC, but had severely diminished IFN-
, TNF, and IL-17 production. Increasing Ag exposure and inflammatory conditions failed to induce EAE in PKC-deficient mice, showing a profound defect in the myelin oligodendrocyte glycoprotein-reactive T cell population. These data provide evidence of a pivotal role for PKC in the generation and effector function of autoimmune Th1 cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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, and TNF-
, and these cytokines are present in the CNS of animals with active EAE or that of MS patients (5). Many efforts are presently directed toward inactivating or eliminating encephalitogenic CD4 T cells or inducing immune deviation away from the Th1 phenotype ((2, 6, 7). For example, blocking activation of CD4 T cells with Abs to CD4, MHC class II, costimulatory molecules, and adhesion molecules, can strongly inhibit the induction and progression of EAE (2, 6, 7). However, these approaches lack Ag specificity and might also result in generalized immune suppression (6). A more useful and clinically applicable approach would be to target molecules that alter the encephalitogenic potential of the autoreactive CD4+ T cell population while at the same time minimizing the effects on other immune responses.
T cell activation requires TCR interaction with MHC-peptide complexes in parallel with engagement of costimulatory molecules such as CD28. Emerging data have suggested that protein kinase C (PKC) may be central to TCR- and CD28-specific signals that lead to T cell activation, proliferation, and cytokine production. Considerable in vitro data have suggested that PKC is essential to many events involved in signaling T cells to become activated (8, 9). Based on these studies, it was widely assumed that T cell activation and differentiation are absolutely dependent on PKC. However, more recent in vivo data have challenged this idea. Using gene-deficient mice, CTL responses to lymphocytic choriomeningitis virus infection and CD4-dependent Ab responses to vesicular stomatitis virus infection were normal in the absence of PKC (10). However, strongly impaired CD8 responses and a form of CTL anergy were observed when soluble peptide Ag was administered in vivo, implying that the requirement for PKC could be dictated by either the subset of T cells responding or the inflammatory milieu that accompanies T cell priming. In support of this, two recent reports showed that PKC was crucially involved in several Th2-type responses, such as that to Nippostrongylus brasiliensis and during asthmatic lung inflammation, but it was dispensable for the protective Th1 response to Leishmania major infection and for Th1-directed lung inflammation (11, 12). This suggested a lesser role for PKC in Th1 immune responses.
In the present study we show, in contrast to previous reports of Th1-dominated responses, that without PKC, the peripheral T cell response to myelin oligodendrocyte glycoprotein (MOG), T cell entry into the CNS, and Th1 cytokine production were strongly impaired, resulting in minimal clinical signs of EAE. These results highlight further complexities in PKC biology and have implications for how MS is controlled.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The studies reported in this paper conform to the principles outlined by the Animal Welfare Act and the National Institutes of Health guidelines for the care and use of animals in biomedical research. PKC-deficient mice (PKC–/–; originally on a 129-C57BL/6J background) were a gift from Dr. D. Littman (New York University, School of Medicine, New York, NY) (13) and were backcrossed for more than five generations onto the C57BL/6J background. These mice have no defect in thymic selection and bear normal numbers of peripheral T cells. Wild-type (wt; PKC+/+) C57BL/6J littermates were used as controls and were bred as previously described (12).
Induction of EAE
MOG (35-MEVGWYRSPFSRVVHLY RNGK-55) was synthesized by A&A Laboratories. Six- to 9-wk-old female and male mice were immunized by s.c. injection at the base of the tail with 150 µg of MOG33–55 peptide in CFA. The mice received injections i.p. with 300 ng of pertussis toxin on days 0 and 2 after immunization. Where indicated, experimental animals received the exact same immunization again 7 or 40 days after the first immunization. Individual animals were scored daily for clinical signs of EAE using the following criteria: 0, no detectable signs of disease; 0.5, distal limp tail; 1, complete limp tail; 1.5, limp tail and hind limb weakness; 2, unilateral partial hind paralysis; 2.5, bilateral partial hind limb paralysis; 3, complete bilateral hind limb paralysis; 3.5, complete bilateral hind limb paralysis and unilateral forelimb; 4, total paralysis of fore and hind limbs; and 5, death. The average day of EAE onset was calculated by adding the first day of clinical signs for individual mice and dividing by the number of mice in the group. The average maximum disease score was calculated by averaging the highest clinical score for each mouse. For those animals that showed no clinical signs of disease, the onset was arbitrarily calculated as 1 d after the experiment was terminated.
Assay of T cell function ex vivo
Spleen and draining lymph nodes (LN; lumbar and inguinal LN) were collected and after lysing RBC with ACK lysis buffer, cells were resuspended in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FCS (Omega Scientific), 1% L-glutamine (Invitrogen Life Technologies), 100 µg/ml streptomycin, 100 U/ml penicillin, and 50 µM 2-ME (Sigma-Aldrich). Cells were plated at 2 x 105/well in round-bottom, 96-well microtiter plates in 200 µl with increasing concentrations of MOG33–55 peptide (0.1–200 µg/ml) for 72 h at 37°C. After 66 h, 1 µCi of [3H]thymidine (ICN Biomedicals) was added, and thymidine incorporation was measured using a Betaplate scintillation counter (Tomtec). Each in vitro stimulation assay was performed in triplicate. Supernatants were harvested after 72 h of in vitro restimulation for cytokine analysis.
Histological assessment for EAE
Mice were anesthetized with sodium pentobarbital and intracardially perfused through the left ventricle with ice-cold PBS. Spinal cords and brains from EAE-induced mice were dissected, fixed in 10% zinc-buffered formalin (Protocal; Fisher Diagnostics), and paraffin embedded, and sections (5 µm) were stained with H&E. To ensure comparable analyses between different groups, six to eight randomly selected sections were analyzed per animal.
Phenotypic and intracellular cytokine staining of T cells
After blocking of FcRs with excess anti-Fc (24.G2), cells were incubated with fluorescently conjugated Abs against CD4 (FITC or allophycocyanin), CD3 (PE), CD8 (FITC), OX40 (PE), CD25 (PE), and CD69 (PerCP). Flow cytometric measurement of cytokine production in T cells was performed after lysing RBCs and LN or splenocytes were plated in round-bottom, 96-well microtiter plates in 200 µl with MOG peptide (100 µg/ml) for 72 h at 37°C. GolgiPlug (BD Biosciences) was added, and the incubation was continued for 8 h. Washed cells were stained with anti-CD4, fixed with Cytofix/Cytoperm (BD Biosciences), and then stained with anti-IL-2 (FITC and PE), anti-IFN- (FITC and allophycocyanin), and anti-TNF- (FITC or allophycocyanin; all from BD Pharmingen). Samples were analyzed after gating on CD4+ T cells on a FACSCalibur flow cytometer using CellQuest (BD Biosciences) and FlowJo software (TreeStar).
RT-PCR
Spinal cords were extruded by flushing the vertebral canal with cold PBS. Quantitative RT-PCR was performed with SYBR Green I dye using an ABI GeneAmp 5700 Sequence BioDetector (PE Biosystems). Total RNA was extracted using TRIzol (Invitrogen Life Technologies) and reverse transcribed using oligo(dT)12–18 (SuperScript II; Invitrogen Life Technologies). Each transcript was analyzed concurrently on the same plate with hypoxanthine phosphoribosyltransferase (HPRT), and levels of mRNA were normalized to HPRT abundance. Sequence-specific primers for murine IFN- (forward primer, 5'-GGATGCATTCATGAGTATTGC-3'; reverse primer, 5'-CCTTTTCCGCTTCCTGAGG-3'), TNF- (forward primer, 5'-CATCTTCTC AAAATTCGAGTGACAA-3'; reverse primer, 5'-TGGGAGTAGACA AGGTACAACCC-3'), IL-17 (forward primer, 5'-GCAAGAGATCCGGTCCTGA-3'; reverse primer, 5'-AGCATCTTCTCGACCCTGAA-3'), CD3-
(forward primer, 5'-TCTCGGAAGTCGAGGACAGT-3'; reverse primer, 5'-TTGAGGCTGGTGTGTAGCAG-3'), CD4 (forward primer, 5'-TCAAGATACCCCAGGTCTCG-3'; reverse primer, 5'-CACCACCAGGTTCACTTCCT-3'), CD8– (forward primer, 5'-ACTGCAAGGAAGCAAGTGGT-3'; reverse primer, 5'-CACCGCTAAAGGCAGTTCTC-3'), F4/80 (forward primer, 5'-GCTGTGAGATTGTGGAAGCA-3'; reverse primer, 5'-AGTTTGCCATCCGGTTACAG-3'), and HPRT (forward primer, 5'-CTGGTGAAAAGGACCTCTCG-3'; reverse primer, 5'-TGAAGTACTCATTATAGTCAAGGGCA-3') were used.
Statistics
Statistical significance was analyzed by Student’s t test. Unless otherwise indicated, data represent the mean ± SD, with p < 0.05 considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-deficient mice are resistant to EAE
To examine whether PKC is involved in the development of autoimmune Th1 responses in vivo, groups of wt (PKC+/+) and PKC–/– mice were immunized with MOG peptide in CFA plus pertussis toxin. The wt mice developed reproducible acute EAE (Fig. 1 and Table I). On the average, clinical signs of disease were evident on day 12 and peaked between days 14 and 18. The acute phase was followed by remission that took place between days 25 and 40. In contrast, PKC–/– mice displayed minimal clinical signs of disease (Fig. 1 and Table I). The disease incidence (52 vs 94%), day of onset (19.83 vs 11.76), and mean maximal disease score (0.91 vs 2.71) were all significantly lower in mice lacking PKC (Table I). These results provide clear evidence that PKC is required for the initiation of clinical EAE.
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-deficient mice show minimal CNS inflammation and Th1 cytokines after EAE induction
Immunization with MOG leads to the generation of encephalitogenic Th1 cells in the local draining LNs, followed by migration of T cells into the CNS. Ag-specific Th1 cells produce cytokines, chemokines, and proinflammatory mediators that initiate inflammation and demyelination, ultimately leading to EAE. To initially try to understand why PKC–/– mice were resistant to EAE, spinal cords and brains were examined (Fig. 2). Before the onset of disease (day 9), histological examination of either wt or PKC–/– tissue revealed no visible signs of inflammatory lesions or cell infiltration (data not shown). Between day 9 and the peak of the disease on day 14, the numbers of inflammatory cells and lesions increased in all wt mice that had clinical signs of EAE. In striking contrast and consistent with the lack of physical signs of EAE, the numbers and sizes of the inflammatory lesions were dramatically reduced in PKC–/– mice (Fig. 2a).
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resulted in fewer leukocytes in the CNS, we examined bulk infiltration at the preclinical (day 9) and acute (day 14) phases of the disease. At each time point, samples from four different spinal cords (Fig. 2b) and brains (data not shown) were subjected to real-time PCR using specific oligonucleotide primers for CD3, CD4, CD8, and F4/80+. All amplifications were calibrated to HRPT, which enabled quantitative analysis of the dynamics of mRNA transcription of CD3, CD4, CD8, and F480+ at the site of inflammation. Fig. 2b shows a representative result from each time point of the experiment. Consistent with the histological data, CD3, CD4, CD8, and F4/80+ mRNA transcript levels were scarcely detectable during the preclinical phase of the disease on day 9, but increased significantly in the spinal cords isolated from wt mice at the acute phase (day 14) of EAE (Fig. 2b). This increase in the transcription of CD3, CD4, CD8, and F4/80+ mRNA correlated well with the rapid onset of clinical disease (compare Fig. 2b and Fig. 1). In sharp contrast, the spinal cords from PKC–/– mice had significantly lower CD3, CD4, CD8, and F4/80+ mRNA transcript levels at the peak of the disease (Fig. 2b). Again, this analysis correlated well with the general lack of inflammation seen in H&E-stained spinal cord tissue sections (Fig. 2a). Similar results were obtained when brains were isolated from each experimental group (data not shown).
Proinflammatory cytokines derived from Th1 cells and macrophages in the CNS play an important role in the pathogenesis of MS and the development of EAE. Consistent with lesser CNS inflammation, mRNA for all the major cytokines implicated in EAE, namely, IL-2, TNF, IFN-, and IL-17, was significantly reduced in the spinal cords (Fig. 2c) and brains (data not shown) of PKC–/– mice compared with wt littermates. These results indicated that PKC affected the amount of mononuclear cell infiltration and Th1 cytokine production in the CNS throughout the course of EAE, implying that this was responsible for the limited disease in PKC–/– mice.
Normal, but delayed, accumulation of activated CD4 T cells in the absence of PKC
To quantify the extent of T cell priming after EAE induction, we performed kinetic evaluation of the number and activation state of CD4 T cells in the draining LN of wt and PKC–/– animals. In the absence of a specific method to track MOG-reactive T cells, cell surface expression of CD44 and OX40, molecules associated with T cell priming and activation, were assessed. The percentages of CD3+CD4+ T cells in the draining LN of PKC–/– mice were comparable to those in wt mice at all time points examined (Fig. 3, a, b, and c, top panel). However, when converted into total CD4 T cell numbers, significantly fewer CD4 T cells were detected in the LN of PKC–/– mice on day 3 after immunization, although comparable numbers were detected on days 8, 14 (Fig. 3, b and c), and 25 (data not shown). Consistent with this, analysis of CD44high-expressing CD4 T cells as marker of activation revealed significantly greater numbers in wt mice on day 3, but not at later time points from day 8 onward. OX40 is selectively expressed on encephalitogenic or Ag-experienced CD4 T cells (16, 17). Analysis of OX40-expressing cells within the CD4+CD44high population also showed a significant reduction in both percentages and absolute numbers induced in PKC–/– mice during the first 3 days after Ag priming, but not thereafter. Comparable results were obtained when CD25- or CD69-expressing cells were analyzed within the CD4+CD44high population (data not shown). Collectively, these data imply that early during Ag encounter, PKC affected T cell priming, but that activated, possibly MOG-specific, T cells were generated with increasing time. Although there are caveats to these phenotypic analyses, this suggests that the lack of clinical signs of EAE and the reduced CNS inflammation seen in PKC–/– mice were not due to an inability of PKC-deficient CD4 T cells to respond in the secondary lymphoid organs.
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deficiency results in Th1 cell hyporesponsiveness
We next considered the possibility that the PKC deficiency resulted in a state of hyporesponsiveness in the responding CD4 T cells and an inability to produce Th1 proinflammatory cytokines. At various time points after immunization, draining LN cells were stimulated with MOG (Fig. 4), and IL-2, IFN-, and TNF production and the ability to proliferate were measured. LN cells isolated from wt mice 3 or 14 days after immunization showed enhanced IL-2 production (Fig. 4a) and concentration-dependent proliferation (Fig. 4b). However, in the absence of PKC, both IL-2 production and proliferation were reduced. As additional proof of hyporesponsiveness, we assessed the production of the effector Th1 cytokines, IFN- and TNF, during the stages of disease (Fig. 4c). In contrast to CD4 cells from wt mice, those from PKC–/– mice exhibited markedly impaired IFN- and TNF production in response to MOG at all stages of disease. This was substantiated when cytokine production was assayed in supernatants from these cultures (data not shown). This demonstrates that the failure to induce EAE in PKC–/– mice was probably due to the inability to generate functionally competent, MOG-specific effector Th1 cells.
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-deficient mice
Lastly, we examined whether the requirement for PKC in the development of EAE could be overcome by increasing Ag signal strength and/or inflammatory conditions. Mice were immunized with MOG in CFA on day 7 as well as day 0, with pertussis toxin given on days 0, 2, 7, and 9. With this protocol all wt mice developed clinical signs of disease, with two mortalities on days 12 and 24 (Fig. 5a). The 50% mortality rate observed with this protocol is significantly higher than that seen in wt mice (11.76%; see Table I) that were immunized once. Under these stringent inflammatory conditions, all PKC–/– mice remained refractory to EAE induction.
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–/– mice showed remission of disease after the first acute episode, and on day 40 after initial immunization reinjected MOG in CFA/pertussis and monitored for signs of EAE for an additional 40 days. As shown in Fig. 5b, in addition to an earlier onset of disease (day 2 compared with day 12), all wt mice developed a chronic-progressive form of EAE (clinical score of 2–2.5) with no signs of remission. In contrast, recall responses in PKC–/– mice were profoundly reduced, as shown by significantly milder signs of EAE.
The development of functionally competent, MOG-specific Th1 cells requires costimulatory signals, such as that from OX40 (18, 19, 20, 21). Therefore, we considered the possibility that signaling through OX40 may overcome the Ag-specific T cell hyporesponsiveness seen in PKC–/– mice and administered agonistic anti-OX40 on day 2 (Fig. 5c) or day 7 (Fig. 5d). The onset of EAE in wt mice that received anti-OX40 was almost identical with that in control mice, but disease progression was accelerated and resulted in significantly more severe clinical signs of disease and increased mortality. In contrast, PKC–/– mice remained largely resistant to EAE induction. Collectively, the data suggest that PKC is absolutely required for the development of functionally competent, MOG-specific Th1 cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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is essential for the development and persistence of Ag-specific Th1 cells in EAE. We show that a PKC deficiency significantly affects the peripheral T cell responses of mice to MOG, and the result is diminished inflammatory cells in CNS tissue and lower Th1 cytokine production, resulting in delayed EAE onset and minimal clinical signs of disease.
Analysis of spinal cords and brains revealed that PKC deficiency significantly affected mononuclear cell (CD4+, CD8+, and F4/80+) accumulation in CNS tissue as well as Th1 (IL-2, IFN-, TNF, and IL-17) cytokine production. Detailed analysis of CD4+ T cells isolated from the peripheral lymphoid organs of PKC-deficient mice suggested normal, but delayed, accumulation of activated T cells in the absence of PKC. When functionality was examined ex vivo in response to MOG, PKC–/– CD4 T cells were impaired in their ability to produce IL-2, to proliferate, and to produce the effector Th1 cytokines, IFN- and TNF, regardless of the time after immunization. These results show that PKC deficiency results in impaired maturation of MOG-reactive CD4 T cells into functionally competent effector Th1 cells. Resistance to EAE is then most likely not simply due to blocking T cells from entering the CNS, although the latter associated with this hyporesponsiveness could also contribute to the phenotype observed.
An important observation from our studies is the demonstration that increasing Ag dose and inflammatory conditions did not overcome the PKC dependence for EAE development. PKC–/– mice, which were resistant to the initial episode of EAE, remained unresponsive to EAE reinduction several months after initial priming, indicating that PKC deficiency results in a long-lasting deficiency in the MOG-specific CD4 T cell population. The phenotype of CD28-deficient mice after single immunization with MOG is very similar to that reported in this paper with PKC-deficient mice (21, 22, 23), potentially correlating with the idea put forward in the literature that PKC is central to CD28 signaling. However, in contrast with our data, CD28-deficient mice were shown to develop EAE with severe Th1 infiltration and demyelination after repeated immunization with MOG (21, 22). Based on the latter results, it was proposed that CD28 costimulation does not regulate immunological anergy in EAE, but, rather, adjusts the threshold for activation of autoreactive Th1 cells. Interestingly, blockade of OX40-OX40L (21) or CD40-CD40L (22) interactions inhibits the development of EAE in repeatedly immunized, CD28-deficient mice, indicating that additional Ag exposure results in up-regulation of alternate costimulatory signaling pathways that can compensate for the lack of CD28. In this regard, we show that coadministration of an agonistic anti-OX40 resulted in the development of a more severe EAE in wt mice, whereas PKC–/– mice remained largely resistant to EAE induction. Thus, the hyporesponsive phenotype observed in PKC–/– CD4 T cells, but not in CD28–/– CD4 T cells, suggests that in addition to CD28, PKC may facilitate the development of encephalitogenic Th1 cells via alternative costimulatory pathways, such as through OX40.
The role of PKC in experimental models of inflammatory diseases has been recently studied in several model systems. PKC was shown to be critical for the development of asthmatic lung inflammatory responses controlled by Th2 cells and also for the Th2 response to the parasite N. brasiliensis (11, 12). Interestingly, PKC-deficient mice on both the C57BL/6 background and the susceptible BALB/c background were able to mount a normal protective Th1 response against L. major infection, implying that PKC might be preferentially involved in the development and effector function of Th2, but not Th1, cells (11). In additional support of this, memory Th1 responses in the lung were normal in PKC–/– mice that were immunized and subsequently challenged via the airways with OVA (12). In the latter study, however, the PKC deficiency was shown to significantly affect early in vivo priming of both Th1 and Th2 cells after immunization. This implied that the requirement for PKC in the generation of Th1 cells might be overcome with time and/or by the overall level of costimulatory and inflammatory signals that accompany Th1 priming. The data presented in this paper extend these studies by showing that PKC is certainly not selective for Th2 inflammation, but can also be critically involved in inflammation driven by some, but obviously not all, Th1 populations.
An interesting question, then, is why PKC–/– mice failed to generate efficient Th1 responses to MOG in the model of EAE, but were largely normal in generating Th1 immunity to Leishmania Ags (11) and to OVA when given via the airways (12). At present we cannot answer this, and the exact parameters that govern the dependence on PKC of any one given T cell response remain to be determined. It is possible that the nature of the Ag plays a key role, whether it is foreign or self, or peptide or protein. Alternatively, the target organ might be crucial to how critical PKC is for the overall response. This could encompass not only the nature of the T cell populations and their affinity for Ag, but also the range of molecules required for trafficking to the organ, and the availability of Ag at the site of T cell priming or at the site of inflammation. All these parameters differ in the three Th1 models that have now been analyzed, so it will be informative to test these and other possibilities in the future. In particular, for those who are interested in therapeutic targeting of autoimmune diseases, it will be particularly important to know whether PKC is essential for the induction of other T cell-mediated autoimmune diseases.
In conclusion, our results indicate that PKC plays a crucial role in the development of the Th1 autoimmune inflammatory process that leads to CNS inflammation and clinical EAE. These findings have important ramifications not only for our understanding of the basic mechanisms of autoimmunity, but also might offer the hope of designing more efficient and highly specific therapeutic strategies for the treatment of autoimmune disorders.
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Acknowledgments |
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-deficient mice. |
Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants AI50498 and CA91837 (to M.C.). This is publication 710 from the La Jolla Institute for Allergy and Immunology. 
2 Address correspondence and reprint requests to Dr. Michael Croft, Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: mick{at}liai.org
3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental allergic encephalomyelitis; HPRT, hypoxanthine phosphoribosyltransferase; LN, lymph node; MOG, myelin oligodendrocyte glycoprotein; wt, wild type.
Received for publication July 6, 2005. Accepted for publication September 27, 2005.
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References |
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: a new essential superstar on the T-cell stage. Immunol. Today 21: 567-573. [Medline]
(PKC): it’s all about location, location, location. Immunol. Rev. 192: 53-63. [Medline]
signals activation versus tolerance in vivo. J. Exp. Med. 199: 743-752. is critical for the development of in vivo T helper (Th) 2 cell but not Th1 cell responses. J. Exp. Med. 200: 181-189. . J. Immunol. 173: 6440-6447. is required for TCR-induced NF-
B activation in mature but not immature T lymphocytes. Nature 404: 402-407. [Medline]
CDR3 motifs associated with BP recognition are enriched in OX-40+ spinal cord T cells of Lewis rats with EAE. J. Neurosci. Res. 44: 562-567. [Medline]
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) and wt (PKC+/+; •) mice were immunized s.c. at the base of the tail with MOG in CFA. Pertussis toxin was given i.p. at the time of immunization and 48 h later. Mice were monitored for clinical signs of EAE for 40 days and were scored on an arbitrary scale of 0–5 as described in Materials and Methods. Results are the mean ± SEM from 12 mice/group.
) and wt (
) mice were immunized with MOG as described in Fig. 1
) and wt (PKC+/+; • and 
) mice were immunized with MOG as described in Fig. 1