αB-Crystallin-reactive T cells from knockout mice are not encephalitogenic

Journal of Neuroimmunology 176 (2006) 51 – 62 www.elsevier.com/locate/jneuroim

aB-Crystallin-reactive T cells from knockout mice are not encephalitogenic Chunhe Wang a,b,*,1, Yuan K. Chou b,1, Cathleen M. Rich a, Jason M. Link a, Michael E. Afentoulis a, Johannes M. van Noort c, Eric F. Wawrousek d, Halina Offner a,b,e, Arthur A. Vandenbark a,b,f a

c

Neuroimmunology Research, Veterans Affairs Medical Center, Portland, 97239 OR, USA b Department of Neurology, Oregon Health and Science University, USA Division of Immunological and Infectious Diseases, TNO Prevention and Health, PO Box 2215, 2301 CE Leiden, The Netherlands d National Eye Institute, National Institutes of Health, Bethesda, 20892 MD, USA e Department of Anesthesiology and Perioperative Medicine, Oregon Health and Science University, USA f Department of Molecular Microbiology and Immunology, Oregon Health and Science University, USA Received 9 March 2006; received in revised form 5 April 2006; accepted 10 April 2006

Abstract Alpha B-crystallin (aB) is a small heat shock protein that is strongly up-regulated in multiple sclerosis (MS) brain tissue, and can induce strong T cell responses. Assessing a potential encephalitogenic function for aB protein in MS and experimental autoimmune encephalomyelitis (EAE) has been challenging due to its ubiquitous expression that likely maintains central and peripheral tolerance to this protein in mice. To address this issue, we obtained aB-knockout (aB-KO) mice in H-2b background that lack immune tolerance to aB protein, and thus are capable of developing aB-specific T cells that could be tested for encephalitogenic activity after transfer into aBexpressing wild type (WT) mice. We found that T cell lines from spleens of aB protein-immunized aB-KO mice proliferated strongly to aB protein itself, and the majority of T cells were CD4+ and capable of secreting pro-inflammatory Th1 cytokines upon restimulation. However, transfer of such aB-reactive T cells back into WT recipients was not sufficient to induce EAE, compared to the transfer of mouse MOG-35 – 55 peptide-reactive T cells from the same donors that induced severe EAE in recipients. Moreover, aB-specific T cells failed to augment severity of actively induced EAE in WT mice that were expressing high levels of aB message in the CNS at the time of transfer. These results suggest that aB-specific T cells are immunocompetent but not encephalitogenic in 129SvEv mice, and that immune tolerance may not be the main factor that limits the encephalitogenic potential of aB. D 2006 Elsevier B.V. All rights reserved. Keywords: Tolerance; EAE/MS; T cells; Knockout mice

1. Introduction Abbreviations: aB, aB-crystallin; APCs, antigen presenting cells; EAE, experimental autoimmune encephalomyelitis; KO, knockout; MS, multiple sclerosis; MOG, mouse myelin oligodendrocyte glycoprotein 35 – 55 peptide; LN, lymph node; MBP, myelin basic protein; PBMC, peripheral blood mononuclear cells; PLP, proteolipid protein; PTX, pertusis toxin; WT, wild type. * Corresponding author. Neuroimmunology Research, Veterans Affairs Medical Center, Portland, 97239 OR, USA. Tel.: +1 503 220 8262x57156; fax: +1 503 721 7975. E-mail address: [email protected] (C. Wang). 1 These authors contributed equally to this work. 0165-5728/$ - see front matter D 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2006.04.010

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by recurrent neuroinflammation, demyelination, and axonal damage (Martin and McFarland, 1995; Steinman, 1996; Adams et al., 1989; Barnett and Prineas, 2004). Several lines of evidence suggested that aB-crystallin (aB), a small heat shock protein, might play a role in the pathogenesis of MS: (1) A predominant T cell response to aB was found in myelin fractions obtained from MS plaque tissues, but not in

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tissues from healthy controls (van Noort et al., 1995). (2) In PBMC of selected MS patients and healthy controls, a large fraction of memory T cells responded to aB protein (van Noort et al., 2000). (3) Its mRNA was identified as the most abundant transcript that is unique to MS plaques (Chabas et al., 2001). (4) A high level of expression of aB was found in oligodendrocytes at the earliest stages during MS lesion development (Bajramovic et al., 2000a,b). (5) Expression of aB in human peripheral lymphocytes could be triggered by virus infection in vitro (van Noort et al., 2000; van Sechel et al., 1999), suggesting that it might serve as a link between microbial infection and myelin-directed autoimmunity in MS patients. Consistent with its hypothesized ‘‘triggering’’ role, aB was found to express at high levels and be presented by antigen presenting cells (APCs) in MS lesions at their earliest developmental stage, but disappear in inactive lesions (Bajramovic et al., 1997; Bajramovic et al., 2000b; van Noort et al., 2000). Yet, it is challenging to directly link the prominent T cell response to the pathogenesis of MS. In other words, the relatively high expression of aB in MS lesions may serve functions other than to promote an encephalitogenic T cell response. Unlike other myelin antigens, aB cannot induce EAE in mice after immunization with syngeneic aB, which is not immunogenic for WT splenocytes, or with xenogeneic aB (van Noort et al., 2000; van Stipdonk et al., 2000a,b) that induced responses to species-specific differences to which the mice were not tolerant (van Stipdonk et al., 2000a,b). In fact, there exists only a single report that immunization with an aB peptide produced mild paralysis in a small percentage of Biozzi ABH (H-2A) mice (Thoua et al., 2000). Further studies demonstrated that aB was absent in the central or peripheral lymphoid organs of humans, but was expressed extensively in animals (van Sechel et al., 1999; Voskuhl, 1998), indicating innate T cell tolerance to aB might account for the inability of aB to induce EAE in animal models. However, it is unknown whether aBreactive T cells might be encephalitogenic in animals, once immune tolerance to aB is circumvented. A line of aB gene knockout (KO) mice created on 129SvJae  129SvEv background might be useful for addressing this issue (Brady et al., 2001). T cells in aBKO mice have been shown not to be tolerant to aB (Charukamnoetkanok et al., 2003), and should respond normally to aB. Moreover, previous studies have shown that adoptively transferred lymphocytes from aB-KO mice can induce inflammation in damaged ocular lenses of wild type 129SvEv mice (Gelderman et al., 2003). Thus, if immune tolerance is the only factor that limits the encephalitogenic potential of aB, transfer of such T cells may possibly cause EAE in mice that express aB. In the current study, functional aB-reactive CD4+ T cells not tolerant to aB were successfully generated in aB-KO mice. However, these T cells were unable to cause passive EAE when transferred into recipient naı¨ve WT mice or mice with actively induced EAE that expressed high levels of aB in

the CNS at the time of transfer. Thus, aB-specific T cells are immunocompetent but not encephalitogenic in 129SvEv mice. In addition, our data show immune tolerance is not the only factor that limits the encephalitogenic potential of aB.

2. Materials and methods 2.1. Mice aB-KO (aB / ) mice with a targeted disruption of the aB gene, and the adjacent HSPB2 gene, was described previously and maintained on a 129SvJae (ES cell line)  129SvEv (mice provide the original chimeras) background (Brady et al., 2001). Briefly, 1.6 kb of 5V flanking region and most of the coding sequence, through the beginning of the last exon, of the aB-crystallin gene was replaced by a PGK/neo selection cassette. Both the aBcrystallin gene and its protein product were shown to be deleted in these KO mice (Brady et al., 2001). PCR was performed according to a protocol published previously (Brady et al., 2001) to confirm that the homozygous KO mice were used as experimental animals and breeders in this study. WT 129SvEv mice, used as controls, were originally purchased from Taconic (Germantown, NY, USA). Our previous study, in which wild type 129SvEv mice receiving aB-reactive T cells from aB-KO mice developed autoimmune lens-associated uveitis (Gelderman et al., 2003), demonstrated that 129SvEv can serve as an appropriate wild type control for aB-KO mice. 129SvJae strain, which has a background close to 129SvEv (Simpson et al., 1997), is not commercially available. The mice were housed in the Animal Resource Facility of Portland Veterans Affairs Medical Center (PVAMC) in accordance with the institutional guidelines. All of animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of PVAMC. 2.2. Antigens Mouse MOG-35– 55 (MOG) peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized using solid phase techniques and was purified by HPLC at Beckman Institute, Stanford University (Palo Alto, CA). Recombinant mouse aB protein was produced and purified in our collaborating lab in Division of Immunological and Infectious Diseases, TNO Prevention and Health, Leiden, The Netherlands. 2.3. Generation of Ag-specific T cell lines To prepare T cell lines specific for MOG peptide or aB protein, aB-KO or WT mice were immunized s.c. in the flanks with 0.2 ml of an emulsion containing 200 Ag of MOG or aB protein in saline, and an equal volume of CFA containing 400 Ag Mycobacterium tuberculosis H37RA

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(Difco Laboratories, Detroit, MI). Ten days after immunization, spleens and draining LN were removed, and single cell suspensions produced by mincing the tissue through a 200-mesh wire screen. The cell suspension was cultured with 20 Ag/ml of MOG peptide or aB protein at 8  106 cells/ml in stimulation medium (RPMI 1640 medium supplemented with 0.1 mM non-essential amino acids, 2 mM l-glutamine, 0.1 mM sodium pyruvate, 0.05 mM 2mercaptoethanol, and 2% FBS) for 48 h. To obtain T cell lines, the splenocytes were expanded in medium containing recombinant IL-2 (5 ng/ml) (R&D Systems, Minneapolis, MN) for 1 week. A proliferation assay was performed in vitro to test the ability of splenocytes and T cells to respond specifically to the immunizing antigens. 2.4. Proliferation assay Splenocytes or T cells were harvested and cultured in a 96-well flat-bottom tissue culture plate at 4  105 cells/ well in stimulation medium in the presence of APC, irradiated (2500 rad) syngeneic thymocytes at a ratio of 1:10 (T/APCs), either without Ag (control), or with MOG peptide or aB protein at varying concentrations. The cells were incubated for 3 days at 37 -C in 7% CO2, and pulsed with 0.5 ACi of [3H] thymidine (Perkin-Elmer, Boston, MA) for the final 18 h of incubation. The cells were harvested onto glass fiber filters, and incorporated radioactivity was measured by a liquid scintillation counter (1205 beta-plate cell culture harvesting and counting system, Wallac, Turku, Finland). The cpm values (mean T S.D.) were calculated from triplicate wells. Stimulation index (SI) was calculated by dividing the experimental cpm with the control cpm. 2.5. Cytokine determination by cytometric bead array (CBA) aB-specific T cells were generally cultured at 4  106 cells/well and stimulated with 2 Ag/ml of aB protein in the presence of APCs (T/APCs at 1:10) in a 24-well flat-bottom culture plate in stimulation medium for 48 h. Supernatants were then harvested and stored at 80 -C until tested for cytokines. The mouse inflammation CBA kit (BD Bioscience, San Diego, CA) was used to detect IL-12, TNF-a, IFN-c, MCP-1, IL-10, and IL-6 simultaneously. Briefly, 50 Al of sample was mixed with 50 Al of mixed capture beads and 50 Al of mouse PE detection reagents. The sample tubes were incubated at room temperature for 2 h in the dark, followed by a wash step. The samples were then resuspended in 300 Al of ‘‘wash buffer’’ before acquisition on the FACScan. The data were analyzed using the CBA software (BD Biosciences). Standard curves were generated for each cytokine using the mixed bead standard provided in the kit, and the concentration of cytokine in the supernatant was determined by interpolation from the appropriate standard curve.

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2.6. Induction of EAE To induce active EAE, aB-KO or WT mice were inoculated s.c. in the flanks with 0.2 ml of an emulsion containing 200 Ag of MOG peptide or 200 Ag of aB protein, and an equal volume of CFA containing 200 Ag of heatkilled M. tuberculosis H37RA (M.Tb.; Difco, Detroit, MI). To induce successful passive EAE by adoptive transfer, as described in our previous publication (15), 1 106 cells/ml MOG-specific T cell lines, as well as aB-specific T cell lines, were pre-cultured with anti-mouse CD3 (1 Ag/ml; clone 145-2C11; BD PharMingen, San Diego, CA) and antiCD28 (1 Ag/ml; clone 37.51; BD PharMingen) Abs immobilized on a tissue culture plate with or without IL12 (20 ng/ml; R&D Systems, Minneapolis, MN) and/or IL18 (25 ng/ml; MBL, Nagoya, Japan) for 24 h. After the cells were washed three times with PBS, they were transferred i.p. into naı¨ve WT 129SvJ recipients (1 –5  106 cells/ mouse). On the same day, and 2 days after the immunization or cell transfer, each mouse was injected i.v. with 67 ng of pertussis toxin (Ptx, List Biological Laboratories, Campbell, CA). The mice were assessed daily for clinical signs of EAE according to the following scale: 0 = normal, 1 = limp tail or mild hindlimb weakness, 2 =moderate hindlimb weakness or mild ataxia, 3 = moderately severe hindlimb weakness, 4 = severe hindlimb weakness or mild forelimb weakness or moderate ataxia, 5 = paraplegia with no more than moderate forelimb weakness, and 6 = paraplegia with severe forelimb weakness or severe ataxia, moribund condition or dead. 2.7. Histology Intact spinal columns were removed from experimental and control groups of mice. The spinal cords were dissected after fixation in 10% phosphate-buffered formalin, dehydrated, and embedded in paraffin before sectioning. For examining neuroinflammation, the sections were stained with hematoxylin and eosin (H&E). For examining demyelination, the sections were stained with either Toluidine blue or Luxol fast blue-periodic acid Schiff (LFB-PAS). The sections were analyzed by light microscopy after staining. 2.8. Detection of aB expression by RT-PCR Similar to the detection methods published previously (9), but with modifications, the tissues from mouse brain, spleen and eyes were homogenized for 15 s using a Tissue Tearor Brand homogenizer and then treated with Qiagen Rneasy Mini Kit (Qiagen, Valencia, CA) to extract mRNA. Using a Beckman UV/Visible Spectrophotometer, the concentrations of mRNA samples were taken from each sample and an equivalent amount of mRNA was taken from each sample for the cDNA synthesis protocol using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Samples were titrated by diluting cDNA 1:10, 1:20,

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and 1:40 in nuclease free water and run by using a ABI Prism 7000 Sequence Detection System with SYBR Green PCR Master Mix (ABI, Foster City, CA) using a standard dissociation protocol recommended by the manufacturer. By graphing the log of the dilution vs. the Ct value obtained from the dissociation protocol, we were able to obtain a standard curve to see where the change in Ct value was the smallest and therefore choose the correct dilution of cDNA for the RT-PCR. The following primers were used in obtaining the relative expression (RE) of aB: (1) Housekeeping gene: mouse ribosomal protein L32-3A (3A) gene. Forward Primer: GGA AAC CCA GAG GCA TTG AC; Reverse primer: TCA GGA TCT GGC CCT TGA AC; GenBank accession Number: K02060. (2) Mouse alpha-B2crystallin gene. Forward Primer: TGC RGT GAC AGC AGG CTT CT; Reverse Primer: GAG AGC ACC TGT TGG AGT CT; GenBank accession Number: M73741. Once RT-PCR was performed using the same amounts of primer and SYBR green listed above at the correct dilution, the Ct values were obtained using ABI Prism software, and the RE was obtained with the following formula for each sample: = POWER(1.8(L32 Ct ABC Ct))*100,000.

3. Results 3.1. WT mice are resistant to induction of active EAE by aB protein We first investigated whether or not aB protein could induce clinical paralysis in 129S6/SvEvTac or aB-KO mice under experimental conditions known to induce active EAE with the encephalitogenic MOG peptide. As shown in Fig. 1A, both WT and aB-KO mice immunized with MOG/CFA/Ptx developed a similar pattern and severity of clinical EAE, whereas mice immunized with aB protein/CFA/Ptx did not develop any discernable clinical signs of EAE. Histological staining showed different, degrees of immune cell infiltration and demyelination in the spinal cords of MOG-immunized WT and aB-KO mice (Fig. 1B), but no obvious pathological changes were found in the spinal cords of aB-immunized WT or KO mice. These results demonstrated that WT and aB-KO mice were resistant to induction of active EAE by immunization with aB protein, even though these mice were susceptible to EAE induced by MOG peptide. 3.2. Differential responses of LN and splenic lymphocytes to aB in WT and aB-KO mice To investigate the mechanisms that prevent aB protein from inducing active EAE in 129S6/SvEvTac, we measured lymphocyte proliferation to aB protein or MOG peptide in both LN and spleen cells from aBimmunized WT and aB-KO mice. Surprisingly, WT LN cells from aB protein-immunized mice proliferated

Fig. 1. Mean clinical EAE scores of aB-KO and WT 129SvJ mice immunized with aB protein or MOG peptide and related pathological findings. (A) Mean clinical scores of EAE. The mice were immunized on day 0 with 200 Ag/ml of aB protein/CFA or mouse MOG-35 – 55 (MOG) peptide/CFA + Ptx on days 0 and 2. Data represent two independent experiments with six mice per group. Student’s t-test, *P < 0.05 or **P < 0.01, compared to aB protein-immunized groups. (B) Toluidine blue stain showed demyelination in the spinal cords of MOG-immunized, but not in those of aB-immunized aB-KO and WT mice.

strongly in response to aB protein (Fig. 2A), whereas splenic cells from the same mice were suppressed to both aB protein and ConA, but not to MOG peptide (Fig. 2B). In contrast, both LN (Fig. 2C) and splenic cells (Fig. 2D) from aB protein-immunized aB-KO mice responded strongly to aB stimulation. In short, LN cells proliferated, but splenocytes were suppressed in response to aB in immunized WT mice that are reported to be tolerant to aB, whereas both lymphocyte populations were responsive in aB-KO mice. 3.3. Production of aB-reactive T cells from aB-KO mice We expected that aB-KO mice would respond to aB and provide a source of functional aB-reactive T cells because negative selection and T cell tolerance toward aB should not occur during T cell development in these mice. Initially, we isolated splenocytes from aB-KO and WT mice immunized with aB protein or MOG peptide in CFA and tested the ability of the splenocytes to

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Fig. 2. Proliferation responses to aB protein of LN cell and splenocytes in immunized WT and aB-KO mice. Immunization with aB protein induced strong proliferation to aB in lymph node cells (A), but suppressed proliferation of splenocytes (B) from WT 129SvJ mice. In contrast, both lymph node cells (C) and splenocytes (D) from immunized aB-KO mice proliferated to aB protein. Student’s t-test, *P < 0.05 or **P < 0.01, compared to control. The data are representative of 2 experiments from cells pooled from 3 mice.

proliferate in response to aB protein or MOG peptide. Pertussis toxin (Ptx) was not included in the inoculum to avoid promoting the migration of lymphocytes into the CNS. As shown in Fig. 3, splenocytes from aB-KO mice immunized with aB protein or MOG peptide showed significant proliferation in response to aB or MOG, respectively. After in vitro restimulation with aB protein, the aB-KO splenocytes of each specificity were cultured in medium containing IL-2 to expand T cells, and 10 days later the specificities of aB- or MOG-selected T cells were revaluated by proliferation assay. The results of these assays demonstrated that the selected Ag-specific T cell lines from aB-KO mice were specific and had increased responsiveness to the selecting antigen (see Fig. 3C and D), and did not cross-react with the nonimmunizing antigen (data not shown). Parallel immunizations of WT mice did not induce specific splenocyte

responses to aB protein (Fig. 3E), but did induce low but significant response to MOG peptide (Fig. 3F). Further expansion of aB stimulated splenocytes in IL-2 failed to produce any viable cells. 3.4. Characterization of aB-reactive T cells We further characterized the phenotype and cytokine profile of aB protein-specific T line cells expanded from splenocyte cultures from immunized aB-KO mice. The majority (99%) of the cells were T cells after expansion in IL-2 as indicated by positive CD3 staining. Among these T cells, 71% were CD4+ T cells and 24% were CD8+ T cells (Fig. 4A). When stimulated with aB protein in the presence of irradiated APCs, aB-reactive T line cells secreted a significant amount of pro-inflammatory Th1 cytokines, including IFN-g, TNF-a, and IL-6,

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in a dose-dependent manner. IFN-g levels exceeded 40,000 pg/ml in supernatants obtained 48 h after the T line cells were stimulated with 25 Ag/ml aB, and TNF-a levels

were also quite high, varying from 1000 to 2500 pg/ml (Fig. 4B). However, there was no measurable IL-12 or IL10 in the culture supernatants (Fig. 4B). In a parallel

Fig. 3. Characterization of aB- and MOG peptide-specific T cell responses from immunized aB-KO donor mice. (A and B) Splenocytes of aB-KO mice proliferated to aB and MOG after immunization with aB or MOG peptide, respectively; (C and D) T cell lines selected from aB-KO mice responded strongly to aB or MOG prior to transfer; (E and F) splenocytes from aB- or MOG peptide-immunized WT 129SvJ mice responded to MOG peptide but not to aB. Student’s t-test, *P < 0.05 or **P < 0.01 compared to control. The data are representative of 2 experiments from cells pooled from 4 mice.

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Fig. 4. The majority of aB-reactive splenic T cells from aB-KO mice were CD4+ T cells that secreted pro-inflammatory cytokines upon aB stimulation in the presence of APCs. (A) Phenotypic analysis of aB-specific T cell line cells by FACScan showed >70% were CD4+. (B) Quantification of pro-inflammatory cytokines (TNF-a, IFN-g, and IL-6) by CBA in the supernatants of aB-specific T cells stimulated with aB in the presence of APCs. Secretion of IL-10 and IL12 was negligible. Student’s t-test, *P < 0.05 or **P < 0.01 compared to control. The data are from cells pooled from 6 mice.

evaluation, we found essentially the same phenotype and cytokine profile for MOG peptide-reactive T cells from immunized aB-KO mice (not shown). These results demonstrated that both aB- and MOG-reactive cells from immunized aB-KO mice were predominantly inflammatory CD4+ T cells, a profile consistent with encephalitogenic T cells. 3.5. aB-reactive CD4+ T cells from aB-KO mice are not encephalitogenic in WT recipients To test the encephalitogenicity of aB protein-specific vs. MOG peptide-specific T cell lines described above, we transferred each specificity into WT mice, in which the gene of aB was not disrupted. As expected, transfer of 15

or 30 million activated MOG-specific T cells induced moderately severe passive EAE in naı¨ve recipients (Fig. 5A and B) with obvious myelin damage and neuroinflammation as revealed by LFB-PAS staining (Fig. 5C) and confirmed by H and E staining (data not shown). In contrast, transfer of the same number of aB-reactive T cells into WT 129SvJ mice under the same exact conditions did not induce EAE (Fig. 5A and B), neuroinflammation, or myelin damage (Fig. 5C). Moreover, aB protein-specific CD4+ T cells failed to transfer EAE even after hyper-activation with anti-CD3 and antiCD28 mAb with added IL-12 and IL-18 (not shown), a protocol demonstrated to enhance encephalitogenic activity of transferred MOG-specific T cells in B6 mice (Brady et al., 1997).

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Fig. 5. Clinical and histological EAE in WT 129SvJ mice that received either MOG- or aB-reactive T cells. (A and B) Clinical EAE scores in mice receiving passive transfer of 30 or 15 million activated MOG- or aB-reactive T cells. The aB-reactive T cells did not induce passive EAE in WT mice even after receiving the higher dose of 30 million aB-reactive T cells per mouse. Data are representative of three independent experiments. Student’s t-test, *P < 0.05 or **P < 0.01 compared to aB-specific T cells-transferred mice. (C) Luxol fast blue-periodic acid Schiff (LFB-PAS) stain showed demyelination and neuroinflammation in WT mice that received MOG-reactive T cells, but not in those that received aB-reactive T cells generated from aB-KO mice or no transferred T cells (naı¨ve WT).

3.6. Failure of aB-reactive CD4+ T cells in boosting severity of MOG-35 – 55-induced EAE As a further test of encephalitogenicity or possible CNS protection, we induced EAE in WT mice by immunization with MOG-35 – 55 peptide, and then transferred aB proteinspecific T cells into mice on the day after onset of active EAE. As shown in Fig. 6A and B, mice immunized with MOG peptide/CFA/Ptx developed EAE characterized by a typical disease course with a peak clinical score of ¨ 4.0, followed by less severe relapses over 40 days. Transfer of 7 million aB-specific CD4+ T cells per mouse at the time of onset of MOG-induced EAE did not increase the clinical severity of disease (Fig. 6A and B). Conversely, it appeared to delay the first disease relapse, even though a statistically significant difference was not reached. Thus, transfer of aBspecific CD4+ T cells did not intensify the severity of active EAE. 3.7. aB expression in the CNS of mice with onset of EAE Since it has been reported that the expression of aB in the CNS is a time-limited event that occurs mostly in the early stages of MS lesion development (Duvanel et al., 2004), conceivably, the failure to transfer or boost EAE with aB-specific T cells might be due to insufficient expression of aB in the CNS of recipient mice. Therefore, we carried out RT-PCR on brain, spleen and eye tissues collected from mice on the first day after onset of MOG-35– 55-induced active EAE to evaluate aB expression in their CNS at the time of passive transfer. The results demonstrated aB expression as 7500 RE units in brain, a level that was 6fold higher than in spleen (Fig. 6C). Comparatively, RTPCR analysis of aB message in eye tissue detected levels

> 70,000 RE units (not shown). Thus, aB was highly expressed in the CNS of recipient mice with EAE when aBspecific T cells were passively transferred.

4. Discussion In general, heat shock proteins have been implicated in both pathogenic and protective roles in human diseases, including neurodegenerative disorders (Sharp et al., 1999; Dabir et al., 2004). aB, a 20-kDa, 175-amino acid residue protein, shares the classical characteristics of heat shock proteins, i.e. stress-inducibility, strong sequence homologies among different species, and molecular chaperone properties (MacRae, 2000; Bhat et al., 1991). Although elevated expression of aB was found to be induced by various conditions of cellular stress (Duvanel et al., 2004; Samali and Orrenius, 1998; Brzyska et al., 1998), the complete physiological role of aB protein is not yet clear. Although a number of reports have suggested a possible pathogenic role for aB protein in MS pathogenesis, whether aB serves as an autoantigen in MS remains unclear, mainly because of its failure to induce EAE in animals. Our results in this study failed to suggest that aB is an encephalitogenic target antigen for induction of EAE in the mouse strain we used. Neither active immunization with aB protein nor passive transfer of inflammatory aB-specific T cells from immunologically non-tolerant aB-KO donors into naı¨ve WT mice resulted in clinical signs of EAE or inflammatory lesions in the CNS. Moreover, transfers of aB-specific T cells into mice with EAE that were ‘‘conditioned’’ to express aB protein in the CNS could not augment EAE severity. The lack of demonstrable encephalitogenic activity of aB might suggest alternative functions associated with its

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Fig. 6. Transfer of aB-reactive T cells into WT 129SvJ mice with EAE did not increase the clinical score of MOG peptide-induced EAE even though aB was highly expressed in the brain and spinal cord at the time of passive transfer. (A and B) Passive transfer of five million activated aB-reactive T cells 13 days after immunization did not boost the clinical score of WT mice immunized with MOG/CFA/Ptx. (C) Relative expression (RE) of aB mRNA in spleen and brain tissues of mice with EAE at the time of passive transfer. Student’s t-test failed to demonstrate significant difference between the clinical scores of the two groups at any time point. Four mice per group were used in this study.

strong induction in early MS lesions, including its postulated role as a heat-shock protein. It has been shown that aB protects cellular proteins against aggregation resulting from abnormal misfolding or unfolding in vitro (MacRae, 2000). Conversely, at high concentrations in ocular lenses, in which its oligomerization partner aA-crystallin has been knocked out, aB itself forms large insoluble inclusion bodies (Brady et al., 1997), suggesting an inherent propensity for this protein to be involved in abnormal protein deposits. In addition, the potential role of aB protein in human diseases including MS has been discussed (Clark and Muchowski, 2000; Duvanel et al., 2004; Sotgiu et al., 2003; van Noort et al., 1995). Most recently, a number of biochemical and cell biological studies have shown that aB protein may be a novel inhibitor of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, which has been shown to suppress the activation of caspase-3 (Kamradt et al., 2005a,b; Mehlen et al., 1996). aB protein can also inhibit UVA-induced abnormal cellular

activation through the RAF/MEK/ERK pathway that leads to cell apoptosis (Andley et al., 2000; Liu et al., 2004). These functions of aB protein could protect oligodendrocytes from apoptosis (Duvanel et al., 2004), thus possibly preventing loss of myelin. The idea that immune tolerance limits the encephalitogenic potential of T cell responses to aB protein was mainly based on observations that aB protein was expressed extensively in lymphoid organs of rodents, but not in those of humans (van Sechel et al., 1999). The relationship between lymphoid tissue expression of an Ag and the resistance of the animal towards experimentally induced autoimmunity is imperfect, and may be influenced by a number of different factors. First, constitutive expression of myelin basic protein (MBP) and proteolipid protein (PLP) observed in the lymphoid tissues of mice, including the thymus, (reviewed by Voskuhl, 1998), did not prevent encephalitogenic T cell responses to these antigens. Secondly, in lymphoid organs, splice variants of myelin

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components can be expressed that are slightly different from those expressed in the CNS. The sequences that happen to be different from the thymic variants may escape from central tolerance induction, as exemplified by encephalitogenicity of the PLP-139– 151 peptide in SJL mice tolerant to the DM20 protein that lacks this determinant (Kuchroo et al., 1994). However, it is unknown if a splice variant of aB protein different from that expressed in the CNS exists in lymphoid organs of 129SvJ mice, the strain used in our current study. Expression of aB protein and induction of T cell responses in 129SvJ mice have not been reported previously, although aB protein was shown to be expressed at a single time point in the spleen and thymus of a closely related strain, 129SvPasIco (van Sechel et al., 1999). Thus, it was expected and confirmed that immune tolerance to aB protein would be strongly apparent in spleen cells from 129SvJ mice, and lacking in spleen cells from aB-KO mice. However, it was surprising to find that LN cells from 129SvJ mice were strongly reactive toward aB protein after immunization, a result that might be related to variation in the expression level of aB in LN vs. spleen. Interestingly, a recent study showed that T cell responses could spread to aB peptide 161 –175 in a humanized transgenic mouse model of spontaneous EAE (Ellmerich et al., 2005), further suggesting that immune tolerance to aB can be circumvented in mice under certain circumstances. Our results showing that aB-reactive T cells from nontolerant mice are not pathogenic does not necessarily conflict with the ‘‘mistaken self’’ model proposed by van Noort et al. (2000). In fact, it has been previously reported that pro-inflammatory memory T cells were present in the PBMC of healthy human controls at high frequencies. According to the ‘‘mistaken self’’ model, stresses or ‘‘danger signals’’ posed by life events, microbial infections in particular, might be crucial for the encephalitogenicity of aB protein-reactive T cells. Microbial infections were found to induce de novo expression of aB protein in APCs and create a local pro-inflammatory environment in the CNS that caused high expression of aB protein in oligodendrocytes. The pro-inflammatory cytokine TNF-a has been shown to induce elevated oligodendrocyte expression of aB protein (Bajramovic et al., 1997; Bajramovic et al., 2000a). In the case of heat shock protein 60 (HSP60), autoreactivity can develop in the face of strong thymic expression that would normally be associated with central tolerance, but only in the presence of ‘‘danger signals’’ (Bajramovic et al., 2000a; Birk et al., 1996). Thus, breaking central tolerance toward aB in the absence of pro-inflammatory cytokines may not be sufficient to induce EAE on it own. Although immunization of WT mice with aB protein and transfer of aB-reactive T cells failed to induce EAE, it was of further relevance to test whether or not the aB-specific T cells from aB-KO mice could augment EAE already induced by a different encephalitogenic determinant. We utilized this paradigm for the first time and found that the prior induction of active EAE with MOG peptide strongly

up-regulated expression of aB message in the CNS. However, transfer of aB-specific T cells did not intensify EAE severity, and if anything, may have reduced EAE severity especially during the first relapse. From these data, we conclude that T cell responses to aB protein were not encephalitogenic, even in the presence of early neuroinflammation, a specific type of ‘‘danger signal’’. However, as a negative result, our study cannot rule out the possibility that encephalitogenic activity could be masked by more complicated unknown interactions between active and passive EAE, or by some aB-related host response involving chaperoning the innate and adaptive immune responses (Srivastava, 2002). Taken together, we showed that 129SvJ mice were resistant to active EAE induced by aB protein, even though LN T cells from these mice were not tolerant to aB. By immunization of aB-KO mice, we successfully produced aB-reactive T cells which failed to induce passive EAE or augment active EAE when transferred back into WT mice that widely express aB protein. Thus, we conclude that aBreactive T cells, in contrast to T cells specific for other classic neuroantigens in EAE such as MOG peptide, do not possess encephalitogenic activity in 129S6/SvEvTac mice, even when central tolerance was circumvented. Our studies have not ruled out the possibility that aB-reactive T cells could be encephalitogenic under different conditions, but the experiments as presented do not support a pathogenic role for aB-reactive T cells. In future studies, we will address additional questions of interest that may shed light on the lack of encephalitogenic activity of aB-specific T cells. These studies include (1) the possible differential expression of cell surface markers and cytokines, including IL-23/IL-17, by encephalitogenic MOG-specific T cells but not by aB-specific T cells; (2) locality of expression of aB protein in the CNS relative to the leukocytic infiltrates; and (3) possible differences in the migration pattern of aBspecific vs. MOG-specific T cells to the CNS. Acknowledgements This work was supported by a Pilot Project Award (PP0898), research grants RD3405A2, RG3400A4, RG3468A from the National Multiple Sclerosis Society, and NIH Grants NS23444, NS47661, AI43960, NS41965, NS46877, NS45445, NS49210, The Nancy Davis MS Center Without Walls, and the Biomedical Laboratory R&D Service, Department of Veterans Affairs. The authors wish to thank Sandhya Subramanian for technical assistance and Eva Niehaus for assistance in preparing this manuscript. References Adams, C.W., Poston, R.N., Buk, S.J., 1989. Pathology, histochemistry and immunocytochemistry of lesions in acute multiple sclerosis. J. Neurol. Sci. 92, 291 – 306.

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