HLA DR and DQ interaction in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in HLA class II transgenic mice

Journal of Neuroimmunology 169 (2005) 1 – 12 www.elsevier.com/locate/jneuroim

HLA DR and DQ interaction in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in HLA class II transgenic mice Meenakshi Khare, Ashutosh Mangalam, Moses Rodriguez, Chella S. David * Department of Immunology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905, USA Received 22 June 2005; accepted 20 July 2005

Abstract Multiple sclerosis (MS) is shown to be associated with the HLA class II genes. The presence of strong linkage disequilibrium between HLA DR and DQ molecules in humans makes it difficult to identify the individual roles of HLA DR and HLA DQ molecule in MS pathogenesis. To address this problem, we used HLA class II transgenic mice and the experimental autoimmune encephalitis (EAE) model. Administration of recombinant MOG (rMOG) induced severe inflammation and demyelination in the central nervous system (CNS) of HLA DRB1*1502 mice (60%), whereas no disease was observed in HLA DQB1*0601(0%) and mild disease was observed in DQB1*0302 mice (13%). Lymphocyte proliferation was blocked by anti HLA antibodies, confirming that the rMOG was functionally presented by the HLA molecules. Introduction of DQB1*0302 into DRB1*1502 mice resulted in the development of chronic progressive clinical disease characterized by severe inflammation and demyelination (90%) in response to immunization with rMOG, whereas mild disease was observed when DQB1*0601 was introduced in DRB1*1502 mice (30%). This would suggest that the presence of more than one susceptible allele, namely HLA DRB1*1502 and DQB1*0302 resulted in enhanced severity of disease in the DRB1*1502/DQB1*0302 mice, possibly due to the additional selection and expansion of potential autoreactive T cells. The use of defined single and double HLA transgenic mice may reveal the intricate interactions between class II molecules in human disease. D 2005 Elsevier B.V. All rights reserved. Keywords: EAE/MS; HLA class II; Transgenic; Pathogenesis; Myelin oligodendrocyte glycoprotein

1. Introduction Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterized by chronic inflammatory demyelination, leading to progressive loss of neurological function (Steinman, 1996). Although the etiology and pathogenesis of MS is poorly understood, both environmental and genetic factors are suggested to play an important role. It is hypothesized that

Abbreviations: EAE, experimental autoimmune encephalomyelitis; LNC, lymph node cells; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; PBMC, peripheral blood mononuclear cell; PLP, proteolipid protein; rMOG, recombinant MOG; SI, stimulation index. * Corresponding author. Tel.: +1 507 284 8182; fax: +1 507 266 0981. E-mail address: [email protected] (C.S. David). 0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2005.07.023

CD4 + T cells play an essential role in the pathogenesis of MS by targeting CNS antigens, such as myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) (Hellings et al., 2002). Experimental autoimmune encephalomyelitis (EAE), a model of MS, can be induced experimentally either by immunization with PLP, MBP or MOG, or by adoptive transfer of myelin antigen-specific activated CD4 + T cells in susceptible strains of laboratory animals(Gold et al., 2000). MOG is a quantitatively minor component of CNS myelin found only in mammals and is highly conserved across species. MOG is the only autoantigen known to induce both an encephalogenetic T cell response as well as a demyelinating antibody response in rodents and non-human primates with EAE (Iglesias et al., 2001; Weissert et al., 1998). This is in contrast to other myelin antigens such as

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M. Khare et al. / Journal of Neuroimmunology 169 (2005) 1 – 12

PLP and MBP, which are encephalogenic but unable to induce demyelinating antibody response. Large concentric areas of macrophage infiltration, autoantibody deposition, and vesicular demyelination are characteristic of MOGinduced EAE (Genain et al., 1999; Kerlero de Rosbo et al., 1997; Raine et al., 1999). In MOG-induced EAE this combination of immune effector mechanisms reproduces demyelinating pathology seen in a subset of MS patients (Lucchinetti et al., 2000). Recent studies in MS patients have indicated that they have a high frequency of MOGspecific autoreactive T cells in their peripheral blood, serum and cerebrospinal fluid (CSF) (Diaz-Villoslada et al., 1999; Hellings et al., 2001; Kerlero de Rosbo et al., 1997; Kerlero de Rosbo et al., 1993) as well as a high titer of anti-MOG antibody in the serum and CSF (Lindert et al., 1999; Reindl et al., 1999; Sun et al., 1991). Moreover, MOG reactive T and B cells from human and EAE animals share common epitopes (Haase and Schmidt, 2001; Kerlero de Rosbo et al., 1997; Khare et al., 2003). As with many autoimmune diseases, linkage and association studies have established that the strongest determinant of genetic susceptibility to MS maps to the HLA region in chromosome 6, specifically to the HLA class II haplotype (Ebers and Sadovnick, 1994; Haines et al., 2002). The association of MS with the HLA class II haplotypes is heterogeneous in different populations. The strongest association of MS is with the HLA DR2 haplotype (DRB1*1501,DRB5*0101,DQA1*0102 and DQB1*0602) found in Caucasians, northern European populations, as well as other non-European ethnic populations (Alvarado-de la Barrera et al., 2000; Hauser et al., 1989; Kankonkar et al., 2003; Kwon et al., 1999; Olerup and Hillert, 1991; SaruhanDireskeneli et al., 1997). However a small proportion of Japanese MS patients possess the DRB1*1502 but not DRB1*1501 allele (Kawamura et al., 2000). MS patients carrying DRB1*1501 in combination with DQB1*0601 (Sejeanston et al., 1992) and DQB1*0603 (Spurkland et al., 1997) alleles had also been reported. A positive association of HLA DRB1*0301 and DRB1*0401 with MS was observed in Sardinian and other Mediterranean populations (Giordano et al., 2002; Marrosu et al., 1997). While it is thought that HLA DR molecules associated with MS present autoantigenic peptides to initiate the disease process (Martin et al., 1992), some studies have suggested that susceptibility to MS may be associated with the HLA-DR and -DQ molecules (Spurkland et al., 1991). Elucidation of the role of individual HLA-DR or -DQ molecule in human MS is difficult due to strong linkage disequilibrium between certain DR and DQ genes and the heterozygosity of affected individuals. The goal of this study was to determine whether presence of susceptible or resistant HLA-DQ allele with susceptible HLA-DR can modulate the outcome of disease severity in HLA class II transgenic mice. We have generated transgenic mice that express individual HLA-DR and HLA-DQ molecules lacking endogenous mouse class II molecules.

Therefore, the only functional class II molecules on APCs are the human class II molecules, mediating the CD4+T cell responses. We have also generated the double transgenic mice carrying both DR as well DQ allele to recreate the coexpression of HLA-DR and -DQ alleles in human MS. EAE, a well-characterized murine model of MS with MOG, was induced in these transgenic mice to explore the contributions of individual as well as dual HLA molecules in disease pathogenesis. We demonstrate here that DRB1*1502 mice were highly susceptible to rMOG induced EAE, where as DQB1*0302 mice showed mild disease and no disease was observed in DQB1*0601 mice. Of interest, introduction of susceptible HLA molecule DQB1*0302 in susceptible DRB1*1502 mice enhances the severity of disease in DRB1*1502 mice, where as introduction of resistant allele DQB1*0601 suppresses the disease severity in DRB1*1502 mice. The result suggests that expression of DR molecule is required for induction of disease. Expression of DQ molecule alone does not have any effect on disease induction, however its presence can modulate disease profile in HLA-DR transgenic mice. This study is the first to correlate the association of disease severity and pathogenesis in MOG-induced EAE with individual human HLA molecules.

2. Methods 2.1. Transgenic mice The production and characterization of transgenic mice expressing HLA-DRB1*1502 (DR2), DQA1*0103/ DQB1*0601 (DQ6) and DQA1*0301/DQB1*0302 (DQ8) genes have been described previously (Khare et al., 2003). The HLA transgenic mice were then mated to class IIdeficient (Abo) mice and the lines generated by a backcrossintercross mating scheme for several generations. Mating of AboDRB1*1502 and AboDQB1*0302 transgenic mice generated double transgenic AboDRB1*1502/DQB1*0302 (DR2/DQ8) mice. The transgene negative littermates were used as controls. Similarly, transgenic mice that expressed Abo.DRB1*1502 and Abo.DQB1*0601 genes were mated to produce Abo.DRB1*1502/DQB1*0601 (DR2/DQ6) mice. Thus, the different transgenic lines have similar background genes and differ from the controls only for the expression of HLA class II genes. All mice used in this study were bred and maintained in the pathogen-free Immunogenetics Mouse Colony at the Mayo Clinic (Rochester, MN). Mice of both sexes were used in this study and were 8– 12 weeks old. All procedures were in accordance with the Mayo Institutional Animal Care and Use Committee. 2.2. Antigens Recombinant MOG (rMOG) corresponding to the extracellular domain of rat myelin oligodendrocyte glyco-

M. Khare et al. / Journal of Neuroimmunology 169 (2005) 1 – 12

protein (aa1-125) was prepared as described previously (Amor et al., 1994). Briefly rMOG was expressed in E.coli. Strain DH5a (generously provided by Dr. C. Linington, Max Plank Institute, Germany) and purified by Nickel chelate affinity chromatography on chelating Sepharose Fast Flow in 6M urea by using a continuous imidazol gradient (40 – 500 mM). This was followed by dialysis against acetate buffer (pH 3.0) and stored at 20 -C. rMOG purity was conducted by SDS-PAGE and Western blot analysis. Amino acid sequence similarity between human and rat MOG was 96% as were the amino acid sequence of human and mouse. For fine mapping of MOG epitopes, a panel of 16 overlapping 20 mer (overlap by 10 amino acids, Khare et al., 2003) peptides encompassing the sequence of human MOG (aa 1-140) were synthesized at the Peptide Core Facility of the Mayo Clinic, Rochester, MN using an automated 430A peptide synthesizer (Applied Biosystems). 2.3. Flow cytometry Expression of HLA-DR, HLA-DQ, CD4, CD8 and mouse H2-A and H2-E molecules on peripheral blood lymphocytes (PBLs) was analyzed by flow cytometry using monoclonal antibodies (mAbs) specific for HLA-DQ a chain (IVD-12), HLA-DR (L227), H2E-h (Y-17), H2Ea (14.4.4), H2A-a (7-16-17), H2A-h (25 5 16), CD4 (GK1.5) and CD8 (53.6.72) prepared in our laboratory (Das et al., 2000). 2.4. T cell proliferation assay Transgenic mice and negative littermates were immunized with 200 Ag of rMOG or individual peptide emulsified (1 : 1) with CFA (Difco Laboratories, Detroit, MI,). T cell proliferative responses were assessed using [3H] thymidine incorporation as described previously(Krco et al., 1992). The results were expressed as the change (D) in cpm and are calculated as Dcpm = (mean cpm of triplicate Ag-containing cultures) (mean cpm of triplicate medium containing cultures) and as stimulation index (SI) = mean cpm of Ag-containing cultures / mean cpm of medium containing cultures. For in vitro inhibition studies, specific mAbs (20 Al of culture supernatant/well) were added to LNCs challenged in vitro with rMOG. 2.5. Cytokine analysis Cytokines were measured in supernatants collected after 72 h from cultured lymph node cells as described previously (Khare et al., 2003). Capture ELISA was done for measuring cytokines IL-4, IL-6, IL-10, IL-12p70 and IFN-g using matched antibody pairs and standards according to the guidelines of manufacturers (Genzyme Diagnostics, Cambridge, MA).

3

2.6. Serum ELISA Serum antibodies were assayed using rMOG or MOG peptide coated plates as described previously (Khare et al., 2003) using alkaline phosphatase-conjugated (AP) goat anti-mouse IgG (Jackson ImmunoResearch) and p-Nitrophenyl phosphate (PNPP; Southern Biotechnology Associates Inc., Birmingham, AL) as substrate. Serum Ab levels were quantified by comparison to purified standards added to each plate. 2.7. Induction and evaluation of EAE All HLA transgenic mice, negative littermates and control mice (Abo) were immunized subcutaneously (s.c.) with 200 Al of an emulsion composed of rMOG (200 Ag/ mouse) in PBS and an equal volume of CFA containing Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI). Mice received 200 ng of pertusis toxin (Sigma Chemicals, St.Louis, MO, USA) in 0.1 ml of PBS (i.v.) on the day 0 following immunization as well as 2 days later. Mice were examined everyday for signs of EAE. The clinical severity was graded into five categories (0, no signs; 1, loss of tail tone; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and forelimb paralysis or weakness; 5, moribund/death). 2.8. Histology-harvesting, and morphology of the CNS 2.8.1. Spinal cord At time of sacrifice mice were perfused with Trump’s fixative via intracardiac puncture. Spinal cords were dissected and cut into 1-mm blocks. Every third block was osmicated and then embedded in glycol methacrylate and stained with a modified erichrome stain with a Cresyl violet counter-stain. The remaining spinal cord blocks were embedded in paraffin for immunoperoxidase staining. Detailed morphologic analysis was performed on 10 – 15 coronal spinal cord sections from each animal. Each Table 1 MOG-induced EAE in HLA transgenic micea Mice strain

Abo C57/BL6 Abo.DR2(1502) Abo.DR2/DQ8 Abo.DR2/DQ6 Abo.DQ6(601) Abo.DQ8

Incidence (%)

Onset day (mean)

Maximum severity score 1

2

3

4

5

0 / 10 (0) 8 / 10 (80) 9 / 15 (60) 18 / 20 (90) 3 / 10 (30) 0 / 12 (0) 2 / 15 (13)

– 12 14 11 19 – 17

– – 2 – 1 – –

– 2 3 1 1 – 1

– 6 4 6 1 – 1

– – – 4 – – –

– – – 7b – – –

a Mice were immunized with 200 Ag of rMOG/400 AgMtb in CFA and Pertusis toxin was administered at 0 and 48 h post immunization. Mice were scored daily for disease(as mentioned in Materials and methods) and maximum severity score is presented for each group. These data are pooled from three independent experiments. b p < 0.01 Mann – Whitney test DR2/DQ8 compared to other mice.

4

M. Khare et al. / Journal of Neuroimmunology 169 (2005) 1 – 12 5

tion without knowledge of genotype or experimental group.

DR2 DQ6

mean clinical score

4

DQ8 DR2/DQ6 DR2/DQ8

3

B6/J Abo 2

1

0 1

4

7

10 13 16 19 22 25 28 31 34 37 40

days postimmunization Fig. 1. EAE induction in HLA transgenic mice with rMOG. Mice were immunized with 200 Ag of rMOG and 400 Ag M.tb in CFA. Pertusis toxin was administered at 0 and 48 h post immunization. Mice were scored daily for disease (as stated in Material and methods) and daily mean clinical score is presented for each group. The data is from three sets of experiments combined.

quadrant from every third spinal cord block from each animal was analyzed for the presence or absence of gray matter disease, white matter inflammation and demyelina-

2.8.2. Brain pathology Following perfusion with Trump’s fixative, two coronal cuts were made in the intact brain at the time of removal from the skull (one section through the optic chiasm and a second section through the infundibulum). This allowed for systematic analysis of the pathology of the cortex, corpus callosum, hippocampus, brainstem, striatum, and cerebellum. The tissue was embedded in paraffin. The resulting slides were then stained with hematoxylin and eosin. Scoring of each part of brain (0, 1, 2, 3 or 4) has been described previously (Drescher et al., 1999). 2.8.3. Isolation and characterization of CNS infiltrating cells Mice were immunized with rMOG for EAE induction as described above followed by pertusis toxin administered at day 0 and 2. Brain and spinal cord were collected from these mice 21 days post immunization as described previously (Johnson et al., 2001) with a slight modification. Mononuclear cells from the brain and spinal cord were extracted by homogenization through a nitex screen into RPMI

Fig. 2. Representative photomicrographs of rMOG-induced inflammatory lesions in spinal cord (A) and cerebellum (B) of HLA-transgenic mice. Transgenic and control mice were immunized with rMOG and sacrificed on day 21. After perfusion with intracardiac injection of 4% paraformaldehyde and 1% glutaraldehyde, the brain and spinal cord were dissected. A) The spinal cord samples were fixed and sectioned coronally into 1 mm blocks, treated with 1% osmium tetroxide and embedded in glycol – methacrylate plastic and stained with a modified erichrome-Cresyl violet stain. The photographs of 2 Am-thick spinal cord sections show inflammation and demyelination in Abo DRB1*1502 and AboDRB1*1502/DQB1*0302 transgenic mice. There was no detectable inflammation or demyelination in Abo or DQB1*0601 mice. DQB1*0302 and DRB1*1502/DQB1*0601 transgenic mice had small focal areas of demyelination and inflammation. Note that the most severe inflammations in the spinal cord of the DRB1*1502/DQB1*0302 mice. The figures are representative of three experiments. B) Brain samples were embedded in paraffin and stained with H and E. Examples of cerebellum pathology are shown. There was parenchymal inflammation and demyelination in DRB1*1502 and DRB1*1502/DQB1*0302 transgenic mice immunized with rMOG. No inflammation or demyelination was observed in transgenic DQB1*0601, DRB1*1502/DQB1*0601, DQB1*0302 and control Abo mice.

M. Khare et al. / Journal of Neuroimmunology 169 (2005) 1 – 12

5

(media). This cell suspension was fractionated using a step gradient consisting of 30% percol (Sigma, St.Louis, MO) diluted in RPMI layered over 75% percol diluted in RPMI. After centrifugation, myelin was aspirated off the top of the 30% percol layer and mononuclear cells were collected and washed with RPMI. RBCs were lysed using ACK lysis buffer and cells were used for FACs or cytokine analysis as described above.

DQB1*0601 played a protective role by reducing incidence and severity of EAE. Histopathology of the brain and the spinal cord revealed moderate degrees of inflammation (37%, Fig. 3A) in the white matter parenchyma, the meninges of the spinal cords, (Fig. 2A) and the brains (Fig. 2B) of the Abo DRB1*1502 transgenic mice. However, the inflammatory infiltrates were

2.9. Statistical analysis

A 100

Percentage (%)

The statistical significance of the differences in mean clinical scores and incidence of EAE among groups was analyzed using Mann – Whitney U test. Elisa data and Ab production data were analyzed by using student’s t test. In all cases, p < 0.05 was considered statistically significant.

3. Results

INFLAMMATION DEMYELINATION

80 60 40 20

8 D

Q

2/ R D

D

R

2/

D

D

Q

6

8 Q

6 D

Q

R D

A

B

bo

6

2

0

3.1. HLA class II transgenic mice are susceptible to EAE induced by rMOG

B 4

2

1

1

0 4

0 4

DQ8

3

3

2

2

1

1 0 4

3

3

2

2

1

1 0

0 4

B6

3 2

Abo

COR

DR2/DQ8

BSTEM

0 4

DR2/DQ6

MEN

2

DQ6

CORP COL

3

HIPP

3

STRIAT

DR2

CER

4

Pathology Score

1 MEN

STRIAT

CORP COL

HIPP

COR

BSTEM

0 CER

To examine the susceptibility of HLA molecules to EAE induced by rMOG, HLA transgenic mice were immunized sub-cutaneously (s.c.) with 200 Ag of rMOG in flanks. Pertusis toxin (200 ng/mouse) was administered at 0 and 48 h post immunization. As shown in Table 1, 60% (9 / 15) of the AboDRB1*1502 mice developed severe EAE after immunization with rMOG (Fig. 1). The mean day of disease onset was 14 T 2, and maximum disease severity score ranged from 1 to 3 (Table 1). No clinical symptoms of disease were seen in transgene negative (Abo) littermate controls, indicating that the presentation of disease was due to the DRB1*1502 molecule. Following the initial acute phase of disease, the majority of AboDRB1*1502 (score 3) went into partial remission (score decreased from 3 to 2), and did not relapse during the remainder of the test period (8– 10 weeks). Immunization of AboDQB1*0601 and AboDQB1*0302 transgenic mice with rMOG resulted in no clinical symptoms or histolopathology characteristic of EAE (Fig. 1, Table 1). In the case of DR/DQ double transgenic mice, 90%( 18 / 20) of Abo DRB1*1502/DQB1*0302 mice developed chronic progressive EAE using the same immunization protocol (Table 1). EAE was characterized by a typical course of ascending paralysis (Fig. 1) with a mean onset of 11 T 2 days and maximum severity scores ranging from 2 to 4 (Table 1). The majority of affected AboDRB1*1502/ DQB1*0302 mice (EAE score 3 or above) never went into remission. In contrast, Abo DRB1*1502/DQB1*0601 mice had a very low incidence of EAE (30%) with only 3 / 10 mice developing mild EAE with mean day onset of 17 T 3 and severity range 1– 2 (Table 1, Fig. 1). This suggested that the presence of DQB1*0302 enhanced the severity of disease in DRB1*1502 mice where as

Fig. 3. Quantitative spinal cord and brain pathology in HLA class II transgenic immunized with rMOG. Transgenic and control mice were immunized with rMOG (200 Ag/mouse) and sacrificed on day 21. After perfusion with intracardiac injection of 4% paraformaldehyde and 1% glutaraldehyde, the brain and spinal cord were dissected. A) Percent of spinal cord quadrants showing inflammation and demyelination (mean T SD). B) Pathology score in brain. Data is shown as a maximal 4-point scale as described in Materials and methods. Each dot represents the histologic score for each mouse.

M. Khare et al. / Journal of Neuroimmunology 169 (2005) 1 – 12

3.2. Kinetics of CNS infiltrating cells in EAE mice To identify the phenotype of cells that cross the blood brain barrier to participate in EAE, cells were isolated from homogenates of brain and spinal cords of diseased and nondiseased mice by Percoll gradient centrifugation, 21 days after rMOG immunization. An increased cellular infiltrate was observed in the CNS of mice, which developed clinical sign of EAE in contrast to the mice that did not develop EAE. The cells isolated from CNS were stained with different antibodies and analyzed by FACs. Higher percentage of CD4 + T cells was present in CNS of mice that developed clinical EAE as compared to the mice who did not develop EAE (Table 2). These CD4 + T cells remained in CNS during the chronic phase of EAE. Further analysis of CD4 + T cells for T cell activation markers revealed an Table 2 CNS infiltrating cells in rMOG-immunized HLA class II transgenic micea MICE

AboDR2 AboDQ6-601 AboDQ8 AboDR2/DQ6 AboDR2/DQ8 Abo a

CD4

EAE No-EAE No-EAE No-EAE EAE No-EAE EAE No-EAE No-EAE

CD8

Mac1

(% of total CD3 + T cells)

(% of total cell population)

39.10 19.00 3.40 9.36 11.27 6.70 52.10 7.91 1.45

36.70 3.80 1.31 1.46 2.10 2.01 38.69 7.12 0.41

26.30 6.20 1.89 3.57 7.89 4.34 31.70 3.58 0.53

B220

29.80 3.10 0.69 0.93 1.31 1.48 33.56 6.89 0.23

FACS analysis of CNS infiltrated cells for different cell phenotypes.

25 Med rMOG

IL-4

20

pg/ml

more extensive (58%, Fig. 3A) in the spinal cords (Fig. 2A) and brains (Fig. 2B) of AboDRB1*1502/DQB1*0302 mice. In Abo DRB1*1502 mice, the infiltrates tended to localize in peri-vascular regions whereas, in the AboDRB1*1502/ DQB1*0302 mice, inflammatory cells infiltrated widely throughout the white matter (Fig. 2A). In the brain, mononuclear cell infiltrates were seen in the meningeal surfaces of the brain stem, cerebellum, cortex and stratum as shown (Fig. 3B). These perivascular and peri-ventricular infiltrates consists of lymphocytes, macrophages and neutrophils. Demyelination was also analyzed in spinal cord sections embedded in plastic. No inflammation or demyelination was observed in the spinal cord of AboDQB1*0601 mice; whereas mild infiltration of inflammatory cells was found in AboDQB1*0302 mice (Fig. 3A, B, 2A, B). In contrast, moderate demyelination (40%) was observed in perivascular lesions of AboDRB1*1502 mice (Fig. 3A). More severe inflammation and demyelination (55%) was observed in AboDRB1*1502/DQB1*0302 mice (Fig. 2A, 3A, B), whereas, only mild inflammation and no demyelination was observed in AboDRB1*1502/DQB1*0601 mice. Therefore the presence of persistent neurologic defects observed in Abo DRB1*1502/DQB1*0302 mice was associated with demyelination in the spinal cord.

15 10 5 0 A

bo

2

8

1

60

R

D

6

Q

6-

D

Q

8

Q

D

2/

Q

D

2/

R

D

R

D

D

1400 Med rMOG

IFN-g

1200 1000

pg/ml

6

800 600 400 200 0 o Ab

01

2

DR

-6

6 DQ

DQ

6

8

8

DQ

D

/ R2

DQ

/ R2

D

Fig. 4. IL-4 and IFN-g secreted by CNS cells in MOG-immunized mice. Mice were immunized as in Fig. 1. Spinal cord and brain infiltrating mononuclear cells (pooled from 4 mice) were isolated using percol gradient and were stimulated with rMOG for 48 h in vitro culture and the cytokine levels were determined as described in the Materials and methods. High IFN-g levels were detected after rMOG stimulation in the HLA DRB1* 1502 and HLA DRB1*1502/DQB1*0302 mice with EAE, and lower levels of IFN-g were observed in DQB1*0601, DQB1*0302 and DRB1*1502/ DQB1*0601 mice ( p < 0.01). There was very little or no IL-4 production by the cells from any of these mice strains.

activated phenotype, as they were CD44 high and CD45Rblow (data not shown). The number of CD8 + T, Mac 1+ and B220+ cells was also found to be increased in mice with clinical EAE as compared to non-diseased mice (Table 2). To identify the Th1 or Th2 phenotype of CNS infiltrating cells, the in vitro production of IFN-g and IL-4 by isolated lymphocytes stimulated with rMOG was measured. Higher level of IFN-g was produced by T cells from Abo DRB1*1502 and Abo DRB1*1502/DQB1*0302 mice as compared to T cells from Abo DQB1*0601, DQB1*0302 or DRB1*1502/DQB1*0601 transgenic mice (Fig. 4). This is consistent with the hypothesis that antigen specific T cells were present in the CNS. 3.3. MOG is presented and recognized by HLA class II molecules: T cell responses to rMOG in HLA class II transgenic mice To examine the immune response of HLA class II transgenic mice against rMOG and to verify that the response is restricted by HLA class II molecules, in vitro LNC proliferation and blocking studies using appropriate monoclonal antibodies were performed. Mice (3 mice/

M. Khare et al. / Journal of Neuroimmunology 169 (2005) 1 – 12

group) were immunized with 200 Ag of rMOG with CFA at the base of tail and hind limbs. Ten days later mice were sacrificed and draining lymph node cells were pooled and stimulated in vitro with the rMOG (20 Ag/ml). As shown in Fig. 5 a strong proliferative response was generated from the lymph node cells (LNCs) of AboDRB1*1502 mice in response to rMOG. This response was inhibited by both antiCD4 and anti-DR mAb (Khare et al., 2003), indicating the response was mediated by CD4 + T cells and restricted by transgenic HLA-DR molecule. T cells from Abo DQB1*0302 also generated a proliferative response, which was weaker than observed in AboDRB1*1502 transgenic mice. As before, both anti CD4mAb and anti HLA-DQ mAb inhibited the T cell response (Khare et al., 2003). Interestingly LNCs from AboDRB1*1502/DQB1*0302 double transgenic mice produced proliferative responses stronger than those seen in either parental transgenic mice (Fig. 5). The proliferative response against rMOG was also assessed in mice expressing HLA DQB1*0601 gene and double transgenic mice expressing HLA DRB1*1502 and DQB1*0601 genes. When immunized with rMOG, the Abo DQB1*0601 mice elicited weak proliferative responses (Fig. 5) against rMOG, whereas the AboDRB1*1502/ DQB1*0601 double transgenic mice mounted moderate proliferative responses (Fig. 5). No proliferative response against rMOG was observed in Abo mice, which are devoid of any mouse or human class II molecule (Fig. 5). Normally proliferative responses were seen in all genotypes except Abo, irrespective of whether or not they developed disease. 70000 60000

delta cpm

50000 40000

DR2 DQ6-601 DQ8 DR2/DQ6 DR2/DQ8 Abo B6

30000 20000 10000 0 0.1

1

5

10

20

50

rMOG µg/ml Fig. 5. T cell proliferative response in HLA transgenic mice with rMOG. Mice were immunized with 200 Ag of rMOG in CFA in the base of the tail and the hind footpads. Ten days later lymph node cells were harvested and cultured (pooled from 3 mice) in the presence of various concentrations of rMOG or medium alone. Proliferation was determined by [3H] thymidine incorporation and was expressed as delta cpm (see Methods). Results are shown as mean T SD from four to five independent experiments conducted on different days. All the transgenic mice showed strong proliferative response with rMOG. The proliferation response was higher in double transgenic mice than in single transgenic mice ( p < 0.01). Using the same conditions no response was found in transgene negative Aß0 mice.

7

This indicated that human HLA class II molecules functionally presented not only pathogenic, but also nonpathogenic epitopes of the rMOG. 3.4. Pathogenesis of EAE in HLA class II transgenic mice: production of both Th1 and Th2 cytokines by MOG-specific T cells and a modifying role for anti-MOG antibodies It has been implicated in various studies that the secretions of particular cytokines by autoreactive T cells play an important role in the development and progression of immune-mediated diseases, such as EAE and MS. To study whether the presence of particular HLA class II transgenes alters the cytokine profile in response to rMOG immunization and thus disease outcome, we measured the cytokines in culture supernatants of LNCs from immunized mice (3 mice/group) that were stimulated in vitro with the immunizing antigen. Culture supernatants were analyzed for both ‘‘pro-inflammatory’’ (IFN-g and IL-12) and ‘‘anti-inflammatory’’ (IL-4, IL-6 and IL-10) cytokines. As in Fig. 6 high amounts of IFN-g and IL-6 were produced by AboDRB1*1502 LNCs, with very low amounts of IL-4, IL-12 and a moderate amount of IL10 (Fig. 6). LNCs of AboDQB1*0302 mice produced low amounts of all cytokines except IL-10. In contrast, LNCs of AboDRB1*1502/DQB1*0302 mice produced the highest levels of all cytokines examined, particularly IFN-g levels. This may explain the increased severity of disease seen in AboDRB1*1502/DQB1*0302 mice compared to AboDRB1*1502 mice, implicating a role for Th1 cytokines such as IFN-g and IL 12 in the pathogenesis of disease. Abo DQB1*0601 mice produce high levels of IL6, IL-10 and IFN-g and moderate levels of IL-4 and IL12, whereas AboDRB1*1502/DQB1*0601 mice produced low levels of all the cytokines examined. Thus, all transgenic mice produced both Th1 and Th2 cytokines after rMOG stimulation, although levels of Th1 cytokines were higher than Th2 cytokines in most of the mice. Altered cytokine levels produced by T cells in these mice could be merely reflecting their increased proliferation response to rMOG. While increase in Th1 cytokines may be correlated to disease severity, it cannot be discounted that the increase in Th2 cytokines (IL-10) may also influence disease development and progression. These data indicated that Th1 cells were the predominantly primed T cell population, although Th2 cells were also stimulated. Anti-MOG Abs have been reported to be present in both MS patients and EAE animals (Genain et al., 1999, 1995). In some cases the severity of MS/ EAE lesions has been attributed to the presence of anti-MOG Ab in lesions (Genain et al., 1999, 1995). To characterize the antibody response to rMOG generated by the HLA transgenic mice, serum was collected by tail bleeding weekly, 2 weeks after immunization with rMOG. A high titer of anti-MOG IgG was detected in all the rMOG immunized transgenic mice,

8

M. Khare et al. / Journal of Neuroimmunology 169 (2005) 1 – 12

Fig. 6. Cytokine profile from peripheral T cells in rMOG immunized HLA transgenic mice. Mice (three per group) were immunized with 200 Ag of rMOG in CFA as in Fig. 1. Draining lymph node cells were collected on the day of sacrifice, and cultured in the presence of rMOG or medium alone for 72 h. Pooled culture supernatants were then analyzed for the presence of cytokines using capture ELISA. Results are from cytokine concentration for one of two representative experiments.

whereas no anti-MOG IgG response was detected in the control Abo mice nor in PBS-immunized HLA class II transgenic mice (Fig. 7). rMOG specific IgG was detected at 2 weeks post-immunization, reached a plateau after 4 – 5

3.5. Mapping of T and B cell determinants of MOG in HLA transgenic mice

2.4 PBS 2

4WKS

1.6

O.D.

weeks, and was still present after 12 weeks post-immunization (data not shown). Both HLA-DR and HLA-DQ transgenic mice generated rMOG-specific IgG, regardless of disease presentation.

1.2 0.8 0.4

R 2/ D

D

R 2/

D

D Q

Q

8

6

8 Q D

1 6-

60

R 2 D Q

D

6 B

A bo

0

Fig. 7. Antibody responses against rMOG in HLA transgenic mice. Transgenic mice were immunized s.c. in the flanks with 200 Ag of rMOG in CFA as in Fig. 1. Sera were obtained from mice after 4 weeks postimmunization. Antibody directed against MOG was analyzed using ELISA as described in Methods. The serum dilutions were 1 : 200 for MOGspecific IgG. Non-specific binding to BSA was subtracted from all the values to calculate the mean absorbance (OD) T SD. Strong responses were found in all transgenic mice.

The data shown above indicate that T cells respond to rMOG in an HLA restricted fashion. Therefore, we examined the epitope recognition of rMOG by the HLA transgenic mice. To do so, transgenic mice were immunized with individual overlapping 20-mer peptides representing entire extracellular region of hMOG protein (aa 1-125) which is 96% identical at the amino acid level to mouse MOG. Ten days post immunization draining LNCs were collected and stimulated in vitro with same hMOG peptides. SI > 3 was considered as positive. T cells from Abo DRB1*1502 mice demonstrated a strong proliferative response to several peptides in N-terminal region of hMOG between aa 1-90 (Table 3). AboDQB1*0302 mice recognized peptides 1 – 30, 61 –80 and 91 – 110. In contrast, T cells from the Abo DRB1*1502/DQB1*0302 mice mounted the strongest response to the previously identified AboDRB1*1502 and DQB1*0302 restricted epitopes. In

M. Khare et al. / Journal of Neuroimmunology 169 (2005) 1 – 12

9

Table 3 A Summary of T and B cell determinants seen by HLA transgenic mice

MOG Peptides

DR2

DQ6-601

DQ8

DR2DQ6

DR2DQ8

1–20 11–30 21–40

T

31–50

B

41–60

T&B

51–70

None

61–80 71–90 81–100 91–110 101–120 111–130 121–140

addition, T cell reactivity to new peptides aa71 – 90 and 111 –130 occurred in AboDRB1*1502/DQB1*0302 transgenic mice (Table 3). T cells from Abo DQB1*0601 mice reacted to epitopes 1– 20, 61 –80 and 91 –110 with a significantly high response to epitope 91 – 110 (SI = 18). The response to hMOG peptide 91 –110 was suppressed by introduction of the DRB1*1502 molecule into the AboDRB1*1502/DQB1*0601 mice, whereas the expression of DQB1*0601 had no effect upon the reactivity of the DRB1*1502 restricted mice (Table 3). These results suggest that combinations of HLA class II genes could differentially regulate peptide-specific T cell responses. We next examined the epitope specificity of anti-MOG antibodies in these transgenic mice by analyzing sera from rMOG-immunized mice (for EAE) using the same set of overlapping 20 mer peptides spanning the N-terminal extracellular portion of hMOG (aa1-125). The main reactivity of anti-MOG antibodies in all mice was directed against three separate regions, 1– 30, 51 –80 and 101 –120 (Table 3). No anti-MOG reactivity was detected in preimmune sera or mice immunized with CFA only (data not shown), indicating that the immune responses were specific for MOG. We found no differences in B cell epitope specificity of MOG peptides in relation to specific DR or DQ molecules. No added specifications were observed in double transgenic mice expressing DR and DQ.

4. Discussion To help define susceptible or resistant HLA class II molecules in MS, we examined EAE induction by rMOG, a well-characterized experimental model of inflammatory demyelination in transgenic mice expressing HLA DRB1* 1502, HLA DQB1*0601 and HLA DQB1*0302 lacking endogenous murine class II molecules. Immunization with rMOG generated a strong CD4 mediated and HLA-DR restricted proliferative response in HLA DRB1*1502 mice, which developed severe EAE (max. severity score — 3) at 13– 14 days post-immunization. Brain sections of HLA DRB1*1502 mice with EAE (score — 3) showed perivascular and periventricular inflammatory infiltrates consisting of lymphocytes and macrophages in various parts of brain (Fig. 3B) including cerebellum (Fig. 2B). FACs analysis showed presence of CD4+, CD8 + T cells, macrophages (MAC1+) and B cells (B220) in CNS infiltrates (Table 2). Mild demyelination was also observed in HLA DRB1*1502 spinal cords (Fig. 2A). A high titer of anti-MOG antibody was also observed in HLA DRB1*1502 transgenic mice (Fig. 7). Both T and B cell responses to MOG were generated by HLA DRB1*1502 mice, resembling the condition of human MS in which both MOG-specific autoreactive T cells and MOG-specific antibodies have been found in peripheral blood as well as in serum and CSF of MS patients

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M. Khare et al. / Journal of Neuroimmunology 169 (2005) 1 – 12

(Lindert et al., 1999; Wallstrom et al., 1998). Similar results were shown in a recent study with MOG peptide 35 –55 in HLA DRB1*1502 mice (Vandenbark et al., 2003). In a previous study HLA DRB1*0401 mice were shown to develop severe EAE upon immunization with encephalogenic peptides of MOG (Forsthuber et al., 2001). Similarly, Finn et al. (2004) showed that MOG 35 –55 induced EAE in both DRB1*1501 as well as DRB1*1502 mice. Whereas, limited studies has been done to investigate the role and effect of HLA DQ genes in MS susceptibility. Immunization of HLA DQB1*0302 with rMOG resulted in a moderate T cell proliferative response and development of mild EAE (max severity score — 2) with low incidence (Table 1). Even though immunization of HLA DQB1*0601 mice also generated a moderate T cell response to rMOG, no clinical or histological signs of EAE were observed. Resistance to EAE in HLA DQB1*0601 mice support the hypothesis that either HLA DQB1*0601 mice lack MOG-restricted autoreactive T cells or that these DQ molecules do not present encephalogenic epitopes. To examine the effects of coexpression of both susceptible and resistant molecules on EAE induction we had introduced DQB1*0302 and DQB1*0601 genes in DRB1* 1502 transgenic mice to generate double transgenic Abo HLA DRB1*1502/DQB1*0601 and HLA DRB1*1502/ DQB1*0302 mice. Even though DRB1*1502/DQB1*0302 genes are not in linkage disequilibrium in the human population they are part of the DRB1*1502/DQB1*0601 and DRB1*0401/DQB1*0302 extended haplotypes found in many individuals prone to MS. Introduction of DQB1*0302 in DRB1*1502 mice resulted in increased T cell proliferative response as compared to those seen in single transgenic mice (DRB1*1502) after immunization with rMOG. Severe clinical signs of EAE with inflammatory and demyelinating lesions in both the brain and spinal cord were observed in a majority of HLA DRB1*1502/DQB1*0302 double transgenic mice, resulting in death 1– 2 week post EAE induction. Even though presence of DQB1*0601 gene in DRB1*1502 mice enhanced the T cell proliferation, it resulted in suppression of severity of EAE in these mice. Thus, the presence of DQB1*0302 enhances the severity of disease in DRB1*1502/DQB1*0302 mice, whereas the presence of DQB1*0601 (DQB1*0601) allele suppresses EAE in DRB1*1502/DQB1*0601 mice. This supports the hypothesis that DQB1*0601 may be playing a protective role against MOG-induced EAE. The next major step is to understand the reason behind the increased severity of MOG-induced EAE in HLA DRB1*1502/DQB1*0302 mice and the decreased severity on DRB1*1502/DQB1*0601 mice. Both T cell autoreactivity and B cell responses to rMOG were higher in HLA DRB1*1502/DQB1*0302 mice in comparison to other transgenic mice. The presence of a higher amount of antiMOG antibody in HLA DRB1*1502/DQB1*0302 transgenic mice may be one possibility for disease severity, as these

antibodies have been shown to enhance disease severity in various animal models of EAE (t Hart et al., 2001). In both MS and the marmoset model of EAE, anti-MOG antibodies have been localized to areas where pathologic changes of white matter occur (Genain et al., 1999). Moreover, a pathologic role for anti-MOG antibodies has been demonstrated in rats (Linington et al., 1988), mice (Morris et al., 1997) and marmosets (Genain et al., 1995). In all three studies, the transfer of antimyelin T cells induced CNS inflammation, but demyelination required the presence of anti-MOG antibodies binding to conformational epitopes on MOG (Brehm et al., 1999). Another possible reason for the increased severity of MOG-induced EAE in DRB1*1502/ DQB1*0302 mice may have been due to a change in the cytokine profile. Increased levels of all the cytokines tested were detected in the HLA DRB1*1502/DQB1*0302 double transgenic mice as compared to parental strains. However, the increased levels of cytokines may have been due to an increased number of T cells in cultures, since a higher T cell proliferation was also observed in the double transgenic mice. A high amount of both ‘‘pro’’ (IFN-g) as well as ‘‘anti’’ (IL10) inflammatory cytokines were produced, which is consistent with several reports in MS/EAE (Okuda et al., 1998). The other possible mechanism for the enhanced severity of disease in HLA DRB1*1502/DQB1*0302 mice may involve antigen presentation by specific HLA molecules as well as TCR repertoire selection. It is possible that the presentation of multiple epitopes of MOG in double transgenic mice as compared to parental mice may have been responsible for the disease severity seen in HLA DRB1*1502/DQB1*0302 mice. Strong T cell responses were found against several human MOG epitopes in HLA DRB1*1502/DQB1*0302 mice (1 –20, 21 –40,31 – 50,61 – 80, 81– 100, 91 – 110 and 111– 130) including responses to epitopes not seen previously in either of the single transgenic DRB1*1502 or DQB1*0302 mice. This would suggest that the presence of both DR and DQ gene resulted in the additional selection and expansion of autoreactive T cells, which may be responsible for the enhanced severity of disease. Interestingly, these mice recognized the immunodominant and encephalogenic T cell epitopes of MOG (1– 22, 34 – 54, 64– 96 and 63– 87), that are observed in MS patients (Kerlero de Rosbo et al., 1997; Wallstrom et al., 1998). Epitope specificity of anti-MOG antibodies was also similar to those found in human MS (1– 26 and 63– 87)(Brehm et al., 1999). However they did not differ between different transgenes, arguing against the role of a unique B cell epitope in disease severity. The data presented in this paper show that presence of DQB1*0302 transgene enhances the disease severity of MOG-induced EAE in HLA DRB1*1502 mice, where as the presence of the DQB1*0601 (DQB1*0601) transgene inhibits EAE development induced by rMOG. These results are consistent with the hypothesis that the expression of the DR molecule is required for the induction of disease.

M. Khare et al. / Journal of Neuroimmunology 169 (2005) 1 – 12

Expression of the DQ molecule alone does not have any effect upon disease induction. However, expression of specific DQ molecules modulates the disease profile. Thus, HLA transgenic mice are novel humanized animal model to study EAE as well as a mean to functionally address a possible role of HLA-DR and/or -DQ molecules in genetic predisposition of MS. Information gained from studies using these transgenic mice can be directly correlated to our understanding of the disease pathogenesis of MS. Studies involving other DR, DQ single and double transgenic mice are in progress and will eventually reveal all the intricate relationship between HLA class II molecules in human disease.

Acknowledgements We would like to thank Dr C. Linington (Max Plank Institute, Germany) for providing the rMOG (amino acids 1 –125), Julie Hanson and her crew for outstanding mouse husbandry, Michele Smart and Susan Demaray for tissue typing, and Dr P. D. Khare for helpful discussions. We also thank Jeff Gomez, Laurie Z. and Louisa Papke for their excellent histologic preparation of tissues. This research was supported by NIH program project grant NS 38468-02 and National Multiple Sclerosis Society Grant FA1578-A-1. The transgenic mice were produced with support from NIH grant AI 14764.

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