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The Journal of Immunology, 2008, 180, 7747 -7756
Copyright © 2008 by The American Association of Immunologists, Inc.
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HLA-DQ6 (DQB1*0601)-Restricted T Cells Protect against Experimental Autoimmune Encephalomyelitis in HLA-DR3.DQ6 Double-Transgenic Mice by Generating Anti-Inflammatory IFN-{gamma}1

Ashutosh Mangalam*, David Luckey*, Eati Basal*, Marshall Behrens*, Moses Rodriguez*,{dagger} and Chella David2,*

* Department of Immunology and {dagger} Department of Neurology, College of Medicine, Mayo Clinic, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The human MHC class II genes are associated with genetic susceptibility to multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the CNS of presumed autoimmune origin. These genes encode for proteins responsible for shaping immune response. The exact role of HLA-DQ and -DR genes in disease pathogenesis is not well-understood due to the high polymorphism, linkage disequilibrium, and heterogeneity of human populations. The advent of HLA class II-transgenic (Tg) mice has helped in answering some of these questions. Previously, using single-Tg mice (expressing the HLA-DR or -DQ gene), we showed that proteolipid protein (PLP)91–110 peptide induced classical experimental autoimmune encephalomyelitis only in DR3.Aβ° mice, suggesting that DR3 (DRB1*0301) is a disease susceptible gene in the context of PLP. Human population studies have suggested that HLA-DQ6 (DQB1*0601) may be a protective gene in MS. To test this disease protection in an experimental model, we generated double-Tg mice expressing both HLA-DR3 and -DQ6. Introduction of DQ6 onto DR3-Tg mice led to a decrease in disease incidence on immunization with PLP91–110 peptide indicating a dominant protective role of DQ6. This protective effect is due to high levels of IFN-{gamma} produced by DQ6-restricted T cells, which suppressed proliferation of encephalitogenic DR3-restricted T cells by inducing apoptosis. Our study indicates that DQ6 modifies the PLP91–110-specific T cell response in DR3 through anti-inflammatory effects of IFN-{gamma}, which is protective for experimental autoimmune encephalomyelitis. Thus, our double-Tg mouse provides a novel model in which to study epistatic interactions between HLA class II molecules in MS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple sclerosis (MS)3 is an inflammatory disease of CNS characterized by demyelinating plaques (1). Although the etiological mechanism is not confirmed, the majority of evidence points toward an autoimmune origin of disease. Both genetic and environmental factors have been shown to play an important role in development of disease. Animal studies as well as data from patients with MS suggest that myelin Ag-specific CD4+ T cells play an important role in development of inflammation and subsequent demyelination in CNS leading to neurological deficit (1). The CD4 T cell response has been observed mainly with myelin proteins such as proteolipid protein (PLP), myelin basic protein, and myelin oligodendrocytic glycoprotein (MOG). The role of CD4+ T cells in disease is further supported by epidemiological studies showing positive association of certain MHC class II genes with MS prevalence. Among the HLA class II haplotypes, the strongest association had been observed with HLA-DR2/DQ6, DR3/DQ2, and DR4/DQ8 (2, 3, 4, 5). The class II association in MS differs in various populations with highest association with HLA-DR2 (DRB1*1501)/DQ6 (DQB1*0602) in Caucasians (2, 5, 6, 7), while DR3 and DR4 genes are more prevalent in MS patients from Jordan, Sweden, Sardinia, and Mexico (2, 4, 8, 9, 10, 11). In contrast, presence of DQB1*0601 and DQB1*0603 has been shown to be negatively associated with development of MS (4, 12, 13, 14). In the human population, it is difficult to analyze the individual role of the DR or DQ alleles in MS due to heterogeneity in MHC genes, linkage disequilibrium, and interaction among different class II genes.

The advent of HLA class II-transgenic (Tg) mice has helped in resolving some of these mysteries. We have shown that PLP91–110 induced experimental autoimmune encephalomyelitis (EAE) in HLA-DR3-Tg mice (15) while MOG and myelin basic protein Ags induced EAE in HLA-DR2- (16, 17) and -DR4-Tg mice (18). None of the studies so far have shown induction of EAE in HLA-DQ-Tg mice. The prevalent data suggest that HLA-DR genes are primarily responsible for susceptibility to MS; however, HLA-DQ genes could play a role in modulating the disease in association with DR genes. The frequency, progression, and severity of disease in human patients differ depending upon the haplotype and heterozygosity. Linkage studies suggest that the DQ6-subtype DQB1*0601 gene may be protective in MS (12, 14). Therefore, we investigated the role of HLA-DQ6 (DQB1*0601) gene in disease-susceptible HLA-DR3-Tg mice. We generated double-Tg mice expressing the protective MS allele DQB1*0601 on disease-susceptible HLA-DR3 background to determine whether presence of DQB1*0601 can lead to a protective phenotype on DR3 background. Because these HLA class II-Tg mice express human class II in the absence of endogenous mouse class II molecule, all the T cell responses in these Tg mice are restricted to human class II molecules. Previously, we have reported that DR3 (DRβ1*0301).Aβ° and DQ6 (DQβ1*0601).Aβ°-Tg mice recognize PLP epitopes similar to human MS patients (15).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Tg mice

The HLA-DQ6 (DQA1*0103, DQB1*0601), HLA-DR3 (DRB1*0301), and HLA-DR3/DQ6-Tg mice were produced, as previously described (19, 20, 21). Briefly, HLA class II transgenes were introduced into (B6 x SWR)F1 fertilized eggs. Positive offspring were backcrossed to B10.M mice for several generations. HLA-Tg mice were then mated to class II-deficient (Aβ°) mice and intercrossed to generate the HLA-Tg lines. To generate double-Tg mice, single-Tg DR3.Abo mice were mated with DQ6.Aβ°-Tg lines to produce HLA-DR3/DQ6-Tg lines. Transgene-negative littermates were used as controls in these studies. All mice were bred and maintained in the pathogen-free Immunogenetics Mouse Colony (Mayo Clinic, Rochester, NY) according to National Institutes of Health and institutional guidelines. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Mayo Clinic.

Flow cytometry

Expression of HLA-DR and HLA-DQ molecules on PBLs/lymph node cells (LNCs)/splenocytes were analyzed by flow cytometry using mAbs L227 and IVD12, specific for HLA-DR and HLA-DQ (22), respectively, as described previously (20). Surface expression of CD4 (GK1.5), CD8 (53.6.72), B cells (RA3-6B2), dendritic cells (DCs) (HL3), monocytes/macrophages (M1/70), NK cells (PK136, CD25 (PC61)), CD127 (A7R34), and CD62L (MEL14) were analyzed using fluorescent-conjugated mAb (BD Biosciences). The TCR Vβ usage of CD4+ T cells was determined on PBLs with mAbs specific for: Vβ2 (B20.6.5), Vβ4 (KT4-10), Vβ5.1,2 (MR9.4), Vβ5.1 (MR9.8), Vβ6 (44.22.1), Vβ7 (TR.310), Vβ8.1,2 (KJ16-133), Vβ8.2 (F23.2), Vβ9 (MR10-2), Vβ11 (RR-153), Vβ14 (14.2), and Vβ17 (KJ23a), as described previously (15).

Peptide

A total of 20-aa-long synthetic peptide PLP91–110 (YTTGAVRQIFGDYKTTICGK) were synthesized at the peptide core facility of Mayo Clinic.

Immunization and T cell proliferation assay

Mice were immunized s.c. with PLP91–110 (100 µg) in CFA. Immunized mice were sacrificed 10 days after immunization; draining lymph nodes were removed and challenged in vitro (19). The results are presented as stimulation indices (cpm of test sample/cpm of the control). For in vitro inhibition experiments, mAbs specific for CD4 (GK1.5), CD8 (TIB 105), HLA-DQ (IVD12), and HLA-DR (L227) were added to LNCs challenged in vitro with human PLP (20 µg/ml).

Intracellular staining for Foxp3

Purified spleen and lymph node cells were stained with PerCp-anti-CD4 together with FITC-anti-CD25 or, after fixation with the fixation/permeabilization buffer, with allophycocyanin-anti-Foxp3 (eBioscience). Stained cells were analyzed using FACSCalibur with CellQuest Pro software (BD Biosciences).

Disease induction

For disease induction, 12- to 14-wk-old Tg mice were immunized s.c. in both flanks with 100 µg of PLP91–110 emulsified in CFA containing Mycobacterium tuberculosis H37Ra (400 µg/mice). Pertussis toxin (Ptx; 100 ng; Sigma-Aldrich) was injected i.v. at day 0 and 48 h postimmunization. Mice were observed daily for clinical symptoms. Disease severity was scored as follows: 0, normal; 1, loss of tail tone; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and forelimb paralysis or weakness; 5, moribundity/death. Mice of both sexes were used in this study.

Cytokine production

Splenocytes were collected 3 wk postimmunization and stimulated with PLP91–110 peptide. Supernatants were collected from culture 48 h after peptide stimulation. The concentration of cytokines (IFN-{gamma}, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, and TNF-{alpha}) in the supernatant was measured by sandwich ELISA using pairs of relevant anti-cytokine mAbs according to the manufacturer’s protocol (BD Pharmingen).

Real-time PCR

Levels of IL-17, IL-21, IL-23, and IL-27 mRNA in vitro cultures were analyzed using real-time PCR. RNA was extracted from cells using RNAeasy columns (Qiagen) and cDNA was prepared using RNase H-reverse transcriptase (Invitrogen). cDNA was analyzed by real-time quantitative PCR in triplicates by using SYBR GreenER qPCR reagent system (Invitrogen). The expression level of each gene was quantified using the threshold cycle (Ct) method normalized for the housekeeping gene GAPDH. The primers for genes encoding IL-17, IL-22, IL-23, IL-27, and GADPH were synthesized as described previously (23, 24).

Neutralization of IFN-{gamma} (anti-cytokine) treatment of EAE

HLA-Tg mice were injected i.p. with 250 µg of anti-IFN-{gamma} (clone H22, mouse IgG), or isotype control (mouse IgG) at days –1 and 10, after immunization (both anti-IFN-{gamma} and isotype control Abs were a gift from Dr. R. Schreiber, Washington University, St. Louis, MO).

Statistical analysis

The statistical significance of the differences in clinical and histological scores between groups was assessed by a one-way ANOVA on ranks (Kruskall-Wallis test) when comparing more than two groups, and by the Mann-Whitney rank-sum test when comparing only two groups. The differences in proliferation or in cytokine levels between groups was assessed by a one-way ANOVA with multiple comparisons of the means when more than two groups were analyzed, or by Student’s t test when only two groups were analyzed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Characterization of HLA-DR3.DQ6-Tg mice

To characterize class II expression on different HLA class II-Tg mice, surface expression of HLA-DR and DQ was measured on mononuclear cells (MNC) from PBLs, LNCs, and splenocytes of naive DR3.Abo, DQ6.Aβ°, DR3.DQ6.Aβ°, and control mice. In PBLs, both HLA-DR and -DQ were expressed on 20–30% of cell population, and a similar level of expression was also observed in LNCs (data not shown). HLA-DR expression (Fig. 1A) was detected on 40–50% of the splenic cell population with highest expression on B cells and DCs in DR3.Aβ°- and DR3DQ6.Aβ°-Tg mice but not in DQ6.Aβ°-Tg or control (Aβ°) mice. Expression of HLA-DQ (Fig. 1B) was also detected on 40–50% of splenocytes from DQ6.Aβ°- and DR3DQ6.Aβ°-Tg mice with highest expression on B cells and DCs while no expression was seen in DR3.Aβ° or class II knockout Aβ° mice. Thus, both HLA-DR and -DQ molecules were expressed at a similar level in DR3.Abo-, DQ6.Aβ°-, and DR3.DQ6.Aβ°-Tg mice.


Figure 1
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  		FIGURE 1. Class II expression and T cell repertoire in single- and double-Tg mice. Normal expression of HLA-DR (A) and/or HLA-DQ (B) was observed in single- and double-Tg mice. Splenocytes were isolated from MHC class II-deficient control mice (Aβ°), DQ6.Aβ°-, DR3.Aβ°-, or DR3.DQ6.Aβ°-Tg mice and analyzed for cell surface markers (HLA-DR/HLA-DQ alone or together with B cells/DC markers) by flow cytometry. Numbers in histograms indicate the percentage of cells positive for the HLA-DR/HLA-DQ marker. Among splenocytes B cells showed maximum class II expression. Data represent one of three experiments performed at different time points. C, Both CD4 and CD8 T cell subsets developed normally in the DR3.DQ6 double-Tg line (similar to DR3.Aβ° and DQ6.Aβ°) whereas a very low CD4+ T cell subset was detected in control Aβ° mice. Splenocytes were isolated from naive MHC class II-deficient mice (Aβ°), DQ6.Aβ°, DR3.Aβ°, or DR3.DQ6.Aβ°-Tg mice and analyzed for cell surface markers (CD4 and CD8) by flow cytometry. Numbers in quadrants indicate percent cells in that gate. Data represent one of four experiments.

 
Cell type analysis showed normal T cell differentiation to both the CD4 and CD8 lineage in DR3.DQ6.Aβ°-Tg mice with a normal number of CD4 and CD8 cells in spleen (Fig. 1C), PBLs, and LNCs (data not shown). The CD4 to CD8 T cell ratio was comparable to other Tg strains (Fig. 1C) and T cells also showed a diverse T cell Vβ repertoire (data not shown). In summary, class II expression and cellular profile of DR3.DQ6.Aβ°-Tg mice was similar to DR3.Abo and DQ6.Aβ° single-Tg mice.

Presence of HLA-DQ decreases disease incidence in susceptible HLA-DR3-Tg mice

The susceptibility and clinical features of single-Tg, double-Tg, and control mice to PLP91–110-induced EAE is presented in Table I and Fig. 2. Administration of PLP91–110 to DR3.Aβ°-Tg mice led to development of chronic progressive clinical disease in 70% (21 of 30) of Tg mice and disease was characterized by ascending paralysis (limp tail followed by hind limb weakness and leading to complete hind limb paralysis). DR3-Tg mice with EAE showed a disease onset of 13 ± 1.5 days and mean clinical disease severity score of 2.3 ± 0.4. Transgene-negative littermates or control Abo mice and DQ6-Tg mice did not develop clinical disease. We observed that immunization of DR3DQ6.Aβ° double-Tg mice with PLP91–110 led to disease development only in 40% (12 of 30) of double-Tg mice, suggesting a protective role of the DQ6 gene. DR3DQ6.Aβ°-Tg mice showed decreased disease severity compared with DR3.Aβ° single-Tg mice (mean clinical score 1.3 ± 0.2 vs 2.4 ± 0.3, p < 0.05). No significant difference between these two groups of mice was detected in the onset of disease. This clinical disease data suggested that DQ6 plays a protective role by inhibiting development of EAE in disease susceptible DR3-Tg mice.


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  		Table I. PLP91–110-induced EAE in HLA-Tg micea

 

Figure 2
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  		FIGURE 2. PLP91–110-induced EAE in single- and double-Tg mice. DR3.Aβ° mice show higher disease incidence as compared with DR3.DQ6.Aβ° double-Tg mice, while no disease was seen in DQ6.Aβ° mice. Five mice per group were immunized with 100 µg of PLP peptide/400 µg of Mycobacterium tuberculosis (Mtb) in CFA. Ptx (100 ng) was administered at 0 and 48 h postimmunization. Mice were scored daily for disease (as stated in Materials and Methods) and the daily mean clinical score for each group is plotted. Error bars represent the SE of mean. The data are from four independent experiments combined.

 
Protective effect of DQ6 is not due to defect in Ag-specific T cell proliferation

The reduced disease incidence in DR3DQ6 mice could be due to Ag presentation and T cell recognition. Therefore, we analyzed PLP91–110-specific immune response in single- and double-Tg mice. As shown in Fig. 3A, PLP91–110-immunized DR3DQ6.Aβ° mice showed a very strong, dose-dependent T cell response to PLP Ag, which were at least 3- to 4-fold higher in magnitude as compared with disease-susceptible DR3.Aβ° mice. HLA-DQ6-Tg mice also showed a very strong T cell-proliferative response as compared with DR3.Aβ°-Tg mice. To confirm that DQ6 molecules bind and activate PLP-specific T cells more strongly then DR3.Aβ°, we performed an Ag-presentation assay with bone marrow-derived DCs (BM-DCs). Bone marrow cells were isolated from DQ6.Aβ° and DR3.Aβ° mice and cultured in presence of IL-4 and GM-CSF as described previously (25) to obtain BM-DCs. CD4+ T cells isolated from PLP91–110-immunized DQ6.Aβ° or DR3.Aβ°-Tg mice, were cultured with 5-day BM-DCs in the presence or absence of Ag. T cells from PLP-immunized DQ6.Aβ° mice showed strong T cell proliferative response as compared with DR3.Aβ° mice even at lower Ag doses (Fig. 3B). The above data indicate that DQ6 molecule can present PLP91–110 Ag better than DR3.Aβ°-Tg mice.


Figure 3
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  		FIGURE 3. DQ6- and DR3DQ6-Tg mice show very strong T cell proliferative response to PLPp91–110 peptide. A, DR3.DQ6.Aβ° and DQ6.Aβ° mice showed 2- to 3-fold higher T cell-proliferative responses to PLP91–110 peptide compared with the T cell response observed in DR3.Aβ°-Tg mice. For measurement of Ag-specific T cell response, draining LNCs from Tg mice were immunized with PLP91–110 peptide and then cultured with or without (control) PLP peptide for 48 h. The proliferative response was assessed by pulsing the cultures with [3H]thymidine for the last 16 h. The data are presented as the mean cpm ± SD and are average of three independent experiments. B, PLP91–110-specific CD4+ T cells from DQ6-Tg mice showed significantly more proliferation as compared with the CD4+ T cells from DR3-Tg mice when cultured with their respective BM-DCs. Bone marrow cells were isolated from DQ6.Aβ° or DR3.Aβ° mice and cultured in presence of IL-4 and GM-CSF to obtain BM-DCs. CD4+ T cells were isolated from draining LNC of PLP91–110 immunized DQ6.Aβ°- or DR3.Aβ°-Tg mice, and were cultured for 48 h with DQ6+/DR3+ BM-DCs in the presence or absence of Ag (0.01 µg to 100 µg/ml). The proliferative response was assessed by pulsing the cultures with [3H]thymidine for the last 16 h. The data are from three independent experiments combined.

 
DR3.DQ6.Aβ° mice produce increased levels of IFN-{gamma}

Because inflammatory cytokines play an important role in development of EAE, we analyzed levels of different Th1, Th2, and Th17 cytokines in PLP91–110-immunized single- and double-Tg mice. For in vitro cytokine analysis, Tg mice were immunized with 100 µg of PLP91–110 and MNCs from draining LNs were stimulated in vitro with 25 µg of PLP91–110 Ag. Disease-susceptible DR3.Aβ°-Tg mice produced moderate to high levels of IFN-{gamma}, TNF-{alpha}, IL-2, IL-6, and IL-12 cytokines (Fig. 4A), showing classical Th1 phenotype. The disease-resistant DQ6 and protected DR3DQ6-Tg mice also produced very high levels of IFN-{gamma}, a cytokine normally associated with development of EAE. Beside high levels of IFN-{gamma}, DR3DQ6.Aβ° double-Tg mice also produced a moderate level of IL-10 and high levels of IL-2. IL-4 levels were below detection limits in all samples from single- and double-Tg mice. DR3.Aβ°-Tg mice also produced IL-17, IL-22, and IL-23, while levels of these cytokines were below detection limits in DQ6 or DR3DQ6 mice (Fig. 4). However, T cells from DQ6.Aβ° and DR3DQ6.Aβ° mice produced a higher level of IL-27 as compared with DR3.Aβ°-Tg mice. Thus, DQ6.Aβ°-Tg mice are disease resistant and DR3.DQ6.Aβ° are protected from EAE despite producing very high levels of IFN-{gamma}.


Figure 4
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  		FIGURE 4. Cytokine levels in single and double-Tg mice immunized with PLPp91–110. A, T cells from PLP91–110 peptide-immunized DR3.DQ6.Aβ° and DQ6.Aβ° mice produced high levels of IFN-{gamma} compared with DR3.Aβ° mice, while T cells from DR3.Aβ° produced increased levels of IL-6, IL-12, and TNF-{alpha}. Cytokines level in culture supernatants were determined by standard sandwich ELISA as described in Materials and Methods. B, T cells from PLP91–110 peptide immunized DR3.DQ6.Aβ° and DQ6.Aβ° mice showed higher expression of IL-27 compared with DR3.Aβ° mice, while T cells from DR3.Aβ° produced increased levels of IL-17, IL-22, and IL-23. Expression of Th17-related cytokines in different Tg mice were quantified by real-time PCR. Expression of GADPH was measured as an internal control. The expression of different cytokines in LNCs stimulated with PLP91–110 relative to that in medium control was calculated by the {Delta}{Delta}Ct method. Data are presented as means ± SD of at least four different mice. *, p ≤ 0.01 as compared with DR3.Aβ°.

 
Increased levels of IFN-{gamma} is produced by DQ6-specific T cells and not by CD8 T cells or NK cells

The above data was consistent with the hypothesis that the protective effect of DR3.DQ6.Aβ°-Tg mice might be due to high levels of IFN-{gamma}. To analyze the source of this IFN-{gamma}, we performed the IFN-{gamma}-ELISPOT assay, a standard assay for analyzing Ag-specific IFN-{gamma} levels. We stimulated LNCs from PLP91–110-immunized DR3.Aβ°-, DQ6.Aβ°-, and DR3.DQ6.Aβ°-Tg mice with PLP peptide in the presence or absence of blocking Abs to CD4 T cells (GK1.5), CD8 T cells (53.6.72), and NK cells (NK1.1) in special ELISPOT plates with membranes. As shown in Fig. 5A, only blocking with anti-CD4 Ab suppressed IFN-{gamma} spots in DR3.DQ6.Aβ° as well as in DR3.Aβ°- and DQ6.Aβ°-Tg mice. Although anti-CD8-blocking Ab had no effect on IFN-{gamma} spot, anti-NK1.1-blocking Ab caused a modest increase in IFN-{gamma} spots, suggesting presence of regulatory NK or NKT cells. These results suggested that the source of IFN-{gamma} was CD4 T cells and not CD8 T cells or NK cells.


Figure 5
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  		FIGURE 5. Source and specificity of IFN-{gamma} in DR3-, DQ6-, and DR3DQ6-Tg mice. A, IFN-{gamma} was mostly produced by PLP91–110-specific CD4+ T cells in in vitro culture. For blocking experiments, LNCs were isolated from PLP91–110 peptide immunized DR3, DQ6, and DR3DQ6-Tg mice and cocultured in special ELISPOT plates with PLP91–110 peptide in the presence or absence of various blocking mAbs specific for CD4 (GK1.5), CD8 (53.6.72), or NK cells (NK1.1). After 48 h in culture, IFN-{gamma} spot was measured as described in Materials and Methods. The results are representative for three independent experiments for all groups. Increased levels of IFN-{gamma} in DR3DQ6-Tg mice were produced by DQ6-restricted PLP91–110-specific CD4+ T cells as blocking mAb specific for DQ (IVD12) inhibited PLP91–110-specific T cells response (B) as well as IFN-{gamma} spot (C) in DR3DQ6-Tg mice. For blocking experiments, LNCs were isolated from PLP91–110 peptide immunized DR3-, DQ6-, and DR3DQ6-Tg mice and cocultured with PLP91–110 peptide in the presence or absence of blocking mAbs specific for HLA-DQ (IVD-12) or HLA-DR (L-227) for 48 h in tissue-culture plates (T cell proliferation) or special ELISPOT plate. The proliferative response was assessed by pulsing the cultures with [3H]thymidine for last 16 h, while IFN-{gamma} spot was measured as described in Materials and Methods. The data are presented as the mean ± SD and are average of three independent experiments.

 
To determine the specificity of CD4 T cells, we stimulated LNCs from PLP91–110-immunized DR3.Aβ°-, DQ6.Aβ°-, and DR3.DQ6.Aβ°-Tg mice with PLP peptide in the presence or absence of blocking Abs to HLA-DR (L-227) or HLA-DQ (IVD-12) and the T cell proliferation was measured using standard thymidine incorporation assay. As expected, HLA-DR Ab inhibited T cell proliferation of DR3.Aβ° (Fig. 5B) but not DQ6.Aβ° mice, and similarly anti-DQ Ab IVD-12 inhibited T cell response in DQ6 mice (Fig. 5B) but not in DR3-specific T cell cultures. In DR3DQ6.Abo-specific culture, the majority of T cell response (>85%) was inhibited by DQ6-specific Ab IVD-12, while anti-DR Ab was able to suppress only 10–15% of T cell response.

ELISPOT assay was done in presence or absence of anti-DR- and anti-DQ-blocking Abs to investigate whether IFN-{gamma} is produced by DQ6-specific T cells or DR-specific T cells in DR3DQ6.Aβ° double-Tg mice. DR3DQ6.Aβ°-Tg mice produced much higher numbers of IFN-{gamma} spots (500 ± 50 vs 75 ± 20, p < 0.01) compared with DR3.Aβ° single-Tg mice (Fig. 5C). These IFN-{gamma} spots were significantly suppressed (>85%) with blocking anti-DQ Ab in DR3.DQ6.Aβ° mice, whereas anti-DR Ab suppressed only 10–15% of IFN-{gamma} spots. However, when a very high amount of PLP91–110 peptide was added to the culture, there was an increase in DR-specific IFN-{gamma} ELISPOTs in DR3DQ6.Aβ°-specific cultures (data not shown). As expected, anti-DR Ab suppressed IFN-{gamma} spot in DR3.Aβ° cultures, whereas anti-DQ Ab suppressed IFN-{gamma} spot in DQ6.Aβ° cultures. These data suggest that high levels of IFN-{gamma} in DR3DQ6.Aβ° double-Tg mice is produced by PLP-restricted DQ6-specific CD4 T cells.

Neutralization of IFN-{gamma} in DR3DQ6.Aβ°-Tg mice abolished the protective effect of the DQ6 molecule

Data from previous experiments suggested that high levels of IFN-{gamma} produced by PLP-specific DQ6-restricted T cells might be responsible for the protective effect of DQ6, leading to low incidence of disease. To confirm role of IFN-{gamma} in disease protection, we investigated whether blocking or reducing this high level of IFN-{gamma} could make these DR3.DQ6.Aβ° mice more susceptible to PLP-induced EAE. Single- and double-Tg mice were immunized with PLP91–110 and treated with 200 µg of neutralizing IFN-{gamma} Ab (clone H-22) or control isotype Ab at days –1 and 10 postimmunization. Double-Tg mice treated with anti-IFN-{gamma} but not with isotype control showed increased disease incidence and severity (Table II and Fig. 6) similar to DR3.Aβ°-Tg mice, confirming a protective role of IFN-{gamma} in this model of EAE. Neutralizing Ab treatment in DQ6.Aβ° mice had no effect.


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  		Table II. Neutralization of IFN-{gamma} in PLP91–110-induced EAE in HLA-Tg micea

 

Figure 6
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  		FIGURE 6. Effect of IFN-{gamma} neutralization in DR3.DQ6.Aβ° and DQ6.Aβ°-Tg mice. Anti-IFN-{gamma} treatment of PLP91–110 peptide-immunized DR3.DQ6.Aβ° mice induce severe EAE compared with DR3.DQ6.Aβ° mice receiving isotype control Ab. DR3.DQ6.Aβ°-Tg mice receiving neutralizing IFN-{gamma} Ab showed higher disease incidence as well as increased disease severity as characterized by higher average clinical score. However, anti-IFN-{gamma} treatment of DQ6.Aβ° mice did not lead to induction of EAE. Isotype Ab had no effect on either strain. For disease induction, DQ6.Aβ°- and DR3DQ6.Aβ°-Tg mice were immunized with 100 µg of PLP91–110 peptide emulsified in CFA containing 400 µg of Mycobacterium. Ptx (100 ng) was administered at 0 and 48 h postimmunization. Five mice per group were also given anti-IFN-{gamma} Ab or isotype control Ab at days –1 and 10 postimmunization. Mice were scored daily for disease (as stated in Materials and Methods) and the daily mean clinical score for each group is plotted. Error bars represent the SE of mean. The data are pooled from two independent experiments.

 
High levels of IFN-{gamma} led to increased T cell apoptosis in DR3.DQ6.Aβ° mice

IFN-{gamma} exerts its anti-inflammatory role through number of pathways including inducing apoptosis of Ag-specific T cells. To investigate whether T cells from DR3DQ6 mice undergo more apoptosis, splenocytes were collected 2 wk after immunization with PLP91–110 (with Ptx) and stimulated in vitro with PLP Ag. T cell apoptosis was assessed by CD4 and annexin V double staining. T cells from DR3DQ6.Aβ°-Tg mice showed a significantly higher number of annexin V-positive staining (Fig. 7, A and B) as compared with T cells from DR3 mice (39 ± 5 vs 12 ± 4, p < 0.001). Similar T cells from DQ6.Aβ°-Tg mice also showed increased T cell apoptosis (Fig. 7, A and B) as compared with DR3DQ6.Aβ°-Tg mice (37 ± 6 vs 12 ± 4, p < 0.001). We also analyzed T cell proliferation by CFSE dilution assay and found that T cells from DR3.DQ6.Aβ° mice underwent more proliferation cycles as compared with T cells from DR3.Aβ° mice (Fig. 7C). Thus, T cells from DR3.DQ6.Aβ° mice underwent increased proliferation and apoptosis as compared with DR3-specific T cells.


Figure 7
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  		FIGURE 7. Apoptosis of Ag-specific T cells in single- and double-Tg mice stimulated with PLP. PLP91–110 peptide-stimulated T cells from DQ6- and DR3DQ6-Tg mice undergo increased apoptosis compared with DR3-Tg mice (p < 0.0001). MNCs isolated from draining LNs of PLP91–110 peptide-immunized DR3.Aβ°, DQ6.Aβ°, or DR3.DQ6.Aβ° mice were cultured with PLP peptide. After 48 h, T cell apoptosis was measured by dual-color flow cytometry using Annexin VFITC and 7-aminoactinomycin D (7AAD). Number in M1 indicates percentage of annexin V+ apoptotic cells in different Tg mice. The data in A are a representative example, whereas B represent the mean ± SD of three individual mice per group. C, T cells from DQ6 and DR3DQ6-Tg mice undergo more proliferation compared with DR3-Tg mice as shown by CFSE dilution assay. LNCs isolated from draining LNs of PLP91–110 peptide-immunized DR3.Aβ°, DQ6.Aβ°, or DR3.DQ6.Aβ° mice were labeled with CFSE and cultured in vitro with PLP91–110. After 48 h in culture, cells were analyzed for CFSE fluorescence after gating on CD4+ T cells. Histogram is a representative example and similar observation was found in three independent experiments.

 
High levels of IFN-{gamma} leads to increased NO production in DR3DQ6.Aβ° mice

IFN-{gamma} can also exert its immune tolerance through increased production of NO or by inducing apoptosis of T cells. Therefore, we assessed the accumulation of NO in vitro cultures of T cells isolated from mice immunized with PLP. In vitro cultures were stimulated with PLP91–110 and NO levels were determined in supernatants using Griess reagents. NO levels were high in DR3.DQ6.Aβ°-Tg mice stimulated with PLP91–110 (Fig. 8), whereas no increase in NO levels were observed in response to PLP in DR3.Aβ° (55 ± 6 Vs 6 ± 2, p < 0.00001). Supernatants from DQ6.Aβ°-Tg mice also showed increased levels of NO after stimulation with PLP91–110. Thus, DR3.DQ6.Aβ° mice showed high levels of NO when stimulated with PLP Ag.


Figure 8
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  		FIGURE 8. PLP91–110-induced NO production in single- and double-Tg mice. LN cells from DQ6- and DR3DQ6-Tg mice produced higher levels of NO after stimulation with PLP91–110 compared with medium alone. DR3-Tg mice did not produce any significant amount of NO when stimulated with PLP91–110. For measurement of NO, MNCs were isolated from draining LNs of PLP91–110 peptide immunized DR3.Aβ°, DQ6.Aβ°, or DR3.DQ6.Aβ° mice and were cultured with PLP peptide. After 48 h, NO levels were measured in cultured supernatant using Griess reagents. The data are presented as the mean± SD and are average of three independent experiments.

 
Disease-resistant DR3DQ6.Aβ° mice show increased levels of CD4+CD25+ T regulatory cells (Tregs)

Finally, we analyzed levels of CD4+CD25+ Tregs in DR3DQ6, DQ6, and DR3 mice. There was no difference in the levels of Tregs in splenocytes from naive mice among different strains. However, after immunization with PLP Ag DR3DQ6 mice as well as DQ6-Tg mice showed higher levels of CD4+CD25+ Tregs compared with DR3 mice. We also analyzed different markers associated with Tregs such as FoxP3 (found on most Tregs), glucocorticoid-induced tumor necrosis factor receptor (GITR), CD62L (high on Tregs), and CD127 (low on Tregs). DR3.DQ6.Aβ° mice not only had increased levels of CD4+CD25+ Tregs, these mice also had a higher percentage of FoxP3+CD4+CD25+ and GITR+CD4+CD25+ cells, suggesting an important role for these Tregs in disease protection (Fig. 9).


Figure 9
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  		FIGURE 9. Levels of CD4+CD25+ Tregs in single- and double-Tg mice. PLP91–110 peptide-immunized DQ6- and DR3DQ6-Tg mice have higher level of CD4+CD25+ Tregs compared with DR3-Tg mice. CD4+CD25+ Tregs from DQ6- and DR3DQ6-Tg mice also show high levels of FoxP3, GITR, and low levels of CD62L. MNCs isolated from draining LNs of PLP91–110 peptide-immunized DR3.Aβ°, DQ6.Aβ°, or DR3.DQ6.Aβ° mice were stained with anti-CD25-PE, anti-Foxp3-allophycocyanin, and anti-CD4-PerCp and either anti-GITR-FITC or anti-CD62L-FITC. The analysis was performed with CellQuest software.

 
DQ6 and DR3 molecules recognize different epitopes within PLP91–110 peptide

Because PLP91–110 is a 20-mer peptide and HLA class II molecule binds to 9- to 12-aa-long peptides, therefore, it was possible that DQ6 and DR3 molecule might recognize different a T cell epitope within PLP91–110. We previously showed that residue 97-108 within PLP91–110 is the minimal epitope required for binding to the DR3 molecule (26). We tested a series of minimal length truncated peptides to identify minimal epitope within the PLP91–110 region, necessary for binding to DQ6 molecule and optimal T cell activation. Draining LNCs were isolated from DQ6 mice immunized with PLP91–110 peptide and challenged with full-length PLP91–110 or individual truncated peptides. Although full-length 20-mer peptide elicited strong T cell response in these Tg mice (stimulation index ≥ 3), removal of amino acid threonine at –NH2 terminal led to significant decrease in the T cell response as compared with those observed with full-length PLP91–110 peptide (Fig. 10). Truncation of additional amino acid from position 94 to 101 resulted in complete loss of T cells response in in vitro culture. In contrast, removal of amino acids from position 101 to 109 at –COOH terminal had no significant effect on proliferation of T cells. Thus, our mapping data suggest that the DQ6 molecule recognizes PLP epitope 91–101 within the PLP91–110 region, which is different from the 97–108 epitope recognized by the DR3 molecule.


Figure 10
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  		FIGURE 10. DQ6 recognizes residues 91-100 within the PLP91–110 peptide. HLA-DQ6-Tg mice were immunized with 100 µg of PLP91–110 peptide and draining LNC were challenged with full length (91-110) or individually truncated peptides for 48 h and the T cell proliferative response was assessed by pulsing the cultures with [3H]thymidine for the last 16 h. The data are presented as the mean ± SD and are the average of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Even though the association of MS with MHC class II genes is well-established, the role of individual DR or DQ genes in MS pathogenesis is not well-understood. Studies with HLA class II-Tg mice established HLA-DR as a major susceptibility gene. However, the role of HLA-DQ in MS pathogenesis is still not clear. It is hypothesized that the HLA-DQ gene might influence disease susceptibility in MS by modulating DR-specific encephalitogenic immune response. In this study, we show experimental proof of the disease modifying effect of a protective class II gene on a MS-susceptible HLA-DR gene. We showed that introduction of DQ6 (DQB1*0601) on disease-susceptible DR3 (DRB1*0301)-Tg mice caused a significant decrease in disease incidence indicating that DQ6 plays a protective role in DR3DQ6 double-Tg mice. Furthermore, we delineated a mechanism for the disease protective phenotype of DR3DQ6.Aβ° and observed that this protective effect of DQ6 was due to anti-inflammatory effect of IFN-{gamma}, which paradoxically is considered a proinflammatory cytokine.

Our results on the protective role of DQ6 (DQB1*0601) are in agreement with MS-linkage studies, where presence of the DQ6 (DQB1*0601) allele has been negatively associated in MS patients (12). In addition, Marrosu et al. (27) reported that the DQB1*0601/0201 gene was associated with decreased disease frequency in Sardinian MS patients. In U.S. Caucasians, MS is linked to DQB1*0602/DRB1*1501(8). Thus, the DQB1*0602 subtype is permissive to MS while DQB1*0601 is protective. Many MS patients are heterozygous for DR2/DR3, therefore also express DQ6 and DQ2. Presence of the DQB1*0601 subtype in these patients should be protective. We have previously shown that DR2 mice are susceptible to MOG-induced EAE while DR2/DQ6 are protected (17). In this study, we show that DR3 mice are susceptible to PLP91–110 induced EAE, whereas DR3DQ6 are protected. The above data indicates that DQB1*0601 can protect EAE mediated by either DR2 or DR3.

How certain MHC class II molecule confers susceptibility in MS while others are resistant or protective is not well-understood. The simplest explanation would be that either T cells from DQ6 and DR3DQ6 mice do not present PLP91–110 peptide, or PLP91–110-specific T cells are deleted during thymic selection. However, our data show that T cells from DQ6 and DR3DQ6 mice are not tolerant to encephalitogenic PLP91–110 peptide and can generate a robust T cell recall response. The Ag-presentation assay using BM-DCs showed that DQ6 molecule induces 3- to 4-fold higher T cell proliferative responses as compared with the DR3 molecule. We also identified that DQ6 bind to residue 91-100 within PLP91–110 peptide as opposed to 97-108 recognized by the DR3 molecule (26). RANKPEP, online software for prediction of peptide binding to the class II MHC molecule also predicted a similar epitope for DQ6 (91–100) and DR3 (98–107). Further RANKPEP software prediction also suggests that DQ6 has a higher affinity for PLP91–110 peptide compared with the DR3 molecule. Our data together with RANKPEP analysis imply that the DQ6 molecule can bind and activates PLP91–110-specific T cells better than the DR3 molecule. These results are in agreement with an earlier study suggesting (28) that alleles such as DQ6 might protect individuals from development of autoimmune diseases as they have higher affinity for self peptide as compared with disease-susceptible alleles.

Another possibility is that DQ6-restricted PLP91–110-specific T cells may be the Th2 phenotype, because it is well-established that T cells of Th1 phenotype secreting IFN-{gamma} are encephalitogenic. Surprisingly, we found that both DQ6 as well as DR3DQ6-Tg mice produced 2- to 3-fold higher levels of IFN-{gamma} as compared with disease-susceptible DR3-Tg mice. IFN-{gamma} can be produced by other cells such as NK cells, which are shown to exert their anti-inflammatory role through IFN-{gamma} (29, 30). The Ab-blocking studies to identify the source of this IFN-{gamma} confirmed that the source of IFN-{gamma} was CD4 T cells because levels of IFN-{gamma} were suppressed only in the presence of anti-CD4 Ab. We analyzed whether the IFN-{gamma} was produced by the DQ6- or DR3-restricted T cells. ELISPOT results indicated that most of the IFN-{gamma} in DR3DQ6 mice were produced by DQ6-restricted T cells. However, addition of larger amounts of peptide led to an increase in DR-specific IFN-{gamma} response in culture from double-Tg mice, suggesting that DQ6 molecule had a higher affinity for PLP91–110 peptide as compared with the DR3 molecule.

Our cytokine data suggest that higher levels of IFN-{gamma} produced by the DQ6-restricted T cells act as an anti-inflammatory cytokine that protect DR3.DQ6 double-Tg mice from EAE. Blocking of IFN-{gamma} in DR3DQ6 double-Tg mice with anti-IFN-{gamma} Ab led to increased disease incidence, confirming our hypothesis. Thus, while low levels of IFN-{gamma} in DR3 mice are proinflammatory, high levels in DR3DQ6 mice are protective. Although IFN-{gamma} is considered a proinflammatory cytokine, the anti-inflammatory role of IFN-{gamma} is well-established (31, 32, 33, 34, 35, 36, 37). Genetic deletion of IFN-{gamma} or IFN-R gene caused exacerbation of EAE in a certain strain of mice (35, 37, 38). A number of other murine studies have shown that IFN-{gamma} confers resistance to EAE development and blockage or deletion of IFN-{gamma} leads to increased disease severity (37, 39, 40). Recently, it was shown that IFN-{gamma} plays an anti-inflammatory role in feedback regulation of murine autoimmunity (31). Neutralization of IFN-{gamma} had no effect on disease induction in DQ6-Tg mice. Minguela et al. (31) showed that production of anti-inflammatory IFN-{gamma} is a dosage effect. They were able to induce EAE using very low doses of the same Ag. However, in our study, even at lower doses (10, 1, and 0.1 µg) of PLP91–110 Ag, none of the DQ6.Aβ°-Tg mice developed disease (data not shown), suggesting that either there are additional suppressive mechanisms in DQ6-Tg mice or that it lacks some inflammatory cytokine such as IL-17, TNF-{alpha}, IL-6, or IL-12 necessary for disease induction. EAE-susceptible DR3.Aβ° mice produced moderate amounts of IL-17 and IL-23, while disease-resistant DQ6.Aβ° mice did not produce any IL-17. IL-17 is a newly described inflammatory cytokine and has been shown to play an important role in disease pathogenesis of EAE. It is possible that down-regulation of IL-17 is one of the mechanisms for the disease-protective effect of IFN-{gamma} in our model. This is in agreement with recent reports that high levels of IFN-{gamma} can restore normoglycemia in NOD mice by down regulating IL-17 levels (41).

IFN-{gamma} can also mediate its anti-inflammatory effect through various mechanisms such as T cell apoptosis, induction of the NO pathway, and through activation of CD4+CD25+ Tregs (33). IFN-{gamma} has been shown to induce apoptosis of T cells in number of studies (42). We found that DR3DQ6-Tg mice showed a higher percentage of annexin V-positive T cells as compared with DR3, suggesting that significant numbers of Ag-specific T cells die in periphery due to high levels of IFN-{gamma}, and very few cells can reach the CNS, leading to low incidence of disease. We also observed that culture supernatants from DR3DQ6 mice produced higher levels of NO as compared with DR3-Tg mice. Previously, it has been shown that IFN-{gamma} down-regulates EAE by inducing inducible NO synthase and subsequent NO production (36). NO can exert its anti-inflammatory effect through a number of pathways such as induction of T cell apoptosis, and interference of Ag presentation (30, 32, 34, 36). Lastly, we found that although there was no difference in the number of Tregs among naive single- and double-Tg mice, DR3.DQ6 mice showed a higher number of CD4+CD25+ Treg cells. These CD4+CD25+ T cells expressed different markers associated with functional Tregs such as FoxP3, GITR, and CD62L (43). A recent study by Wang et al. (33) has shown that IFN-{gamma} can induce Foxp3 expression on Tregs and convert CD4+CD25– T effector cells into CD4+CD25+ Tregs.

We propose that the disease-resistant DQ6 allele modulates disease in DR3.Aβ°-Tg mice through the anti-inflammatory effects of IFN-{gamma}. IFN-{gamma} mediates its effect through the suppression of IL-17 levels, induction of apoptosis of T cells, increased production of NO, and generation of Tregs. We propose that moderate levels of IFN-{gamma} act as a proinflammatory cytokine, inducing disease development, while at very high levels it causes feedback inhibition. Our studies imply that incidence, progression, severity, and modulation of EAE are dependent on epistatic interactions between MHC class II molecules. Similarly, interactions between HLA class II genes in human will determine the course of MS. Knowledge of such interactions could aid in designing individualized therapy for MS patients.


   Acknowledgments
 
We thank Julie Hanson and her staff for mouse husbandry and Michele Smart for tissue typing of Tg mice. We also thank Lauri Zoecklein and Louiza Papke for excellent technical assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institute of Health Grants NS 0521732, NS24180, and NS32149 and National Multiple Sclerosis Society Grants CA1011-03 and RG3172. Back

2 Address correspondence and reprint requests to Dr. Chella David, Department of Immunology, College of Medicine, Mayo Clinic 200, 1st Street SW, Rochester, MN 55905. E-mail address: david.chella{at}mayo.edu Back

3 Abbreviations used in this paper: MS, multiple sclerosis; PLP, proteolipid protein; MOG, myelin oligodendrocytic glycoprotein; Tg, transgenic; EAE, experimental autoimmune encephalomyelitis; LNC, lymph node cell; DC, dendritic cell; Ptx, pertussis toxin; Ct, cycle threshold; BM-DC, bone marrow-derived DC; Treg, T regulatory cell; Mtb, M. tuberculosis; MNC, mononuclear cell; GITR, glucocorticoid-induced tumor necrosis factor receptor. Back

Received for publication August 8, 2007. Accepted for publication March 31, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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