MHC variant peptides are analogues of immunogenic peptides involving alterations of the MHC-binding residues, thereby altering the affinity of the peptide for the MHC molecule. Recently, our laboratory demonstrated that immunization of WT B6 mice with 45D, a low-affinity MHC variant peptide of MOG35–55, results in significantly attenuated experimental autoimmune encephalomyelitis (EAE), yet IFN-γ production is comparable to myelin oligodendrocyte glycoprotein (MOG)35–55-immunized mice. In light of these findings, we asked whether IFN-γ was required for the reduced encephalitogenicity of the weak ligand 45D in EAE. In this study, we report that immunization of mice deficient in IFN-γ or its receptor with 45D exhibit significant EAE signs compared with 45D-immunized wild-type B6 mice. Moreover, 45D-immunized IFN-γ−/− and IFN-γR−/− mice demonstrate MOG tetramer-positive CD4+ T cells within the CNS and display substantial numbers of MOG-specific CD4+ T cells in the periphery. In contrast, wild-type mice immunized with 45D exhibit reduced numbers of MOG-specific CD4+ T cells in the periphery and lack MOG tetramer- positive CD4+ T cells in the CNS. Importantly, the increased encephalitogenicity of 45D in mice lacking IFN-γ or IFN-γR was not due to deviation toward an enhanced IL-17-secreting phenotype. These findings demonstrate that IFN-γ significantly attenuates the encephalitogenicity of 45D and are the first to highlight the importance of IFN-γ signaling in setting the threshold level of responsiveness of autoreactive CD4+ T cells to weak ligands.

Experimental autoimmune encephalomyelitis (EAE)3 is an experimental model of multiple sclerosis mediated primarily by autoreactive CD4+ T cells against myelin-producing oligodendrocytes in the CNS. Over the years, various approaches have been used to develop Ag-specific therapies for autoimmune diseases. The ultimate goal of such efforts is the induction of clonal anergy, thereby leaving nonautoreactive T cell clones unaffected. Such strategies include the use of peptides containing substitutions at the self-Ag TCR contact residues, termed altered peptide ligands (APLs). APLs vary in their potency for T cell activation and are categorized as full agonists, weak agonists, partial agonists, or antagonists (1). APLs are capable of inducing T cell anergy (2, 3) as well as T cell antagonism (4). However, the clinical use of this approach has been hampered by several issues, including the inability to induce anergy in a polyclonal T cell population (5), generation of APL-reactive T cell clones (5, 6, 7), and immune deviation to a Th2 phenotype and hypersensitivity reactions (7, 8, 9).

An alternate strategy involves the alteration of the self-peptide MHC anchor residues, which we refer to as MHC variant peptides. These variant peptides have altered affinity for the MHC molecule with little to no effect on the TCR contact regions, thereby allowing the selective targeting of all Ag-reactive T cells in a polyclonal population (i.e., complete anergy of all Ag-reactive cells). Previously, we demonstrated the ability of myelin-associated MHC variant peptides to induce anergy in myelin-reactive CD4+ T cells, which results in reduced encephalitogenicity in several EAE models (5, 10). In the B6 MOG35–55 model, substitution of the MHC anchor residue at position 45 from serine to aspartic acid (termed 45D) results in a 200-fold reduction in the affinity for I-Ab (5) without altering TCR recognition (C. Beal and B. Evavold, unpublished results). The reduced half-life of 45D for the MHC molecule inhibits the proliferation and IL-2 secretion of myelin oligodendrocyte glycoprotein (MOG)35–55-reactive CD4+ T cells without altering the Th1 phenotype (5). Moreover, immunization of B6 mice with 45D does not result in the development of EAE (5), yet CD4+ T cells from these mice secrete significant amounts of IFN-γ (11).

The maintenance of IFN-γ by anergized MOG-reactive CD4+ T cells is highly interesting given the numerous studies demonstrating a protective role for IFN-γ in EAE (12, 13, 14, 15, 16, 17, 18, 19, 20). In light of these findings, we reasoned that IFN-γ may be required to prevent EAE induction by the tolerizing variant peptide 45D. In this study, we report that, in contrast to wild-type (WT) B6 mice, which have been shown to be extremely resistant to EAE induction by 45D, IFN-γ−/− and IFN-γR−/− mice were highly susceptible to the induction of EAE by 45D. Consistent with these findings, MOG tetramer-positive CD4+ T cells were found within the CNS of 45D- immunized IFN-γ−/− and IFN-γR−/− mice, but were not found in the CNS of 45D-immunized WT mice. Moreover, significantly higher absolute numbers of MOG-specific CD4+ T cells were found in the draining lymph nodes of IFN-γ−/− and IFN-γR−/− mice footpad-primed with 45D compared with WT mice. Lastly, immunization with 45D in an IFN-γ-deficient environment did not cause skewing to an enhanced Th17 phenotype, demonstrating that the increased encephalitogenicity of 45D in IFN-γ−/− and IFN-γR−/− mice was mediated by the increased responsiveness of autoreactive CD4+ T cells. Thus, this illustrates the importance of IFN-γ in negatively regulating the response of autoreactive CD4+ T cells to weak ligands for myelin Ags and suggests a crucial role for IFN-γ in the induction of peripheral tolerance.

Female WT, IFN-γ−/− and IFN-γR−/− C57BL/6 mice (H-2b) were purchased from The Jackson Laboratory. Mice were housed in an Emory University Department of Animal Resources facility and used in accordance with an Institutional Animal Care and Use Committee-approved protocol. Mice were used for experiments at 6–8 wk of age.

MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) and 45D (MEVGWYRSPFDRVVHLYRNGK) were purchased from Biosynthesis and the Emory University Microchemical Core Facility (Atlanta, GA). Peptides were analyzed by mass spectrometry and HPLC.

EAE was induced by s.c. immunization in the hind flanks on days 0 and 7 using 100 μg of MOG35–55 or 45D emulsified in CFA containing 2.5 mg/ml heat-inactivated Mycobacterium tuberculosis (Difco). Mice also received 250 ng of pertussis toxin (List Biological Laboratories) i.p. on days 0 and 2. Disease severity was assessed according to the following scale: 0, no disease; 1, flaccid tail; 2, hind limb weakness; 3, hind limb paralysis; 4, forelimb weakness; and 5, moribund.

Culture medium consisted of RPMI 1640 medium (Mediatech) supplemented with 10% FBS (HyClone), 2 mM l-glutamine (Mediatech), 0.01 M HEPES buffer (Mediatech), 100 μg/ml gentamicin (Mediatech), and 2 × 10−5 M 2-ME (Sigma-Aldrich). Flow cytometry was performed on a BD FACSCalibur and data were processed using FlowJo software (Tree Star).

Ex vivo splenocytes from MOG35–55 or 45D-immunized mice (5 × 105/well) were incubated in a 96-well plate with the indicated concentration of peptide. After 48 h in culture, cells were labeled with 0.4 μCi/well [3H]thymidine. After 18–24 h, the plates were harvested on a FilterMate harvester (Packard Instrument) and analyzed on a 1450 LSC Microbeta TriLux counter (PerkinElmer).

Mice were euthanized with CO2 and perfused through the left ventricle with PBS. The brain and spinal cord were removed from each animal and homogenized on a 0.2-μm filter. Mononuclear cells were isolated over Percoll (Sigma-Aldrich,), washed, and stained with anti-CD45.2 FITC and anti-CD11b PerCP (BD Biosciences).

Constructs encoding the extracellular domains of murine I-Ab α- and β-chains were cloned into a bicistronic expression cassette. The β-chain N terminus contains a tethered MOG38–49 peptide and flexible linker; the β-chain C terminus contains the acidic strand of the leucine zipper (21) followed by a “ribosome skippage sequence” from the insect virus Thosea asigna. The ribosome skippage sequence directs chain stop and restart during translation, thereby separating the I-A β- and α-chains (22). The α-chain contains C-terminal modifications comprising the basic leucine zipper strand, the biotinylation signal peptide LNDIFEAQKIEWHE (23), and a 6X histidine tag. Protein expression and tetramers were generated by the National Institute of Allergy and Infectious Diseases Tetramer Core facility (the detailed method for class II tetramer production will be described elsewhere). The bicistronic expression construct was cloned into the SIN lentiviral vector pCMJJ (a gift from J. Jacob, Department of Microbiology and Immunology, Emory University, Atlanta, GA). Lentiviral particles were generated and used to transduce HEK 293T cells. Transduced cells were cultured in standard DMEM supplemented with 10% FCS, l-glutamine, and Pennstrep. Cultures were expanded on a Bellocell bioreactor (Bellco) and recombinant monomers were purified by Ni2+ column chromatography followed by affinity purification on Sepharose-conjugated mAb 2H11 against the leucine zipper. Purified monomers were biotinylated and tetramerized to streptavidin-conjugated to PE.

Cells were incubated with either MOG38–49 or OVA323–339 I-Ab tetramer at a concentration of 4 μg/ml for 20 h at 37°C. The cells were washed with buffer containing 1× PBS, 0.1% BSA, and 0.05% sodium azide. Cells were stained with anti-CD4-allophycocyanin (RM4.5; BD Biosciences) and 7-aminoactinomycin D (7-AAD) for 30 min on ice. The percentage of tetramer-PE-positive cells was determined in live (7-AAD-negative) CD4-positive populations.

WT, IFN-γ−/−, and IFN-γR−/− mice were primed in the hind footpads and tail base using 100 μg of MOG35–55 or 45D emulsified in 1.25 mg/ml CFA. The inguinal and popliteal lymph nodes (LNs) were isolated 12–14 days after priming, pooled together for each mouse, and stained with MOG:I-Ab or OVA:IAb tetramers (see above). The percentage of live tetramer-positive cells was multiplied by the absolute number of LN cells determined by flow cytometric analysis using TruCOUNT tubes (BD Pharmingen). The following formula was used to calculate the absolute number of LN cells per mouse: ((number of cells collected/number of beads collected) × (number of beads per tube/test volume)).

Cells from the spleen and CNS of immunized mice were stimulated for 5 h with 20 nM PMA (Fisher Biotech) and 1 nM ionomycin at 37°C. After the first 30 min of stimulation, 10 μg/ml brefeldin A was added. Cells were first stained extracellularly with anti-CD4 Ab (RM4-5, CD4 PerCP) and were then fixed and permeabilized using the Caltag Laboratories Fix & Perm Cell Permeabilization Kit (Invitrogen Life Technologies) according to the manufacturer’s protocol. Cells were stained for intracellular cytokine using anti-IFN-γ-FITC (DB-1; BD Pharmingen), anti-IL-17-allophycocyanin (eBio f7B7; eBioscience), and anti-IL-4-PE (11B11; BD Pharmingen) for 30 min on ice.

Statistical analyses were performed using GraphPad Prism 4 (Software for Science). Mean clinical scores were compared using a one-tailed Wilcoxon-matched pairs test and disease incidence was analyzed using a one-tailed paired t test. Absolute numbers of MOG tetramer-positive CD4+ T cells were compared using a one-tailed t test.

To determine the role of IFN-γ in regulating the encephalitogenicity of 45D, WT, IFN-γ−/−, and IFN-γR−/− B6 mice were immunized with MOG35–55 or 45D and were monitored for EAE signs. Confirming our previous findings (5, 11), WT mice immunized with MOG35–55 developed significant EAE signs (average maximum score of 3.2 ± 0.3), in contrast to WT mice immunized with 45D, which displayed little to no signs of clinical EAE (average maximum score of 0.6 ± 0.3) (Fig. 1 and Table I). Moreover, IFN-γ−/− and IFN-γR−/− mice immunized with MOG35–55 developed rapid and severe EAE (average maximum score of 3.5 ± 0.2 and 4.3 ± 0.2, respectively), consistent with previous reports (14, 18). Surprisingly, 45D-immunized IFN-γ−/− and IFN-γR−/− mice developed delayed, but considerable EAE signs (average maximum EAE score of 1.8 ± 0.6 and 1.5 ± 0.4, respectively) with incidences of 60 and 67%, respectively (Fig. 1 and Table I). The disease courses of IFN-γ−/− and IFN-γR−/− 45D-immunized mice were significantly more severe than WT 45D-immunized mice in terms of both EAE score (p = 0.0017 and p = 0.0013, respectively) and incidence (p = 0.0052 and p = 0.0121, respectively). Thus, the loss of IFN-γ enables the induction of EAE by the weak myelin Ag 45D.

FIGURE 1.

IFN-γ−/− and IFN-γR−/− mice develop significant EAE following 45D immunization. WT B6, IFN-γ−/−, or IFN-γR−/− mice were immunized with 100 μg of MOG35–55 or 45D on days 0 and 7. Mice also received 250 ng of pertussis toxin on days 0 and 2. Mice were monitored for disease signs (A) and incidence (B) as described in Materials and Methods. Compared with WT mice immunized with 45D, 45D-immunized IFN-γ−/− and IFN-γR−/− mice developed EAE with significantly greater severity (p = 0.0017 and p = 0.0013, respectively) and incidence (p = 0.0052 and p = 0.0121, respectively). Data for each group are the average of two to three independent experiments.

FIGURE 1.

IFN-γ−/− and IFN-γR−/− mice develop significant EAE following 45D immunization. WT B6, IFN-γ−/−, or IFN-γR−/− mice were immunized with 100 μg of MOG35–55 or 45D on days 0 and 7. Mice also received 250 ng of pertussis toxin on days 0 and 2. Mice were monitored for disease signs (A) and incidence (B) as described in Materials and Methods. Compared with WT mice immunized with 45D, 45D-immunized IFN-γ−/− and IFN-γR−/− mice developed EAE with significantly greater severity (p = 0.0017 and p = 0.0013, respectively) and incidence (p = 0.0052 and p = 0.0121, respectively). Data for each group are the average of two to three independent experiments.

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Table I.

EAE incidence, average severity, and day of onset in WT, IFN-γ−/−, and IFN-γR−/− mice immunized with MOG or 45D

GroupDisease IncidenceaAverage Maximum Severityb (±SEM)Average Day of Onset (±SEM)
WT MOG 5/5 3.20 ± 0.26 16.4 ± 1.0 
WT 45D 3/12 0.58 ± 0.31 21.7 ± 6.3 
IFN-γ−/− MOG 6/6 3.50 ± 0.18 15.8 ± 1.1 
IFN-γ−/− 45D 6/10 1.80 ± 0.56 19.5 ± 1.3 
IFN-γR−/− MOG 4/4 4.25 ± 0.25 13.3 ± 0.5 
IFN-γR−/− 45D 8/12 1.54 ± 0.41 19.1 ± 0.9 
GroupDisease IncidenceaAverage Maximum Severityb (±SEM)Average Day of Onset (±SEM)
WT MOG 5/5 3.20 ± 0.26 16.4 ± 1.0 
WT 45D 3/12 0.58 ± 0.31 21.7 ± 6.3 
IFN-γ−/− MOG 6/6 3.50 ± 0.18 15.8 ± 1.1 
IFN-γ−/− 45D 6/10 1.80 ± 0.56 19.5 ± 1.3 
IFN-γR−/− MOG 4/4 4.25 ± 0.25 13.3 ± 0.5 
IFN-γR−/− 45D 8/12 1.54 ± 0.41 19.1 ± 0.9 
a

p = 0.0846 between WT 45D and IFN-γ−/− 45D and p = 0.0417 between WT 45D and IFN-γR−/− 45D.

b

p = 0.0536 between WT 45D and IFN-γ−/− 45D and p = 0.0573 between WT 45D and IFN-γR−/− 45D.

To verify that the development of disease signs in IFN-γ−/− and IFN-γR−/− mice immunized with 45D was due to inflammation within the CNS, CNS mononuclear cell infiltration was assessed by flow cytometry. All MOG-immunized mice demonstrated significant numbers of lymphocytes (CD45.2+CD11blow/ − in the CNS 20 days postimmunization, whereas these cell populations were minimally present in WT mice immunized with 45D (Fig. 2). Consistent with the delayed, but significant onset of disease signs in 45D-immunized IFN-γ−/− and IFN-γR−/− mice, few lymphocytes were found in the CNS of these mice at day 15 (data not shown), but high percentages were present by day 20. Similar percentages (4.8–8.8%) of infiltrating macrophages (CD45.2+CD11bhigh) were found in all groups of mice (Fig. 2). Thus, these results indicate that the development of EAE signs in 45D-immunized IFN-γ−/− and IFN-γR−/− mice correlated with substantial amounts of infiltrating lymphocytes in the CNS.

FIGURE 2.

45D induces significant lymphocyte infiltration in the CNS of IFN-γ−/− and IFN-γR−/− mice. CNS tissue was isolated 20 days postimmunization and stained with CD45.2 and CD11b to assess percentages of infiltrating lymphocytes (CD45.2+CD11blow/−), macrophages (CD45.2+CD11bhigh), and resident microglia (CD45.2intCD11bhigh). Results shown are representative of two independent experiments with each group containing CNS tissue pooled from two mice.

FIGURE 2.

45D induces significant lymphocyte infiltration in the CNS of IFN-γ−/− and IFN-γR−/− mice. CNS tissue was isolated 20 days postimmunization and stained with CD45.2 and CD11b to assess percentages of infiltrating lymphocytes (CD45.2+CD11blow/−), macrophages (CD45.2+CD11bhigh), and resident microglia (CD45.2intCD11bhigh). Results shown are representative of two independent experiments with each group containing CNS tissue pooled from two mice.

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The observation that 45D-immunized IFN-γ−/− and IFN-γR−/− mice develop significant EAE signs implies that primed CD4+ T cells from these mice are responsive to endogenous myelin components within the CNS. To test this, our laboratory has developed an I-Ab tetramer specific for MOG38–49, which spans the dominant Th epitope of MOG40–48 (24). The DNA sequence for MOG38–49 was inserted into a construct for I-Ab, such that the resulting protein contains the MOG epitope tethered to the I-Ab molecule. This MOG tetramer is highly specific because it stains MOG-reactive CD4+ T cells, which show minimal staining with a control I-Ab tetramer specific for OVA323–339. Conversely, the OVA tetramer stains OVA-reactive, but not MOG-specific CD4+ T cells (data not shown).

Class II tetramer staining revealed the presence of MOG-specific CD4+ T cells within the CNS (2–4%) of MOG35–55-immunized WT, IFN-γ−/−, and IFN-γR−/− mice beginning at the onset of disease signs (data not shown). By day 20, MOG tetramer-positive CD4+ T cells of MOG-immunized WT, IFN-γ−/−, and IFN-γR−/− mice reached levels of 7.4, 10.1, and 9.6%, respectively (Fig. 3) and remained detectable in substantial amounts through day 30 (data not shown). MOG tetramer-positive CD4+ T cells were not detected at any time point in the CNS of WT mice immunized with 45D. In contrast, increased frequencies of MOG-specific CD4+ T cells were identified in IFN-γ−/− and IFN-γR−/− mice immunized with 45D beginning at day 20 (2.6 and 3.4%, respectively; Fig. 3) and persisted through day 30 (data not shown). Notably, the percentages of MOG-specific CD4+ T cells (7.4%) at the peak of disease in WT B6 mice were very similar to those detected with a recently reported MOG35–55 I-Ab tetramer (25), indicating that the core epitope used for the tetramer is sufficient for tracking MOG-reactive CD4+ T cells. These findings convincingly demonstrate that the induction of EAE by 45D in IFN-γ−/− and IFN-γR−/− mice is mediated by MOG-reactive CD4+ T cells in the CNS.

FIGURE 3.

MOG tetramer-positive CD4+ T cells are found in the CNS of 45D-immunized IFN-γ−/− and IFN-γR−/− mice. Mononuclear cells were isolated from the CNS of mice 20 days postimmunization as described in Materials and Methods. CD4+ T cells were stained with a MOG38–49 I-Ab tetramer or an OVA323–339 I-Ab control tetramer. Tetramer-positive cells are gated on CD4- positive, 7-AAD-negative cells. Results shown are representative of two independent experiments with CNS tissue pooled from two mice in each group.

FIGURE 3.

MOG tetramer-positive CD4+ T cells are found in the CNS of 45D-immunized IFN-γ−/− and IFN-γR−/− mice. Mononuclear cells were isolated from the CNS of mice 20 days postimmunization as described in Materials and Methods. CD4+ T cells were stained with a MOG38–49 I-Ab tetramer or an OVA323–339 I-Ab control tetramer. Tetramer-positive cells are gated on CD4- positive, 7-AAD-negative cells. Results shown are representative of two independent experiments with CNS tissue pooled from two mice in each group.

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Given the marked increase in encephalitogenicity of 45D in IFN-γ−/− and IFN-γR−/− mice, the proliferative responses of T cells from WT, IFN-γ−/−, and IFN-γR−/− mice immunized with either MOG35–55 or 45D were compared. As expected, WT MOG T cells proliferated in response to MOG35–55 restimulation, whereas WT 45D T cells did not (Fig. 4,A). IFN-γ−/− and IFN-γR−/− MOG T cells showed very robust proliferation in response to MOG35–55 restimulation compared with WT MOG T cells. Strikingly, both IFN-γ−/− and IFN-γR−/− 45D T cells also proliferated strongly in response to MOG35–55 restimulation (Fig. 4,A), which was much greater than WT 45D T cells. Consistent with the increased proliferative capacity to MOG35–55, IFN-γ−/−, and IFN-γR−/− 45D T cells proliferated significantly in response to 45D (Fig. 4 B). Interestingly, splenocytes from 45D-immunized IFN-γ−/− and IFN-γR−/− mice proliferated in response to concentrations of 45D as low as 10−4 to 10−3 μM. These results therefore indicate that loss of IFN-γ increases the responsiveness of MOG-reactive CD4+ T cells from 45D-immunized mice.

FIGURE 4.

Splenocytes from 45D-immunized IFN-γ−/− and IFN-γR−/− mice proliferate significantly in response to MOG35–55. Fifteen days postimmunization, 5 × 105 splenocytes (ex vivo) were cultured for 48 h with the indicated concentrations of MOG35–55 (A) or 45D (B). Proliferation was assessed by [3H]thymidine incorporation over the next 18–24 h. Data shown are representative of three independent experiments.

FIGURE 4.

Splenocytes from 45D-immunized IFN-γ−/− and IFN-γR−/− mice proliferate significantly in response to MOG35–55. Fifteen days postimmunization, 5 × 105 splenocytes (ex vivo) were cultured for 48 h with the indicated concentrations of MOG35–55 (A) or 45D (B). Proliferation was assessed by [3H]thymidine incorporation over the next 18–24 h. Data shown are representative of three independent experiments.

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The increased responsiveness of splenocytes from 45D-immunized IFN-γ−/− and IFN-γR−/− mice to MOG35–55 suggests that the loss of IFN-γ may lower the activation threshold of MOG-reactive CD4+ T cells in response to the weak ligand 45D. As a result, we would predict IFN-γ deficiency would allow the generation of increased numbers of MOG-reactive CD4+ T cells in response to 45D in the periphery. To test this, WT, IFN-γ−/−, and IFN-γR−/− mice were immunized in the hind footpads with MOG35–55 or 45D and the absolute number of MOG:I-Ab-positive CD4+ T cells from the draining LNs were quantified on days 12–14. There were greater numbers of MOG-specific CD4+ T cells in the LNs of MOG-primed IFN-γ−/− and IFN-γR−/− mice (31,355 ± 11,622 and 41,533 ± 8,722, respectively) compared with WT mice (14,716 ± 5,548; Fig. 5 A). As predicted, significantly fewer MOG-specific CD4+ T cells were isolated from the LNs of 45D-primed WT mice (3263 ± 991) compared with 45D-primed IFN-γ−/− (6645 ± 1145) and IFN-γR−/− mice (7276 ± 1119) (p = 0.0379 and p = 0.0221, respectively). These results therefore indicate that loss of functional IFN-γ allows the increased generation of MOG-reactive CD4+ T cells in response to the normally tolerizing weak ligand 45D.

FIGURE 5.

Loss of IFN-γ increases absolute numbers of MOG:I-Ab tetramer-positive CD4+ T cells in the periphery. WT, IFN-γ−/−, and IFN-γR−/− mice were primed in the footpad and tail base with MOG35–55 (A) or 45D (B) and draining LNs (popliteal and inguinal) were harvested 12–14 days later. The percentage of MOG tetramer- positive CD4+ T cells was multiplied by the absolute number of cells from the LNs of each mouse as described in Materials and Methods. The absolute number of MOG-specific CD4+ T cells from IFN-γ−/− 45D and IFN-γR−/− 45D mice was significantly higher compared with WT 45D mice (p = 0.0379 and p = 0.0221, respectively).

FIGURE 5.

Loss of IFN-γ increases absolute numbers of MOG:I-Ab tetramer-positive CD4+ T cells in the periphery. WT, IFN-γ−/−, and IFN-γR−/− mice were primed in the footpad and tail base with MOG35–55 (A) or 45D (B) and draining LNs (popliteal and inguinal) were harvested 12–14 days later. The percentage of MOG tetramer- positive CD4+ T cells was multiplied by the absolute number of cells from the LNs of each mouse as described in Materials and Methods. The absolute number of MOG-specific CD4+ T cells from IFN-γ−/− 45D and IFN-γR−/− 45D mice was significantly higher compared with WT 45D mice (p = 0.0379 and p = 0.0221, respectively).

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In contrast to a prototypic Th1-mediated disease process, a number of recent reports have implicated myelin-reactive IL-17-producing CD4+ T cells (Th17 cells) as the key encephalitogenic T cell population in EAE (26, 27, 28). IFN-γ has also been shown to inhibit the development of Th17 cells (27, 29); therefore, we investigated whether the increased encephalitogenicity of 45D in the absence of IFN-γ might be due to an enhanced Th17 phenotype. Splenocytes and CNS mononuclear cells were isolated at various time points postimmunization and restimulated with PMA and ionomycin to compare the production of IFN-γ, IL-17, and IL-4 by CD4+ T cells from each group of mice.

Markedly increased frequencies of CD4+ T cells producing IFN-γ (27.8–28.2%, includes IFN-γ+IL-17 and IFN-γ+IL-17+) and IL-17 (20.2–26.3%, includes both IL-17+IFN-γ and IL-17+IFN-γ+, were detected at day 20 within the CNS of all groups of mice, except from WT 45D-immunized mice (Fig. 6). No IL-4 production was detected in any of the mice analyzed (data not shown). Similar frequencies of IL-17-producing CD4+ T cells were detected in the spleen (data not shown) and CNS of 45D-immunized IFN-γ−/− (20.7%) and IFN-γR−/− (22.8%) mice compared with MOG-immunized mice (20.2–26.3%; Fig. 6). Thus, although CD4+ T cells from 45D-immunized IFN-γ−/− and IFN-γR−/− mice produced substantial amounts of IL-17, there was no significant increase over the other groups that developed EAE. This indicates that the ability of 45D to induce EAE in IFN-γ−/− and IFN-γR−/− mice is not due to an enhanced IL-17-secreting profile of encephalitogenic CD4+ T cells.

FIGURE 6.

CD4+ T cells from 45D-immunized IFN-γ−/− and IFN-γR−/− mice do not demonstrate an enhanced IL-17-secreting phenotype. Splenocytes and CNS mononuclear cells were isolated at 20 days postimmunization and stimulated for 5 h with PMA/ionomycin or medium alone in the presence of brefeldin A. Displayed flow plots are gated on CD4-positive cells.

FIGURE 6.

CD4+ T cells from 45D-immunized IFN-γ−/− and IFN-γR−/− mice do not demonstrate an enhanced IL-17-secreting phenotype. Splenocytes and CNS mononuclear cells were isolated at 20 days postimmunization and stimulated for 5 h with PMA/ionomycin or medium alone in the presence of brefeldin A. Displayed flow plots are gated on CD4-positive cells.

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45D is an MHC variant peptide of MOG35–55 that has an ∼200-fold reduction in the affinity for I-Ab and is weakly encephalitogenic in WT B6 mice (5). In addition, treatment of MOG-reactive T cells with 45D in vitro results in clonal anergy as measured by decreased proliferation and IL-2 production (5), suggesting that the decreased affinity of 45D for the MHC molecule leads to negative signaling and a permanent change in the phenotype of MOG-reactive CD4+ T cells. Interestingly, the anergized CD4+ T cells retain normal IFN-γ levels upon restimulation with MOG or 45D (5, 11), indicating the retention of certain cytokine effector functions. The importance of the preservation of IFN-γ production by 45D is highlighted by numerous studies revealing a protective, anti-inflammatory role of IFN-γ in EAE (12, 13, 14, 15, 16, 17, 18, 19, 20). Moreover, it has been reported that the poor encephalitogenicity of a high-affinity myelin-associated MHC variant peptide can be reversed by anti-IFN-γ Ab (30). Given the protective role of IFN-γ in EAE and the ability of 45D to maintain IFN-γ production by MOG-reactive CD4+ T cells, we hypothesized that IFN-γ was required to inhibit the responsiveness of myelin-reactive CD4+ T cells to weak MHC variant peptides and thereby prevented the induction of EAE.

In this study, we demonstrate that loss of either IFN-γ or IFN-γR results in a significant increase in the susceptibility of B6 mice to EAE induction by 45D (Fig. 1 and Table I), confirming our hypothesis that IFN-γ reduces the encephalitogenicity of this weak ligand. That 45D induces EAE in the absence of functional IFN-γ suggests that this weak ligand activates myelin-reactive CD4+ T cells that are responsive to endogenous MOG35–55 within the CNS. Indeed, the increased encephalitogenicity of 45D in IFN-γ−/− and IFN-γR−/− mice is reflected by the detection of significant percentages of MOG38–49 tetramer-positive CD4+ T cells in the CNS of these mice (Fig. 4). Importantly, we have ruled out deviation toward an enhanced Th17 phenotype as a mechanism for the increased encephalitogenicity of 45D in the absence of IFN-γ because similar frequencies of IL-17-producing CD4+ T cells were found in the spleen and the CNS of all mice that developed EAE, regardless of the presence of IFN-γ signaling or the peptide used for immunization (Fig. 6).

The detection of MOG-specific CD4+ T cells in the CNS of IFN-γ−/− and IFN-γR−/− mice immunized with 45D was mirrored by the demonstration of MOG-reactive CD4+ T cells in the periphery of these mice (Figs. 4 and 5). Due to the time frame of the LN priming experiments, primed CD4+ T cells have not entered the CNS to mediate the release of endogenous CNS Ags. Thus, the increased frequency of MOG-specific CD4+ T cells in 45D-primed IFN-γ−/− and IFN-γR−/− mice is likely due solely to increased expansion in response to local Ag. Nonetheless, differences in naive precursors cannot be excluded. The increased numbers (∼2-fold) of MOG-specific CD4+ T cells in the periphery of IFN-γ−/− and IFN-γR−/− mice compared with WT mice primed with 45D suggests that loss of IFN-γ allows the generation of numerous MOG-reactive CD4+ T cells that can subsequently traffic to the CNS to elicit their effector functions. The number of MOG-specific CD4+ T cells in the 45D-primed IFN-γ-deficient mice, however, is much lower than those found in MOG-primed mice. Although this number of myelin-reactive CD4+ T cells is lower than that induced with MOG35–55, this level of response is still sufficient to induce EAE, albeit at a slower rate.

There are several potential mechanisms we are actively pursuing to explain the increased expansion of MOG CD4+ T cells in response to a nonencephalitogenic peptide in the absence of IFN-γ. IFN-γ could limit CD4+ T cells responses via the induction of NO (20, 31, 32) or the tryptophan-catabolizing enzyme IDO on relevant APCs (33). It is also possible that loss of IFN-γ signaling protects against IFN-γ-induced apoptosis (15, 32, 34), thereby allowing greater CD4+ T cell expansion. Although it is intriguing that the increased EAE severity in IFN-γ−/− mice was correlated with decreased CD4+CD25+Foxp3+ regulatory T cells (18), our preliminary studies thus far have not revealed decreased frequencies of Foxp3+ CD4+ T cells in 45D-immunized IFN-γ−/− and IFN-γR−/− mice; however, this will require further investigation.

We purport that functional IFN-γ deficiency increases the encephalitogenicity of 45D by permitting the expansion of MOG-reactive CD4+ T cells in response to priming with the weak ligand in the periphery. Nonetheless, it is conceivable that IFN-γ may regulate the encephalitogenicity of 45D by effects in the CNS in addition to effects on the CD4+ T cell proliferation. Despite well-known proinflammatory effects of IFN-γ in the CNS (16, 35), IFN-γ also has protective effects on mature oligodendrocytes in the CNS (36, 37). Consequently, it is possible that the loss of IFN-γ or IFN-γR may increase oligodendrocyte apoptosis leading to the enhanced release of myelin Ags, thereby allowing the enhanced activation and/or CNS recruitment of CD4+ T cells primed with 45D.

In this study, we demonstrate that loss of IFN-γ or IFN-γR endows 45D, a myelin variant peptide of MOG35–55 that is normally poorly encephalitogenic, with the ability to induce EAE. We have gone on to show that the increased EAE susceptibility of IFN-γ−/− and IFN-γR−/− mice following immunization with 45D is due to the expansion of MOG-reactive CD4+ T cells in the periphery. Although numerous studies have suggested a protective role for IFN-γ in EAE, we have provided the first evidence suggesting that loss of functional IFN-γ increases the responsiveness of autoreactive CD4+ T cells to weak ligands, thereby enabling the induction of autoimmune disease in response to normally tolerizing peptides. Moreover, our findings suggest a fundamental role of IFN-γ in setting the activation threshold of autoreactive CD4+ T cells, thereby affecting the tolerizing abilities of weak ligands. Consequently, this suggests that loss of IFN-γ converts a negative, anergizing signal into a positive proliferation-inducing signal in myelin-reactive CD4+ T cells responding to weak ligands. Finally, given that the absence of IFN-γ significantly increases the encephalitogenicity of 45D, these findings imply that IFN-γ is required for the induction of anergy by low-affinity MHC variant peptides.

The authors have no financial conflict of interest.

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 Institutes of Health Grant AI056017.

3

Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; APL, altered peptide ligand; MOG, myelin oligodendrocyte glycoprotein; 7-AAD, 7-aminoactinomycin D; LN, lymph node; WT, wild type.

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