Immunodominant T-cell epitopes of MOG reside in its transmembrane and cytoplasmic domains in EAE

Native MOG contains multiple T-cell determinants in C57BL/6 mice.

Overlapping 15 and 20 aa peptides spanning the entire 218 aa sequence of murine MOG were tested for their capability to elicit T-cell proliferative responses in C57BL/6 mice (table 1). In addition to p35-55, p111-130, p181-195, and p186-200 stimulated recall proliferative responses (figure e-1A). Following immunization with full-length MOG, responses could be detected (stimulation index > 2.5) for each of these peptides. The MOG 111-130 sequence contains aa residues from the distal extracellular and proximal transmembrane domains.² Immunization with rMOG 1-117 stimulated a recall response to p35-55 but not to 111-130 (figure 1A), indicating that the core of this novel T-cell determinant is located within the transmembrane region. Further, MOG 111-130 and 35-55 sequences are not homologous, and the proliferative responses induced by immunization with either p35-55 or p111-130 were not cross-reactive (figure e-1A). Residues 181-200 correspond to a hydrophobic sequence within the cytoplasmic domain that is thought to be associated with the cell membrane.¹⁷ Although overlapping, T cells primed to p181-195 or p186-200 responded only to the peptide used for immunization (figure e-1A). Thus, our results indicated that each of the 4 MOG peptides contained unique T-cell determinants.

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Figure 1 Identification of the encephalitogenic MOG determinant, p119-132

(A) Immunization with rMOG 1-117 elicited a recall proliferative response to p35-55 but not to p111-130. (B) Proliferation was detected to MOG p116-130 but not to p111-125 in mice immunized with p111-130. (C) Testing proliferative responses to truncated peptides after immunization with MOG p116-130 identified the core N-terminal boundary, F¹¹⁹. (D) Recall proliferative responses to MOG p119-132 and truncated peptides after immunization with MOG p119-132 identified the core C-terminal boundary, T¹²⁸. The proliferative response was maximal for p119-132. Lymph node cells were harvested 12 days after immunization. Results shown in panels A–D are representative of 3 separate experiments with 4 mice/group. (E) Mice were primed with MOG p119-132. Lymph node cells were isolated on day 10 and restimulated with MOG p119-132 in the presence of anti-MHC class II (M5/114), anti-MHC class I (28-14-8), or isotype control antibodies. Proliferation was evaluated after 72 hours by thymidine incorporation. (F, G) Mice were immunized with MOG p35-55, MOG p119-132, p181-195, and p186-200 for experimental autoimmune encephalomyelitis (EAE) induction. (F) EAE clinical course was similar after immunization with p35-55 and p119-132, but no signs of disease were observed with MOG p181-195 and p186-200. Data are representative of 5 separate experiments (5 mice/group) and represent mean clinical scores ± SEM. (G) Histologic analysis was performed on mice 14 days after immunization. Mice immunized with p35-55 (a, b) and p119-132 (c, d) developed EAE lesions in spinal cord white matter (arrows in a and c). (b, d) Meningeal and parenchymal mononuclear cell inflammation and demyelination were observed at higher magnifications. No evidence of histologic disease was observed in spinal cords of mice immunized with MOG p181-195 (e) or p186-200 (f). Luxol fast blue-hematoxylin & eosin; scale bars 100 μm (a, c, e, and f), 50 μm (b and d). (H) Mice were immunized with p35-55 or p119-132 and CNS-infiltrating cells were isolated 4 days after disease onset. Cells were stained with markers specific for CD4⁺ T cells, CD8⁺ T cells, B cells (CD19⁺B220⁺), monocytes (CD11b⁺, CD45^high), and dendritic cells (CD11c⁺). (I) Adoptive transfer EAE was induced by MOG p119-132–specific and p35-55–specific CD4⁺ T cells but not by p181-195–specific and p186-200–specific T cells. MOG epitope-specific CD4⁺ T cells were isolated from mice primed with individual MOG peptides and adoptively transferred into naïve recipient mice by intraperitoneal injection. Data shown represent mean clinical scores ± SEM of 5 recipient mice/group. EAE incidence was 100% in recipients of MOG p119-132–specific or p35-55–specific T cells. No clinical EAE was detected in recipient mice that received donor MOG p181-195–specific or p186-200–specific T cells. Results are representative of 3 independent experiments. MHC = major histocompatibility complex; MOG = myelin oligodendrocyte glycoprotein.

Overlapping synthetic peptides corresponding to the sequence of full-length MOG were tested for EAE induction in C57BL/6 mice (table 1). MOG p111-130 induced EAE in a manner similar to p35-55. In contrast, although MOG p181-195 and p186-200 had induced T-cell proliferative responses, neither clinical (table 1) nor histologic (table e-1) signs of EAE were observed after immunization with any peptide doses tested (data not shown).

Identification of the encephalitogenic MOG T-cell determinant, p119-132.

In general, T cells recognize linear peptide determinants containing 9–14 aa.^18,19 Thus, we characterized the encephalitogenic region further by testing shorter overlapping peptides. MOG p116-130 elicited proliferation after immunization with MOG p111-130 (figure 1B) and was encephalitogenic (table 2), whereas MOG p111-125 did not stimulate proliferation or cause EAE.

The minimal encephalitogenic T-cell determinant within MOG p116-130 was identified using nested peptides truncated by individual residues from its N-terminus or C-terminus. While MOG p119-130 stimulated proliferation (figure 1C) and caused EAE (table 2), p120-130 did not stimulate proliferation or cause EAE, suggesting that the Phe at residue 119 is critical for recognition of this determinant. As the inability to stimulate proliferation with p120-130 could simply have reflected a requirement for length of this determinant, we immunized mice with MOG p120-131 and p120-132. These 2 peptides did not stimulate proliferation (figure e-1A) or cause EAE (table 2), confirming the importance of hydrophobic residue, Phe, at position 119 of this T-cell epitope. Peptide 119-128, which was the shortest peptide to stimulate proliferation (figure 1D), was also the smallest one to cause EAE (table 2). Peptide 119-130 induced clinical and histologic EAE as potently as p35-55. The addition of residues 131 and 132 to this sequence created peptides that had slightly greater stimulatory capacity. Increasing length at the C-terminus beyond residue 132 (e.g., p119-133) did not increase the stimulatory capacity (figure e-1A) or encephalitogenic potential (table 2). Collectively, these results indicated that p119-132 contains the complete encephalitogenic T-cell epitope of this region.

MOG p119-132–specific T cells are CD4⁺ and MHC II (I-A^b)–restricted, produce proinflammatory T cells, and transfer EAE to naive recipient mice.

In most EAE models, encephalitogenic myelin autoantigen-specific T cells are CD4⁺ and MHC II–restricted.²⁰ In this regard, T cells that proliferated to MOG p119-132 or p119-130 expressed CD4 but not CD8 molecules. A limited repertoire of TCR genes may be utilized for recognition of certain encephalitogenic myelin epitopes.²¹ For example, nearly 80% of MBP Ac-11–specific T clones use the same TCR Vβ gene. Although there was heterogeneity within the population of MOG p119-132–specific T cells as measured by TCR Vβ usage, 40% of CD4⁺ T cells for this determinant utilized Vβ8.3, a Vβ that was rarely used by p35-55–specific CD4⁺ T cells (table e-2). Proliferation to MOG p119-132 was inhibited by anti–MHC II but not anti–MHC I antibodies (figure 1E), indicating that T-cell recognition of MOG p119-132 is restricted by I-A^b MHC II molecules. Besides inducing encephalitogenic responses in C57BL/6 mice, MOG peptides p119-130 and p119-132 also caused EAE in C57BL/10 and 129/Sv, 2 other H-2^b (I-A^b) mouse strains (table 3). However, MOG p119-132, like p35-55, did not induce EAE in C57BL/6 MOG^−/− mice,²² indicating that p119-132 did not elicit encephalitogenic responses through cross-reactivity with another CNS autoantigen (table e-3). In contrast to recombinant human MOG, which is a T-cell and B-cell dependent autoantigen and does not cause EAE in B-cell deficient (JHT) mice,¹⁵ p119-132 caused clinical and histologic EAE in JHT mice (data not shown). MOG p119-132 did not induce clinical or histologic EAE in NOD (H-2^g7), Biozzi (H-2^dq1), or PL/J (H-2^u) mouse strains that are susceptible to EAE induced by MOG p35-55,^2,23 or in strains of other MHC (H-2) haplotype genes that are susceptible to EAE induced by different myelin antigens.²⁰

The clinical course of EAE induced by either p119-132 or p35-55 in C57BL/6 mice was similar at all equivalent peptide doses tested (figure 1F, table e-4). Like MOG p35-55, p119-132 induced parenchymal and meningeal inflammation as well as demyelination. Optic neuritis was also observed. Composition and distribution of the CNS inflammatory lesions, as well as the frequencies of CNS-infiltrating CD4⁺ and CD8⁺ T cells, B cells, monocytes, and dendritic cells, were similar in EAE induced by p119-132 and p35-55 (figure 1, G and H).

Encephalitogenic myelin-specific T cells produce proinflammatory cytokines.³ Therefore, we examined various cytokines produced by T cells following immunization with MOG p119-132 and during acute EAE. T cells primed to p119-132 in complete Freund's adjuvant produced IFN-γ and interleukin (IL)-17 in a manner that was similar to mice immunized with p35-55 (figure e-1B). Percentages of Th1 and Th17 cells in the periphery and in CNS-infiltrating cells were similar when EAE was induced by MOG p119-132 or p35-55 (figure 2A, figure e-1B). When used as donor cells for adoptive transfer, proinflammatory MOG p119-132–specific T cells induced EAE in naive recipient C57BL/6 mice, demonstrating the pathogenicity of those T cells (figure 1I). Incidence, onset, and severity of acute and chronic EAE were similar when induced by MOG p119-132–specific T cells or p35-55–specific T cells.

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Figure 2 MOG p119-132 is the immunodominant T-cell determinant of full-length mouse MOG and elicits a proinflammatory bias

(A, B) Mice were immunized with myelin oligodendrocyte glycoprotein (MOG) p35-55, p119-132, p181-195, and p186-200. (A) Fourteen days after immunization with p35-55 and p119-132, spleen cells and CNS-infiltrating cells were isolated and analyzed for interleukin (IL)-17 and interferon (IFN)-γ production by intracellular staining after stimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin for 5 hours. Results are representative of 2 experiments that tested 4 individual mice for each peptide immunization. (B) Ten days after immunization with p119-132, p181-195, and p185-200, spleen cells were isolated and analyzed for IL-17 and IFN-γ production by intracellular staining after stimulation with PMA/ionomycin for 5 hours. Data summarize 2 experiments that tested 5 individual mice for each peptide immunization. (C) Frequencies of MOG peptide-specific IFN-γ–secreting cells were determined by ELISPOT as described in the methods. Lymph node cells from mice immunized with full-length MOG (left panel) or the extracellular domain of MOG (rMOG 1-117) (right panel) were cultured with the respective antigen used for immunization. Quantification of MOG peptide-specific T cells was performed at the time of restimulation with MOG peptides (25 μg/mL). Results are representative of 3 separate experiments (3 mice/group). (D) Schematic anatomic localization of T-cell epitopes within full-length (218 amino acid) mouse MOG (indicated in red).⁴¹ Amino acid 117, the last residue of extracellular domain, is indicated. T-cell determinants are highlighted in blue.

Of interest, donor T cells specific for p181-195 or p186-200 that produced proinflammatory cytokines did not induce clinical (figure 1I) or histologic (table 4) EAE in recipient WT mice. RAG1^−/− mice, which do not contain either mature T or B cells, are more susceptible to EAE induction by donor encephalitogenic T cells than WT recipient mice.^24,25 When using this sensitive EAE measure, MOG p186-200–specific T cells rarely caused clinical (1/20 mice tested) or histologic (2/20 mice tested) EAE. Donor T cells specific for p181-195 did not induce clinical or histologic signs of EAE (table 4), confirming that p181-195 is indeed a nonencephalitogenic MOG T-cell determinant. Notably, frequencies of IL-17⁺, IFN-γ⁺, and IL-17⁺IFN-γ⁺ T cells were statistically lower after immunization with the nonencephalitogenic T-cell determinant MOG p181-195 (figure 2B).

p119-132 is an immunodominant T-cell epitope of native MOG.

In general, multideterminant protein antigens, like native MOG, are processed by APC.^6,15 By definition, immunodominant T-cell epitopes are those that are recognized more frequently among all T cells responding to the naturally processed protein.²⁶ Even though MOG p119-132 caused EAE as severe as MOG p35-55, it was possible that p119-132 represented a subdominant or cryptic determinant. Thus, we immunized mice with native MOG and examined recall to itself, p35-55, p119-132, p181-195, and p186-200. ELISPOT analysis of IFN-γ–secreting cells, a highly sensitive measure to examine the repertoire of antigen-specific T cells,^27,28 was used to quantitate the frequency of MOG-specific T cells. Of responses to the peptide determinants, the frequency of IFN-γ–secreting T cells was highest for MOG p119-132 (figure 2C) and was significantly greater than for MOG p35-55. These results indicated that p35-55 is not the immunodominant T-cell epitope of MOG. In parallel, we examined the response to these peptides after immunization with the extracellular MOG domain. Immunization with MOG 1-117 elicited a robust response to MOG p35-55 (figure 2C, left panel) but not to the novel determinants located in the transmembrane and cytoplasmic domains (figure 2C, right panel). Similar results were obtained when testing a different concentration of MOG peptides in recall to immunization with full-length MOG or rMOG (figure e-1C).