Endogenous myelin basic protein inactivates the high avidity T cell repertoire

Abstract

To study the contribution of endogenous myelin basic protein (MBP) to the positive and/or negative selection of the MBP-specific T cell repertoire, we studied the T cell response to MBP in MBP-deficient shiverer and MBP-expressing congenic C3H mice. Immunization with MBP induced a vigorous T cell response in shiverer mice directed against a single I-Ak- restricted immunodominant determinant, the core of which is peptide MBP:79-87 (DENPVVHFF). Injection of this peptide induced a high avidity T cell repertoire in shiverer mice that primarily consisted of clones capable of recognizing the native MBP protein in addition to the peptide itself. These data show that endogenous MBP is not required for the positive selection of an MBP-specific T cell repertoire. C3H mice, in contrast, were selectively unresponsive to the MBP protein and injection of MBP:79-87 peptide induced a low avidity repertoire that could be stimulated only by the peptide, not by the protein. Therefore, endogenous MBP induced profound inactivation of high avidity clones specific for the immunodominant determinant making that determinant appear cryptic.

Figure 1

Mapping of the MBP response in shiverer mice. (A) Fine specificity of the response. Shiverer mice (open bars) and C3H mice (filled bars) were immunized with 50 μg mMBP in CFA, subcutaneously. 10 d later, the draining LN and spleen cells of three mice in each group were pooled and tested by IFN-γ ELISA spot assay using a nonamer peptide series that covered the entire murine MBP sequence. The results were read with an automated ELISA spot image analyzer. The medium background in this experiment was 6 ± 3 spots. Because no significant spot formation (SI < 2) was seen with peptides 1–9 to 69–77 or 89–97 to 183–191, these results are represented in a single bar. The results shown are from one experiment, which was reproduced four times. (B) MHC restriction of peptide 79–87. A shiverer mouse–derived 79-87–specific T cell hybridoma 2G9.4 was tested in the presence of the indicated MHC-disparate APC. The inserted table shows the MHC alleles expressed by these APC, which were either spleen cells from H-2 recombinant mice or the I-A^k transfected cell line, C3F6. IL-2 production was measured by the CTLL assay.

Figure 2

Functional avidity of the MBP:79-87–specific T cell repertoire induced by immunization with this peptide. (A) Dose–response curves of peptide-induced IFN-γ production in freshly isolated draining LN cells as measured by ELISA spot. Shiverer mice (open symbols) and C3H mice (filled symbols) were immunized with the dose of MBP:79-87 peptide indicated (in CFA, subcutaneously) and tested on day 9 for the frequency of IFN-γ–producing cells at various concentrations of the peptide. Data shown are mean ± SD of triplicate wells from individual mice. Where not visible, the SD where smaller than the size of the symbols. Unimmunized or HEL-immunized mice did not respond to this peptide by cytokine production or proliferation (data not shown). These data are fully representative of 16 individual mice of both strains tested in four independent experiments. (B) Response characteristics of MBP:79-87–specific T cell hybridomas from shiverer (open symbols) and C3H mice (filled symbols). Mice were immunized with MBP:79-87 as in A; day 9 LN cells were fused and cloned as described in Materials and Methods. Individual T cell hybrid clones with characteristic response patterns are shown as measured in CTLL proliferation assays using C3H spleen cells as APC.

Figure 3

Recognition of naturally processed MBP and the MBP:79-87 peptide after immunization with this peptide. (A) The IFN-γ–recall response in primary draining LN cells measured by ELISA spot. Shiverer or C3H mice were immunized with 50 μg MBP:79-87 and tested on day 9 for reactivity to the peptide (5 × 10⁻⁵ M) and to MBP (50 μg/ml). The data show responses of individual mice and are fully representative of 20 mice tested in five experiments. (B) The IL-2 response of mMBP:79-87– reactive T cell hybridomas. Hybridoma clones generated from MBP:79-87–immunized shiverer or C3H mice (see legend to Fig. 2 A and the text) were tested in a standard CTLL assay for their response to 5 × 10⁻⁵ M of peptide 79–87 and 50 μg/ml mMBP protein. Two response patterns were seen in shiverer mice, represented by clones 2G9.4 and 3D8.5, respectively. All seven C3H-derived clones displayed the response pattern of the 2G8.10 clone shown. The same results were obtained in A and B when the MBP concentration was increased by as much as 10-fold, to 500 μg/ml (data not shown).