A public T cell clonotype within a heterogeneous autoreactive repertoire is dominant in driving EAE

Abstract

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis. Immunization of B10.PL mice with the Ac1-9 peptide, the immunodominant determinant of myelin basic protein (MBP), produced a single episode of EAE followed by recovery and resistance to reinduction of disease. Using the CDR3 length spectratyping technique, we characterized the clonal composition of the Ac1-9-specific T cell repertoire from induction through onset and resolution of disease. Two clonally restricted subsets within a heterogeneous self-reactive repertoire were found in mouse lymph nodes, spleen, and spinal cord soon after immunization, before any sign of EAE. These clonotypes, designated BV8S2/BJ2S7 and BV16/BJ2S5, were present in all mice examined and thus considered public. BV8S2/BJ2S7 was found in far greater excess; was exclusively Th1 polarized; disappeared from the spinal cord, spleen, and lymph nodes concomitantly with recovery; and transferred disease to naive recipients. In contrast, BV16/BJ2S5 and numerous private clonotypes were either Th1 or Th2 and persisted following recovery. These results are consistent with the hypothesis that the public clonotype BV8S2/BJ2S7 is a driver of disease and necessary for its propagation.

Figure 1. EAE in B10. PL mice follows a monophasic course.

Splenocytes were harvested for measuring the dose-dependent proliferative response, as shown by the stimulation index (SI), to Ac1–9 at the onset of disease (A) and during recovery (B), as measured by ³H-thymidine uptake. The mean ± SEM for 8 mice is shown.

Figure 2. The BV/BJ usage of cells responding to Ac1–9 is diverse.

Immunoscopic analysis of (A) draining lymph node cells 8 days after immunization followed by in vitro stimulation with Ac1–9 and (B) spinal cord–infiltrating cells without in vitro stimulation. cDNA was amplified in RT-PCR as described in Methods. Results represent clonotypic CDR3 expansions found among the BV/BJ families of 4 mice. Shown are expansions in which the same CDR3 length was found in 1 of 4 mice (Private), in 2–3 of 4 mice (Semi-private), and in all mice analyzed (Public). Results are representative of 3 independent experiments. SC, spinal cord.

Figure 3. Immunologic features of public clonotypic expansions.

Analysis of mRNA expression of the public expansions by real-time PCR was performed (A) from draining lymph node cells of individual mice collected 4 and 8 days after immunization, followed by restimulation in vitro with Ac1–9 for 3 days, or (B) from spinal cord cells of individual mice collected 8 and 14 days after immunization without in vitro stimulation. Symbols represent individual samples; mean expression is denoted by horizontal bars, and mean values are shown. After 6 (C) or 48 (D and E) hours in culture, pooled cells from the draining lymph nodes and spleen of individual mice, 8 days after immunization, were sorted with antibodies to different surface markers using magnetic beads and analyzed by immunoscope, as described in Methods. Results are representative of 4 independent experiments. mFasL.1, mouse FasL.1.

Figure 4. CD4⁺ BV8S2/BJ2S7-DAGGGY clonotypic T cells induce EAE.

(A) Cells from mice immunized with Ac1–9 8 days earlier were stimulated in vitro with the peptide and IL-12; sorted into CD4+, CD4+BV8.2+, and CD4+BV8.2– populations; and adoptively transferred into naive mice. (B) The BV8.2⁺ T cell clone bearing the DAGGGY motif in the CDR3 region and the Ac1–9–specific T cell line depleted of BV8.2⁺ T cells were stimulated in vitro with the peptide and IL-12, washed, and adoptively transferred into naive mice. The numerical range of cells transferred was adjusted for different populations as described in Methods. Mice were monitored daily for clinical signs of EAE.

Figure 5. BV8S2/BJ2S7 expansion parallels disease.

At the indicated time points after immunization, draining lymph node cells were harvested and stimulated in vitro with Ac1–9. cDNA was amplified by RT-PCR as described in Methods. Shown are expansion indexes (EIs) of the single CDR3 expansions, (A) BV8S2/BJ2S7(L9) and (B) BV16/BJ2S5(L9). An EI of 1 represents the particular expansion peak compared with its expansion within the Gaussian distribution; values above or below this limit show positive or negative expansions of the peak, respectively.

Figure 6. The CDR3 region of the public expansion is conserved at onset of EAE, but not during recovery.

BV8S2/BJ2S7 PCR samples from individual mice were directly cloned and sequenced (A) at the onset of EAE on day 14, (B) in remission on day 25, and (C) later in the remission phase on day 30. Concordances with the DAGGGY motif are shaded gray. The figure is representative of the results obtained from restimulated splenocytes and directly from the spinal cord because both samples showed similar profiles.

Figure 7. BV8S2/BJ2S7 expansions after recovery.

In vitro–stimulated cells from lymph nodes (LN), spleen (Sp), blood (Bl), lung (Lu), bone marrow (BM), liver (Li), mesenteric lymph nodes (mLN), and thymus (Thy) were analyzed immunoscopically at different time points after immunization and recovery. Shown are EIs of the single CDR3 expansion BV8S2/BJ2S7(L9). An EI of 1 represents the particular expansion peak compared with its expansion within the Gaussian distribution. Values above or below this limit indicate expansions or contractions of the peak, respectively. Each symbol represents the EI value from organs of individual mice; mean expression is denoted by horizontal bars. ND, not determined.

Figure 8. Cells responding to Ac1–9 produce a low level of IFN-γ after recovery.

IFN-γ production by cells obtained from different sites at 30 days (gray bars) or 60 days (white bars) after recovery. IFN-γ production by lymph node cells 8 days after immunization is shown as a reference (black bars).

Figure 9. BV/BJ usage of cells responding to Ac1–9 is diverse after recovery.

Immunoscopic analyses were performed on stimulated lymph node cells 75 days after recovery as described in the Figure 2 legend. Results are shown for 4 mice and are representative of 2 individual experiments. The 7-aa and 9-aa CDR3 lengths found for BV8S2BJ2S7 and BV16BJ2S5 expansions are denoted. Note that the CDR3 length found for the BV8S2BJ2S7 expansion in this figure is different from the conserved 9-aa sequence found in the previous figures.