Regulatory CD8+ T cells fine-tune the myelin basic protein-reactive T cell receptor V beta repertoire during experimental autoimmune encephalomyelitis

Abstract

A significant number of self-reactive T cell clones escape thymic negative selection and are released into the periphery, where some are potentially pathogenic. The clonal expansion of self-reactive T cells is known to be limited during initial antigen encounter by apoptotic or anergic mechanisms, regulatory CD4+ T cells, and cytokines. Here we report that superimposed on these mechanisms, during the evolution of autoimmunity in experimental autoimmune encephalomyelitis (EAE), CD8+ T cells are induced, which fine-tune the peripheral self-reactive T cell receptor (TCR) repertoire. We assayed the myelin basic protein-reactive TCR repertoire in naive, EAE-recovered mice as well as EAE-recovered mice depleted of CD8+ T cells by TCRV beta surface expression, complementarity-determining region 3 length distribution, and complementarity-determining region 3 sequencing analysis. In EAE-recovered mice, certain myelin basic protein-reactive CD4+V beta 8.2+ clones are significantly decreased and this decrease is not observed if CD8+ T cells were depleted from these mice. The clones that persist in CD8+ T cell-intact mice are highly diverse in contrast to the clones expanded in CD8+ T cell-depleted mice, which are dominated by the significant outgrowth of a few clones. Importantly, the T cell clones that expand in the absence of CD8+ T cell control are enriched in potentially pathogenic self-reactive T cell clones capable of inducing EAE in vivo.

Fig. 1.

TCR Vβ8.2 and Vβ6 surface expression of MBP-reactive CD4⁺ T cells derived from control, EAE, and CD8^-/EAE mice. The CD4⁺ T cells were prepared and assayed for TCR Vβ expression by two-color FACS analysis as described.

Fig. 2.

Distribution of the TCRβ-chain CDR3 length of CD4⁺ T cells isolated from control, EAE, and CD8^-/EAE mice. The CD4⁺ T cells were prepared and the CDR3 length distribution of all Vβ families was assayed as described in Materials and Methods. The CDR3 length distributions for Vβ8.2, Vβ13, and Vβ8.1 families are shown. The data depict representative results of four independent experiments.

Fig. 3.

CD8⁺ T cells control the clonal outgrowth of CD4⁺ Vβ8.2⁺ MBP-reactive T cells in EAE mice. Each point represents one Vβ8.2 clone with its outgrowth index. Outgrowth index = [(the number of Vβ8.2 clones with the same CDR3 sequence)/(total clones analyzed) × (CD4⁺ Vβ8.2⁺ T cells/total CD4⁺ T cells assayed by FACS)] × 100%. The cloning and sequencing were performed as described in Materials and Methods.

Fig. 4.

Regulatory CD8⁺ T cells adoptively transferred into naive mice fine-tune the MBP-reactive TCR Vβ repertoire. CD8⁺ T cells isolated and adoptively transferred into naive B10PL mice as described and recipient mice were immunized with 1–9Nac MBP 1 day after the T cell transfer. CD4⁺ T cells were isolated from draining lymph nodes from recipient mice, and CDR3 length distribution was performed as described.