CD8+ regulatory T cells generated by neonatal recognition of peripheral self-antigen

Abstract

In comparison with CD4+ regulatory T cells, the generation and function of immunomodulatory CD8+T cells is less well defined. Here we describe the existence of regulatory anti-Kb-specific CD8+ T cells that are rendered tolerant during neonatal life via antigen contact exclusively on keratinocytes. These regulatory T cells maintain tolerance during adulthood as they prevent Kb-specific graft rejection by naïve CD8+ T cells. Third-party immune responses remain unaffected. Up-regulation of TGF-beta1 and granzyme B in the regulatory CD8+ T cell population suggests the involvement of these molecules in common suppressive pathways shared with CD4+ regulatory T cells. In summary, CD8+ regulatory T cells can be induced extrathymically through antigen contact on neonatally accessible parenchymal cells and maintain tolerance throughout adult life.

Fig. 1.

Keratinocytes establish long-lived CD8⁺ T cell tolerance during the neonatal phase in the absence of CD4⁺ T cells. Shown is the acceptance of K^b-positive skin grafts on tolerant mice. Tx Des-TCR × 2.4KerIV-K^b.Rag-2^−/− (n = 9; ■) and Tx Des-TCR.Rag2^−/− (n = 6; ×) mice were grafted with C57BL/6 tail skin on their lateral thoracic wall. The transplants were monitored for 30 days. Results were confirmed in three independent experiments.

Fig. 2.

Regulation of naïve Des-TCR⁺ CD8⁺ T cells in tolerant mice. Acceptance of K^b-positive C57BL/6 skin grafts on Tx Des-TCR × 2.4KerIV-K^b.Rag-2^−/− (n = 5; ■) but not on Tx Leish-TCR × 2.4KerIV-K^b.Rag-2^−/− (n = 4; ×) mice after i.v. transfer of 3 × 10⁵ spleen cells from naïve Des-TCR.Rag2^−/− mice. The transplants were followed up for 30 days.

Fig. 3.

Tolerant T cells do not inhibit lymphopenia-induced division of naïve Des-TCR⁺ CD8⁺ T cells. A total of 3 × 10⁵ CFSE-labeled spleen cells from naïve Des-TCR.Rag2^−/− mice were transferred i.v. into Tx Des-TCR × 2.4KerIV-K^b.Rag-2^−/− mice (a) and Tx Leish-TCR × 2.4KerIV-K^b.Rag-2^−/− mice (b). After 4 days, total lymph node cells pooled from two or three individual mice were analyzed by flow cytometry for CFSE-positive cells after gating on Des-TCR⁺ CD8⁺ T cells. The filled peak indicates the staining intensity of undivided cells. Representative data from three independent experiments are shown.

Fig. 4.

Third-party immune reactivity remains intact within the K^b-tolerant mice. Tx Des-TCR × 2.4KerIV-K^b.Rag-2^−/− and Tx Leish-TCR × 2.4KerIV-K^b.Rag-2^−/− mice received i.v. a mixture of naïve CD8 T cells from TCR-transgenic Rag1^−/− mice reacting against Tag and from Des-TCR.Rag2^−/− mice (3 × 10⁵ spleen cells from each donor). All mice were challenged s.c. with a Tag-transfected P815 tumor (P815.Tag.K^k.B7). Then, after rejection of this tumor was seen in all animals, the P815.K^b.B7 tumor was inoculated into the same mice. Tumor growth was observed in five of five of the Tx Des-TCR × 2.4KerIV-K^b.Rag-2^−/− mice but in none of the Tx Leish-TCR × 2.4KerIV-K^b.Rag-2^−/− mice.