Positive selection of MHC class Ib-restricted CD8(+) T cells on hematopoietic cells

Abstract

Unlike conventional CD8(+) T cells, major histocompatibility complex (MHC) class Ib-restricted CD8(+) T cells show an activated phenotype in uninfected mice and respond rapidly to foreign invaders. The underlying factors that contribute to these differences are not well understood. We show here that the activated phenotype of MHC class Ib-restricted CD8(+) T cells was partially acquired as a result of interactions in the thymus and reflected an increased capacity to be selected via interactions with MHC molecules on hematopoietic cells. Using bone marrow-chimeric mice, we have shown that MHC class Ib-restricted, but not MHC class Ia-restricted, CD8(+) T cells specific for Listeria monocytogenes were efficiently selected when MHC class I was expressed only on hematopoietic cells. Thus, the distinct functional properties of MHC class Ib-restricted versus MHC class Ia-restricted CD8(+) T cells may result, at least in part, from the different ways in which they are positively selected in the thymus.

Figure 1. Phenotype of CD8⁺ T cells in K^b−/−D^b−/− and B6 → β₂M^−/− chimeric mice

Surface expression of activation markers, gated on CD8⁺ T cells from lymph nodes. (a) Flow cytometry histograms show CD8⁺ T cells from B6 (thin black lines) versus K^b−/−D^b−/− mice (red lines). Mice were aged 13 weeks. (b) B6 → B6 (thin black lines) versus B6 → β₂M^−/− chimeras (blue lines). Mice were examined 88 days after irradiation.

Figure 2. Phenotype of thymic CD8⁺ T cells in K^b−/−D^b−/− mice

Surface expression of activation markers gated on CD24⁻CD8⁺ thymocytes in (a) K^b−/−D^b−/− and (b) C57BL/6 mice. The percentages of each CD24⁻CD8⁺ population within total thymus cells and the percentages of CD24⁻CD4⁻CD8⁺ cells displaying each activation marker are shown. Mice were aged 13 weeks.

Figure 3. Selection of MHC class Ib–restricted CD8⁺ T cells on hematopoietic cells

(a) Surface expression of activation markers gated on TCRβ⁺CD8⁺ spleen cells in K^b−/−D^b−/− → K^b−/−D^b−/−, K^b−/−D^b−/− → β₂M^−/− and β₂M^−/− → K^b−/−D^b−/− chimeric mice. The percentages of the TCRβ⁺CD8⁺ populations within total spleen cells and the percentages of TCRβ⁺CD8⁺ cells displaying each activation marker are shown. Chimeric mice represented age-matched mice analyzed 15 weeks after irradiation. (b) K^b−/−D^b−/− → K^b−/−D^b−/− or K^b−/−D^b−/− → β₂M^−/− chimeric mice were infected intravenously with 2000 CFU L. monocytogenes strain 10403S or were mock infected. Spleen cells were analyzed 6 days after infection. The percentages of CD44⁺tetramer⁺ cells within the total CD8⁺ population are shown. Results are representative of two mice per group.

Figure 4. Preferential selection of H2-M3–restricted T cells on hematopoietic cells

(a) Tetramer staining: B6 → B6 or B6 → β₂M^−/− chimeric mice were infected intravenously with 2000 rLM-OVA and spleen cells were analyzed 6 days after infection. The percentages of CD44⁺, K^b-OVA or M3–f-MIGWII tetramer⁺ cells within the total CD8⁺ population are shown. (b) Intracellular IFN-γ analysis: spleen cells were stimulated with medium, OVA peptide or f-MIGWII and intracellular IFN-γ was analyzed by flow cytometry. The percentages of IFN-γ–expressing cells within the CD8⁺ population are shown. (c) Cytolysis assay: specific killing of OVA peptide-loaded (open squares) or f-MIGWII–loaded (filled circles) RMA target cells are shown. Analyses in a–c were done at the same time with spleen cells from the same mice. Results are representative of two separate experiments; two mice were used per group in each experiment.