Positive selecting cell type determines the phenotype of MHC class Ib-restricted CD8+ T cells

Abstract

Several studies have demonstrated an apparent link between positive selection on hematopoietic cells (HCs) and an "innate" T-cell phenotype. Whereas conventional CD8(+) T cells are primarily selected on thymic epithelial cells (TECs) and certain innate T cells are exclusively selected on HCs, MHC class Ib-restricted CD8(+) T cells appear to be selected on both TECs and HCs. However, whether TEC- and HC-selected T cells represent distinct lineages or whether the same T-cell precursors have the capacity to be selected on either cell type is unknown. Using an M3-restricted T-cell receptor transgenic mouse model, we demonstrate that not only are MHC class Ib-restricted CD8(+) T cells capable of being selected on either cell type but that selecting cell type directly affects the phenotype of the resulting CD8(+) T cells. M3-restricted CD8(+) T cells selected on HCs acquire a more activated phenotype and possess more potent effector functions than those selected on TECs. Additionally, these two developmental pathways are active in the generation of the natural pool of M3-restricted CD8(+) T cells. Our results suggest that these two distinct populations may allow MHC class Ib-restricted CD8(+) T cells to occupy different immunological niches playing unique roles in immune responses to infection.

Fig. 1.

Characterization of CD8⁺ T cells in D7 Tg mice. (A) Splenocytes and thymocytes from D7⁺Rag^−/− and B6 mice were stained with mAb against various lymphocyte activation markers. Data shown are representative of three to six mice in each group. (B) Ex vivo stimulation of CD8⁺ T cells from D7⁺Rag^−/− and B6 mice. CD8⁺ T cells were stimulated with PMA and ionomycin and then subjected to intracellular IFN-γ staining. Data shown are representative of two independent experiments with two mice in each group. (C) PLZF expression on D7 thymocytes, iNKT cells (TCRβ⁺CD1d/α-galactosylceramide tetramer⁺), and conventional CD8⁺ T cells from B6 mice. The expression of PLZF on conventional CD8⁺ T cells was indistinguishable from isotype control. Data shown are representative of two independent experiments.

Fig. 2.

Selection of M3-restricted CD8⁺ T cells on HCs and TECs. Thymocytes and splenocytes from indicated chimeric mice were stained with antibodies specific for CD8β, CD4, and Vβ5. (A) (Upper) Numbers represent percentages of total thymocyte population within each quadrant. (Lower) Percentages of CD8⁺Vβ5⁺ cells within the total splenocyte population. Because both donor and recipient mice are on the Rag^−/− background, all Vβ5⁺ cells in chimeric mice are D7 T cells. (B) Percentage of CD8 single positive cells within the total thymocyte population from various BM chimeras. (C) Mean percentage of CD8⁺Vβ5⁺ cells in the total lymphocyte populations from the lymph node, spleen, and small intestine ± SEM. *P < 0.05; **P < 0.01. Data shown are representative of four independent experiments with two to three mice per group. IEL, intestinal epithelial lymphocytes.

Fig. 3.

D7 CD8⁺ T cells selected on HCs are more activated and possess more potent effector function than those selected on TECs. Expression of activation markers on lymphocytes isolated from chimeric mice. The percentages of CD8⁺Vβ5⁺ cells (A; lymph node) and HSA^loCD8⁺ cells (B; thymus) displaying each activation marker are shown. Data shown are representative of four independent experiments with two mice per group. (C) Splenocytes from D7_TECs and D7_HCs were stimulated with LemA. Intracellular IFN-γ production and CD107a expression were analyzed. Data shown are representative of two independent experiments with three mice per group.

Fig. 4.

D7 T cells selected on TECs and HCs respond with similar kinetics but different magnitudes during LM infection. Sorted D7_TECs and D7_HCs (CD45.2⁺) were adoptively transferred into CD45.1 B6 congenic mice and recipient mice infected with LM. (A) Seven days postinfection, splenocytes and hepatic leukocytes from recipient mice were stimulated with LemA. The percentages of cells producing IFN-γ within the CD45.2⁺CD8⁺ LemA-specific D7_TEC or D7_HC population are shown. (B) Percentages of Vβ5⁺CD45.2⁺ cells detected in the blood of recipient mice at different time points following primary and secondary LM infection. *P < 0.05. Data shown are representative of two independent experiments with three to four mice per group.

Fig. 5.

 D7 T cells selected on HCs express higher levels of Eomes than D7 T cells selected on TECs. (A) Expression of CD122 and Eomes on heat stable antigen^hi-depleted thymocytes from D7_TEC and D7_HC chimeric mice. (B) Expression of Eomes on hepatic leukocytes from D7_TEC and D7_HC chimeric mice, compared with isotypye control (gray filled). Histograms are gated on CD8⁺ cells. Data shown are representative of two separate experiments with four mice per group. (C) Real-time RT-PCR of Eomes mRNA from D7 T cells sorted from D7_TEC and D7_HC chimeric mice. Bar graph represents the mean ± SEM of triplicate determinants of Eomes expression normalized to GAPDH expression levels.

Fig. 6.

Natural (polyclonal) pool of M3-restricted CD8⁺ T cells can be selected by both HC- and TEC-mediated pathways. (A) Surface phenotypes of M3-restricted and conventional CD8⁺ T cells in naive B6 mice. (B and C) BM chimeric mice were immunized with LemA-pulsed BMDCs. Seven days later, the M3-restricted LemA-specific T-cell response was analyzed. (B) Percentages of CD8⁺M3/LemA tetramer⁺ cells in DC-immunized chimeric mice. (C) Splenocytes from day 7 immunized chimeric mice were cultured with unpulsed or LemA-pulsed BMDCs isolated from B6 mice. IFN-γ production was quantified via IFN-γ enzyme-linked immunospot assay. SFU, spot forming unit. *P < 0.05; **P < 0.01; ***P < 0.001. Results are representative of three independent experiments with two to three mice per group.