SAP protein-dependent natural killer T-like cells regulate the development of CD8(+) T cells with innate lymphocyte characteristics

Abstract

CD8(+) T cells are selected via low-affinity interaction with MHC class I molecules on thymic epithelial cells (TECs). However, compromised T cell receptor signaling was proposed to force CD8(+) T cell selection on hematopoietic cells through a SLAM-associated protein (SAP)-dependent mechanism similar to NKT cells. The outcome is an unconventional CD8(+) T cell with phenotypic and functional characteristics of innate lymphocytes. Here we showed that Id3(-/-) CD8(+) T cells had an innate-like phenotype and required SAP for their development. However, like conventional CD8(+) T cells, Id3(-/-) CD8(+) thymocytes were selected on TECs. The requirement for SAP and the innate-like phenotype was not intrinsic to Id3(-/-) CD8(+) thymocytes. Rather, an expanded population of NKT-like cells induced the innate phenotype on CD8(+) T cells through production of interleukin-4. Our findings reveal that accumulation of NKT-like cells promotes conventional CD8(+) thymocytes to acquire innate lymphocyte characteristics.

Figure 1

Id3^{−/ −} CD8 thymocytes have an innate-like phenotype. (A) WT and Id3^{−/ −} thymocytes were analyzed by flow cytometry for TCRβ . The frequency of TCRβ ⁺ cells is indicated. (B) CD4 and CD8 expression on TCRβ ⁺ cells. The frequency of CD4⁺ or CD8⁺ cells among TCRβ ⁺ cells is indicated. (C) Expression of CD122, CD44 or CD24 on WT (grey) or Id3^{−/ −} (black) TCRβ ⁺CD8⁺ thymocytes. (D) Number of TCRβ ⁺CD8⁺CD122⁺ thymocytes in WT and Id3^{−/ −} mice. n = 13 for each genotype. Graphs show mean +/− s.d. (E) Expression of CD122 or CD44 on WT (grey) or Id3^{−/ −} (black) TCRβ ⁺CD8 splenocytes. Data are representative of 10 experiments. (F) Relative expression of Eomes mRNA in Id3^{−/ −} CD4⁺CD8⁺ or TCRβ ⁺CD8⁺ thymocytes compared to WT. Graphs show mean +/− s.d. Representative of 3 experiments (G) Expression of CD44 and intracellular IFNγ in WT or Id3^{−/ −} CD8 thymocytes 5 hours after stimulation with PMA + ionomycin. Data is representative of 3 experiments. For data on neonates see Figure S1.

Figure 2

Id3^{−/ −} innate-like CD8 T cells were selected on MHC class Ia. (A) WT, H2-K^bH2-D^{b−/ −} or Id3^{−/ −}H2-K^bH2-D^{b−/ −} thymocytes were analyzed by flow cytometry for TCRβ . The frequency of TCRβ ⁺ cells is indicated. (B) CD4 and CD8 expression on TCRβ ⁺ cells. The frequency of CD4⁺ or CD8⁺ cells among TCRβ ⁺ cells is indicated. (C) Expression of CD122, CD44 or CD24 on H2-K^bH2-D^{b−/ −} (grey) or Id3^{−/ −} H2-K^bH2-D^{b−/ −} (black) TCRβ ⁺CD8 thymocytes. Data are representative of >5 experiments. (D) Average number (+ standard deviation) of TCRβ ⁺CD8⁺ cells in the thymus of WT, Id3^{−/ −}, H2- K^bH2-D^{b−/ −} or Id3^{−/ −}H2-K^bH2-D^{b−/ −}mice. n > 5 for each genotype, p-values are shown for the comparison of populations at the end of each line.

Figure 3

Id3^{−/ −} innate-like CD8 T cells were selected on non-hematopoietic cells. (A–D) Flow cytometric analysis of thymocytes derived from chimeric mice 6 weeks after transplantation. (A and C) CD4 and CD8 expression on total thymocytes (upper panels), TCRβ expression on total thymocytes (middle panels) and CD4 and CD8 expression on TCRβ ⁺ thymocytes (lower panels). Expression of CD122, CD44 or CD24 on TCRβ ⁺CD8 thymocytes in (B) WT to B2m^{−/ −} chimeras (grey) or Id3^{−/ −} to B2m^{−/ −} chimeras (black) or (D) H2-K^bH2-D^{b−/ −} to WT (grey) or Id3^{−/ −}H2-K^bH2-D^{b−/ −} to WT chimeras. (E) Average number (+ standard deviation) of CD122⁺TCRβ ⁺CD8 thymocytes in chimeric mice from the indicated donor and recipient combinations. n> 3 for each combination. P-values are shown for populations at the end of each line. Three independent experiments were performed. See Figure S2 for analysis of the innate-like phenotype on Id3^{−/ −} to WT chimeras.

Figure 4

SAP was required for the innate-like phenotype of Id3^{−/ −} CD8 thymocytes. (A) WT, Id3^{−/ −} or Sh2d1a^{−/ −} and Id3^{−/ −}Sh2d1a^{−/ −} thymocytes were analyzed by flow cytometry for TCRβ (upper) and TCRβ ⁺ cells were analyzed for CD4 and CD8 (lower). The frequency of TCRβ +cells and CD4⁺ or CD8⁺ cells among TCRβ ⁺ cells is indicated. (B) Average number of TCRβ ⁺ CD8 thymocytes in the thymus of mice with the indicated genotype. n > 6 for each genotype, p-values are shown for the comparison of populations at the end of each line. (C) Expression of CD122, CD44 or CD24 on TCRβ ⁺CD8 thymocytes from WT (grey) or mutant mice (black). (D) Average number of CD122⁺TCRβ ⁺ CD8 thymocytes in the thymus of mice with the indicated genotype. n > 5 for each genotype, p-values are shown for the comparison of populations at the end of each line. Data are representative of > 5 experiments.

Figure 5

SAP-dependent PLZF⁺ T cells were required for the innate-like CD8 phenotype in Id3^{−/ −} mice. (A) WT and Id3^{−/ −} thymocytes were examined by flow cytometry for intracellular PLZF (upper, grey histogram is isotype control) and PLZF⁺ cells were analyzed for TCRβ and TCRγ δ . PLZF⁺TCRβ ⁺ cells were analyzed for CD4 and CD8. PLZF⁺TCRβ ⁺CD4⁺ cells analyzed for staining with CD1d tetramer loaded with PBS57 (lower, grey histogram is unloaded tetramer). (B) Average number (+ standard deviation) of PLZF⁺TCRβ ⁺ cells in the thymus of WT and Id3^{−/ −} mice. n > 5 for each genotype. (C) CD4 thymocytes were analyzed for intracellular expression of IL4 and IFNγ 5 hours after stimulation with PMA + ionomycin. (D) Intracellular staining for PLZF (black line) in mice of the indicated genotype. Isotype control (grey). (E) WT, Zbtb16^lu/lu, Id3^{−/ −} and Id3^{−/ −}Zbtb16^lu/lu thymocytes were analyzed by flow cytometry for TCRβ (upper) and TCRβ ⁺ cells were analyzed for CD4 and CD8 (lower). The frequency of TCRβ + cells and CD4 or CD8 cells among TCRβ ⁺ cells is indicated. (E) Expression of CD122, CD44 or CD24 on TCRβ ⁺CD8 thymocytes from WT (grey) or mutant mice (black). Data is representative of 3 experiment.

Figure 6

Unequal chimeras reveal that the innate-like phenotype was not CD8 cell autonomous. (A) Mixed bone marrow chimeras were established using an excess of Id3^{−/ −} cells (>85%, CD45.2) and a minority of WT cells (<15%, CD45.1) (left) or an excess of WT cells (>90%, CD45.1) and a minority of Id3^{−/ −} cells (<10%, CD45.2) (right). Cells were injected into irradiated CD45.2 mice. (B) TCRβ ⁺CD8 thymocytes (black line) from the majority and minority populations were analyzed for CD122 (upper), CD44 (middle) and CD24 (lower). Isotype control (grey). (C) Mixed bone marrow chimeras were established using an excess of WT cells (>70%, CD45.2) and a minority of Sh2d1a^{−/ −} cells (<30%, CD45.1) (left) or an excess of Id3^{−/ −} cells (>75%, CD45.1) and a minority of Sh2d1a^{−/ −} cells (<25%, CD45.2) (right). (D) TCRβ ⁺CD8 thymocytes (black) from the majority and minority populations were analyzed for CD122 (upper), CD44 (middle) and CD24 (lower). Isotype control (grey). Data are representative of > three experiements. See Figure S3 for experimental controls and analysis of PLZF expression.

Figure 7

IL4 was necessary for the innate-like phenotype on Id3^{−/ −} CD8 thymocytes. (A) FACS analysis for IL4Rα on CD8 thymocytes from Id3^{−/ −} (left), Id3^{−/ −} Zbtb16^lu/lu (middle) and Id3^{−/ −}Il4^{−/ −} (right) mice compared to WT (grey). (B) IL4Rα expression on WT minority cells that developed with a majority of WT (left) or Id3^{−/ −} (right). Isotype control (shaded histogram). (C) Intracellular PLZF in CD8 thymocytes from mice of the indicated genotype compared to isotype control (shaded). (D) WT, IL4^{−/ −}, Id3^{−/ −} and Id3^{−/ −}IL4^{−/ −} thymocytes were analyzed for TCRβ (upper) and CD4 versus CD8 on TCRβ ⁺ thymocytes (lower). (E) CD8 thymocytes from mice of the indicated genotype (black) or WT mice (grey) were analyzed for CD122 (upper), CD44 (middle) or CD24 (lower). Grey histogram is WT CD8 thymocytes. Data are representative of > 4 experiments.