Themis, a T cell-specific protein important for late thymocyte development

Abstract

During positive selection, thymocytes transition through a stage during which T cell antigen receptor (TCR) signaling controls CD4-versus-CD8 lineage 'choice' and subsequent maturation. Here we describe a previously unknown T cell-specific protein, Themis, that serves a distinct function during this stage. In Themis(-/-) mice, thymocyte selection was impaired and the number of transitional CD4(+)CD8(int) thymocytes as well as CD4(+) or CD8(+) single-positive thymocytes was lower. Notably, although we detected no overt TCR-proximal signaling deficiencies, Themis(-/-) CD4(+)CD8(int) thymocytes showed developmental defects consistent with attenuated signaling that were reversible by TCR stimulation. Our results identify Themis as a critical component of the T cell developmental program and suggest that Themis functions to sustain and/or integrate signals required for proper lineage commitment and maturation.

Figure 1

Structure and expression of E430004N04Rik (THEMIS). a) Schematic representation of mouse THEMIS and its human ortholog. NLS, Nuclear Localization Signal; PRR, Proline rich region. Percent identities of the indicated domains are shown. b) Northern blot analysis of THEMIS expression in mouse tissues. c) Immunoblot analysis of THEMIS expression in thymocytes and peripheral lymphoid cells. IEL, intraepithelial lymphocytes. d) Analysis of THEMIS expression in mouse thymocyte subsets by intracellular staining. Bars show THEMIS expression in gated thymocyte populations from Themis^+/+ or Themis^-/- mice. Δgeo. mean = geometric mean of THEMIS staining in Themis^+/+ thymocytes – geometric mean of THEMIS staining in Themis^-/- thymocytes. Graphic is representative of three independent experiments.

Figure 2

Development of SP thymocytes is impaired in Themis^-/- mice. Analysis of Themis^+/- and Themis^-/- thymocytes (a) and lymph node T cells (b) by flow cytometry. Two parameter dot plots show CD4 versus CD8 surface staining. Numbers indicate percentage of cells in the indicated quadrant. Bar graphs represent average cell numbers of the indicated thymocyte subsets (normalized to 10 cells/thymus) or lymphocyte subsets calculated from six mice per group. (t-test, *P < 0.05). c) CD3 versus CD24 surface staining of gated CD4SP or CD8SP thymocytes. Numbers are percentage of cells within the indicated gate. Bar graphs show ratio of mature (CD24^lo/-) to immature (CD24^hi) cells calculated from four mice per group. (t-test, *P < 0.05).

Figure 3

Positive and negative selection are impaired in Themis^-/- mice. a) Positive selection of thymocytes in Themis^+/- or Themis^-/- mice expressing MHC class II-restricted (AND) or MHC class I-restricted (H-Y) TCR transgenes. Two parameter plots show CD4 versus CD8 staining on total thymocytes. Bar graphs show average cell numbers of DP or SP thymocytes calculated from 4-6 mice per group. (t-test, *P < 0.05). Single parameter histogram plots show CD5 staining on DP thymocytes. b) Negative selection of thymocytes in male Themis^+/- and Themis^-/- mice expressing the class I-restricted H-Y TCR transgene. Numbers below plots show percent of cells within each quadrant. Bar graphs are average cell numbers of total thymocytes or DP thymocytes calculated from 4-6 mice per group. (t-test, *P < 0.05).

Figure 4

The generation of CD4⁺CD8^int thymocytes is impaired in Themis^-/- mice. a) Two parameter plots show CD3 versus CD69 surface staining on total thymocytes from Themis^+/- or Themis^-/- mice. Bar graphs represent average cell numbers of the indicated thymocyte subsets calculated from 4 mice per group. (t-test, *P < 0.05). b) Flow cytometry analysis of thymocytes from H2Ab1^-/-Themis^+/+ and H2Ab1^-/-Themis^-/- mice. Two parameter plots show CD4 versus CD8 staining on gated CD3^hi thymocytes. Bar graphs are average numbers of CD3^hiCD4⁺CD8^int thymocytes calculated from 4 mice per group. (t-test, *P < 0.05). c) DP thymocytes from Themis^+/+ or Themis^-/- mice were left unstimulated (medium) or stimulated overnight with anti-TCRβ + anti-CD2. Thymocytes were analyzed immediately by flow cytometry (stimulatory) or washed and incubated in medium without stimulation for an additional 24 h prior to FACS analysis (recovery). Two parameter plots show CD4 versus CD8 staining profiles. Data are representatives of four independent experiments.

Figure 5

Evidence of a signaling defect in Themis^-/- thymocytes at the CD4⁺CD8^int stage. a) Comparison of surface expression of CD3, CD5, CD69 and IL-7 receptor on gated CD4⁺CD8^int thymocytes from Themis^+/+ and Themis^-/- mice. b) MHC class II-restricted thymocytes are redirected to the CD8 lineage in Themis^-/- mice. Two parameter plots show CD4 versus CD8 staining of thymocytes or lymph node T cells from B2m^-/-Themis^+/- or B2m^-/-Themis^-/- mice. Single parameter histograms show CD5 and CD69 surface staining on CD4⁺CD8^int thymocytes from B2m^-/-Themis^+/- or B2m^-/-Themis^-/- mice. Data are representatives of five independent experiments.

Figure 6

Gata-3 and Th-POK expression are reduced in Themis^-/- CD69⁺CD4SP thymocytes. a) Cell extracts from purified CD69⁺ thymocytes from Themis^+/+ and Themis^-/- mice were analyzed by immunoblot with antibodies specific for the indicated proteins. b) Gata-3 intracellular (IC) staining of the indicated thymocyte subsets from Themis^+/+ and Themis^-/- mice. c) Tox and Th-POK gene expression in purified CD69⁺CD4SP thymocytes from Themis^+/+ and Themis^-/- mice measured by real-time PCR. All data are representatives of three independent experiments.

Figure 7

Gata-3 and Th-POK are induced in Themis^-/- thymocytes by strong TCR signals. a) Two parameter plots of thymocytes from H2Ab1^-/-Themis^+/- or H2Ab1^-/-Themis^-/- mice four days after intraperitoneal injection of PBS or anti-TCRβ. b) Intracellular staining for Gata-3 and surface staining for CD5 on gated CD4⁺CD8^int thymocytes isolated from mice four days after anti-TCRβ injection. c) Expression of Th-POK in total thymocytes four days after anti-TCRβ injection determined by real-time PCR. Data are representative of two independent experiments (three mice for each group).

Figure 8

Forced expression of Th-POK or Gata-3 does not rescue CD4SP development in Themis^-/- mice. a) Flow cytometry analysis of thymocytes and lymph node cells from Themis^+/- and Themis^-/- mice with or without the Gata-3 transgene. Data are representative of four independent experiments. b) Flow cytometry analysis of thymocytes and lymph node cells from Themis^+/- and Themis^-/- mice with or without the Th-POK transgene. Data are representative of three independent experiments.

Figure 9

Evidence for a survival defect in immature CD8SP thymocytes. a) Two parameter dot plots show CD4 versus CD8 staining of total thymocytes or CD24 versus TCRβ staining of gated CD8SP TCR^hi thymocytes from Themis^+/- and Themis^-/- mice with or without the Bcl-2 transgene. Numbers in plots indicate percentage of cells within each quadrant or gate. Data are representative of three independent experiments. b) Bar graphs show numbers of CD8SP, CD4SP and CD4⁺CD8^int thymocytes in the indicated mice. NT, non-transgenic. Three mice were analyzed for each group.