Themis controls thymocyte selection through regulation of T cell antigen receptor-mediated signaling

Abstract

Themis (thymocyte-expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis had high expression in thymocytes between the pre-T cell antigen receptor (pre-TCR) and positive-selection checkpoints and low expression in mature T cells. Themis-deficient thymocytes showed defective positive selection, which resulted in fewer mature thymocytes. Negative selection was also impaired in Themis-deficient mice. A greater percentage of Themis-deficient T cells had CD4(+)CD25(+)Foxp3(+) regulatory and CD62L(lo)CD44(hi) memory phenotypes than did wild-type T cells. In support of the idea that Themis is involved in TCR signaling, this protein was phosphorylated quickly after TCR stimulation and was needed for optimal TCR-driven calcium mobilization and activation of the kinase Erk.

Figure 1

Themis structure, expression and localization in wild-type mice. (a) Alignment of predicted amino acid sequence of murine (Mm) and human (Hs) Themis proteins. Sequence alignments and comparisons were performed using Multalin, and rendered using ESPript. Polyproline region is underlined. Amino acid identity shown as white-on-black. (b) Northern blot of Themis RNA from different tissues. Representative of three independent experiments. (c)Themis mRNA expression analyzed by real-time RT-PCR of FACS-sorted subsets of B6 thymocytes. CD8m refers to mature CD8SP cells. Data shown relative to β-actin expression. Representative of three experiments. Error bars denote s.d. (d)In situ hybridization of mouse thymus with Themis probe. Top left, brightfield image showing Themis expression as black dots. Original magnification ×10. Top right, same image in darkfield showing Themis expression as white dots. Bottom, brightfield image of corticomedullary junction (cortex on right, medulla on left). Original magnification ×40. Representative of 2 independent experiments, 3 mice each. (e) OT-I TCR tg Tap1^−/− mice were injected i.v. with PBS, OVA or VSV peptide. Thymocytes were isolated and Themis expression analyzed by real-time RT-PCR. Note that OT-I tg Tap1^−/− mice do not have any mature T cells or thymocytes specific for the injected peptide, so they would not be expected to undergo apoptosis due to cytokine release. FACS analysis did not show any increase in the percentage of dead cells in thymocytes from the treated mice, nor was there any change in the percentage of DP or SP subsets (data not shown). Representative of 4 experiments.

Figure 2

Defective positive selection in Themis^−/− mice. (a) Thymocytes from Themis^+/+ and Themis^−/− mice (intercross from line backcrossed ×10 to B6) were analyzed by FACS. Bar graph shows percentages of different thymocyte sub-populations from 7 mice analyzed in one experiment. Dot plots show representative staining. Results representative of >3 experiments. (b) Differential surface expression of CD69 and CD3 was used to identify thymocyte populations of different maturity in Themis^+/− (upper) and Themis^−/− (lower) mice. Plots on the right are gated on subpopulations indicated in the left-most plot. Percentage of thymocytes in each of the subpopulations shown above each panel. Representative of 2 mice each in 2 independent experiments. (c) Thymocyte sub-populations from irradiated Thy1.2Ly5^a mice were reconstituted with a 1:1 mixture of BM from Thy1.2,Ly5^bThemis^−/− and Thy1.1,Ly5^bThemis^+/+ mice. Donor populations were identified by Thy1.1 or Thy1.2 expression of Ly5^b+ cells. Two chimeric mice per group were analyzed in 3 separate experiments with similar results. (d,e) Thymocytes from Themis^+/+ and Themis^−/− mice expressing the AND TCR (d) or the OT-I TCR (e) were analyzed by FACS. Significant differences (P < 0.0005, t-test) were found between the percentage of CD4 SP (d) and CD8 SP (e) cells in Themis^+/+ and Themis^−/− thymi (data pooled from >3 separate experiments each); Themis^+/+ n=4 (d), n=8 (e); and Themis^−/− n=6 (d), n=12 (e). (f) Thymus sections from Themis^+/+ and Themis^−/− mice were stained with hematoxylin. Medulla shows lighter staining. Original magnification ×10. Representative of two experiments.

Figure 3

Negative selection defect in Themis^−/− mice. B6 mice (H-2^b) bearing wild-type or mutated Themis alleles were backcrossed or not onto B10.D2 (H-2^d) mice and the expression of indicated V_β elements in CD4 or CD8 peripheral blood T cells was analyzed by flow cytometry. V_β5, V_β11, and V_β12 are deleted by the Mtv-8 and Mtv-9 superantigens expressed in B6 and B10.D2, but only when I–E is also expressed. Thus deletion occurs on H-2^b/d but not H-2^b/b backgrounds. V_β6 is not deleted by these Mtv superantigens. Representative of three independent experiments.

Figure 4

Peripheral T cell phenotype in Themis^−/− mice. (a,b) CD4 and CD8 T cells in the spleen of B6 Themis^+/+ and Themis^−/− mice of indicated ages were quantified by flow cytometry. Bar graph shows average percentage of each population in seven 6 week old mice analyzed in one experiment. Dot plots show representative stainings. Data are representative of >3 experiments. (c,d) CD4 and CD8 expression on spleen cells from mice expressing AND (c) and (d) OT-I TCR transgenes. Differences between AND Themis^+/+ and Themis^−/− CD4 T cell percentages (P < 0.0009, n=4 mice of each genotype), and Themis^+/+ and Themis^−/− OT-I CD8 T cell percentages (P < 0.003, Themis^−/− n=7, Themis^+/+ n=5) were significant and representative of at least 3 individual experiments. (e) Increased percentage of CD25⁺Foxp3⁺ cells in Themis^−/− compared to Themis^+/+ splenocytes. Dot plots are gated on CD4⁺ cells. (P = 2.8×10⁻⁵, n=4 mice of each genotype, representative of 2 independent experiments). (f) Expression of CD62L and CD44 on lymph node T cells from Themis^+/+ and Themis^−/− mice. In graphs each dot represents an individual mouse. Dot plots show representative staining, and are gated on CD4⁺ or CD8⁺ cells as indicated. Representative of 3 independent experiments.

Figure 5

Defective activation of Themis-deficient T cells. Themis^−/− cells are shown in blue and Themis^+/+ cells in red. (a) CD3 expression on gated CD4⁺ (CD25⁻) and CD8⁺ spleen cells. (b) Spleen cells were activated by cross-linking with anti-CD3 for 5 hours, after which CD69 upregulation was analyzed on gated CD4⁺ or CD8⁺ cells. Med, unstimulated cells. (c) Spleen cells were labeled with CFSE and stimulated in vitro with anti-CD3 for 3 days. CD4⁺ and CD8⁺ splenocytes were gated and CFSE dilution was analyzed by flow cytometry. Each panel representative of at least 3 independent experiments.

Figure 6

Themis is part of the TCR signal cascade. Jurkat (a) or fresh human PBL T cells (b) were stimulated with anti-CD3 for the indicated times (min, US is unstimulated), lysed and immunoprecipitated with anti-THEMIS serum or anti-p-Tyr mAb. Blots were probed with the same Abs. (a) and (b) are representative of five and two experiments, respectively. (c) Rested Themis^+/+ and Themis^−/− thymocytes were stimulated with anti-CD3 plus anti-CD4, and whole cell lysate (WCL) or anti-p-Tyr immunoprecipitates were blotted and probed with anti-Themis. Representative of three experiments. (d) Freshly isolated B6 thymocytes were activated with anti-CD3 and lysates were immunoprecipitated with anti-Themis. The blot was probed with anti-PLC-γ1 or anti-Themis. Representative of two experiments. (e) Freshly isolated Themis^+/+ or Themis^−/− thymocytes were stimulated with anti-CD3, lysed and precipitated with anti-Itk, then blotted and probed with anti-PLC-γ1, anti-Themis and anti-Itk. In some experiments, mature thymocytes were depleted by MACS using anti-CD53, and cells were stimulated with anti-CD3 plus anti-CD4, with similar results. Representative of three experiments. (f) Rested B6 thymocytes were activated with anti-CD3 plus anti-CD4, lysed and immunoprecipitated with anti-Itk. Immunoprecipitate or WCL blots were probed with anti-PLC-γ1, anti-Themis and anti-Itk. Representative of two experiments. (g) Rested Themis^+/+ and Themis^−/− thymocytes were stimulated with anti-CD3 plus anti-CD4, and WCL or anti-p-Tyr immunoprecipitates were blotted and probed with anti-p-ZAP70, anti-Itk, anti-p-PLC-γ1, and anti-p-p42/44 (Erk1/2) or anti-p42/44. Representative of three experiments.

Figure 7

Signaling in Themis-deficient thymocytes. (a) Pre-selection DP thymocytes from Themis^+/+ or Themis^−/− OT-I Tap1^−/− mice were stimulated by crosslinking biotinylated anti-CD3 and -CD4 with streptavidin (SAv) (left and center) or with OVA-K^b tetramer (right) in Ca²⁺-free medium. CaCl₂ was added later. Cells were surface-labeled with CFSE, Cy5 or nothing, mixed and assayed in one tube so that both Themis^+/+ or Themis^−/− cells were tested at the same time in the same environment. Left; 3 samples of Themis^+/+ cells tested against each other, center and right; Themis^+/+ cells tested against cells from two different Themis^−/− mice. Representative of three independent experiments. (b) OT-I T cells or pre-selection DP thymocytes, Themis^+/+ or Themis^−/−, were incubated with EL4 cells loaded with OVA peptide and stained to detect polymerized actin. Amount of f-actin in the immunological synapse versus the rest of the T cell membrane was calculated from imaging data and expressed in the left graph as the actin signal at the membrane within or outside of the synapse (boxes, s.e.m.; crossbar, median; plus sign, mean; bars, 5–95 percentile range; dots, data points outside this range). Right graph shows ratio of f-actin in the synapse to that in the non-synapse membrane. Each circle represents a single data point, bold lines show the mean, thinner lines show ± s.e.m. ***P < 0.05. Representative of two experiments. (c) Pre-selection DP thymocytes from OT-I Tap1^−/−Themis^+/+ or Themis^−/− mice were stimulated in vitro with OVA presented by EL4 cells and CD69 upregulation analyzed,. Representative of three experiments.