Signaling by intrathymic cytokines, not T cell antigen receptors, specifies CD8 lineage choice and promotes the differentiation of cytotoxic-lineage T cells

Abstract

Immature CD4(+)CD8(+) (double-positive (DP)) thymocytes are signaled via T cell antigen receptors (TCRs) to undergo positive selection and become responsive to intrathymic cytokines such as interleukin 7 (IL-7). We report here that cytokine signaling is required for positively selected thymocytes to express the transcription factor Runx3, specify CD8 lineage choice and differentiate into cytotoxic-lineage T cells. In DP thymocytes genetically engineered to be cytokine responsive, IL-7 signaling induced TCR-unsignaled DP thymocytes to express Runx3 and to differentiate into mature CD8(+) T cells, completely circumventing positive selection. We conclude that TCR-mediated positive selection converts DP cells into cytokine-responsive thymocytes, but it is subsequent signaling by intrathymic cytokines that specifies CD8 lineage choice and promotes differentiation into cytotoxic-lineage T cells.

Figure 1

Impaired CD8⁺ T cell generation in Stat5a- and Stat5b-deficient mice. (a) The E8_III-Cre transgene consists of enhancer and promoter elements from Cd8a that drive expression of Cre cDNA. (b) STAT5 protein content of thymocytes from wild-type (WT) and STAT5-cKO mice, assessed by intracellular staining with anti-STAT5 (shaded histograms) or control antibodies (open histograms). Data are representative of seven experiments. (c) Flow cytometry analysis (top) and frequencies of TCR^hiCD4⁺ and TCR^hiCD8⁺ SP thymocytes (bottom) in wild-type and STAT5-cKO mice. Numbers above plots indicate total thymocytes; numbers in outlined areas indicate percent cells in each. P value, Student’s two-tailed t-test; NS, not significant. Data are a summary of seven independent experiments with at least seven mice of each genotype (bottom; mean and s.e.m.). (d) Intracellular staining of phosphorylated STAT5 (p-STAT5) and phosphorylated STAT6 (p-STAT6) in lymph node T cells from wild-type, STAT5-cKO and STAT5-6-DKO mice after overnight stimulation with medium alone or with IL-7 (1 ng/ml) or IL-4 (10 ng/ml). Data are representative of three experiments.

Figure 2

The generation of CD8⁺ T cells requires STAT-mediated cytokine signaling. (a) Thymic profiles of wild-type, STAT6-KO, STAT5-cKO and STAT5-6-DKO mice (top row) and absolute numbers of TCR^hiCD8⁺ SP, TCR^hiCD4⁺ SP and total thymocytes (bottom row). P value, Student’s two-tailed t-test. Data are from five independent experiments (mean and s.e.m.). (b) Thymic profile (below) of mice expressing a Myc-tagged SOCS1 transgene (tag expression, top plot). Number above bracketed line (top) indicates percent cells expressing Myc tag; numbers in outlined areas (below) indicate percent cells in each. Data are representative of two experiments. (c) Intracellular expression of phosphorylated STAT5 in TCR^hi wild-type and TCR^hi SOCS1-Tg thymocytes after 30 min of stimulation with IL-7 or medium (right). Left, TCRβ expression by wild-type and SOCS1-Tg thymocytes; dashed lines indicate control antibody staining; numbers above bracketed lines indicate percent TCR^hi cells. Data are representative of three independent experiments.

Figure 3

IL-7 signaling induces Runx3, which specifies CD8 lineage choice. (a) Experimental setup for the generation of intermediate thymocytes from preselection thymocytes (left): purified DP thymocytes (population 1) were stimulated overnight with phorbol 12-myristate 13-acetate plus ionomycin (PMA+Iono) and were allowed to differentiate into intermediate cells (population 2), which were further cultured in either medium alone (population 3) or with IL-7 (population 4). Right, RT-PCR analysis of cultured thymocytes with primers specific for Runx3 mRNA transcribed from the Runx3 distal promoter. WT LN T cells (far right), RNA from freshly isolated B6 CD4⁺ and CD8⁺ lymph node T cells (tissue specificity control). (b) RNA-hybridization analysis of Runx3 mRNA expression in CD8⁺ lymph node T cells cultured overnight (O/N) in medium, IL-7 or IL-4, as well as in freshly isolated CD8⁺ and CD4⁺ lymph node T cells (Fresh). (c) Expression of CD4 and CD8 in TCR cells from wild-type, Runx3-Tg and SOCS1-Tg–Runx3-Tg mice. (d) Expression of CD4 and CD8 in V_α11^hi cells from MHC class II–specific AND–transgenic (AND), AND SOCS1-Tg, AND Runx3-Tg, and AND SOCS1-Tg–Runx3-Tg mice. (e) Thymic profiles of SOCS1-Tg, SOCS1-Tg–Bcl-2-Tg and wild-type mice (top), and RT-PCR analysis of Runx3 mRNA expression in CD8⁺ SP thymocytes sorted from SOCS1-Tg–Bcl-2-Tg and wild-type mice (bottom). Numbers above bracketed lines (top, c,d) indicate percent TCR^hi cells (c) or Vα11^hi cells (variable α-region 11; d); numbers in outlined areas (bottom, c,d; top, e) indicate frequency of cells in each subset. Data are representative of six (a), three (b,d) or two (c,e) experiments.

Figure 4

Effect of in vitro IL-7 signaling on cytokine-responsive preselection DP thymocytes. (a) Intracellular content of phosphorylated STAT5 in preselection DP thymocytes from 7SZ and wild-type mice stimulated for 30 min with IL-7 (1 ng/ml) or medium. (b) Quantification of Runx3 mRNA expression in 7SZ DP thymocytes freshly isolated or stimulated for 1 or 5 d with IL-7 or medium alone, presented in arbitrary units relative to endogenous HPRT mRNA expression (encoding hypoxanthine guanine phosphoribosyl transferase). CD8 LNT (far left), purified wild-type CD8⁺ lymph node T cells (control). (c) Expression of CD4 and CD8 by 7SZ DP thymocytes purified by anti-CD8 panning and cultured for 1 or 5 d with IL-7 (10 ng/ml), IL-4 (10 ng/ml) or medium alone. Numbers in parentheses above plots indicate number of cells at end of culture. (d) Expression of TCRβ and Qa-2, assessed by surface staining, and expression of granzyme B and perforin, assessed by intracellular staining, in freshly purified 7SZ DP thymocytes and after 5 d of stimulation with IL-7 (10 ng/ml). Dashed lines indicate control antibody staining. Top, expression of CD4 and CD8 before and after IL-7 stimulation. Data are representative of four (a,c,d), or two (b) experiments.

Figure 5

In vivo IL-7 signaling of 7SZ thymocytes induces their differentiation into mature CD8⁺ T cells. Expression of TCRβ, CD24, Qa-2 and CD4 on CD8⁺ thymocytes from 7SZ and Zap70-KO mice after 14 d of in vivo administration of recombinant mouse IL-7 (10 μg/day) via a subcutaneously implanted osmotic pump (middle columns) or thymocytes from unstimulated 7SZ and Runx3-Tg–Zap70-KO mice (far left and far right). Numbers in outlined areas indicate percent cells in each. Data are representative of four experiments.

Figure 6

Transgenic IL-7 expression induces efficient CD8⁺ T cell differentiation in 7SZ mice. (a) Absolute number of mature TCR^hiCD8⁺ thymocytes in 7SZ, IL-7-Tg 7Z, IL-7-Tg 7SZ and wild-type mice. Data are the summary of two independent experiments with two mice of each genotype (error bars, s.e.m.). (b) Expression of CD4 and CD8 in TCRβ^hi thymocytes from 7SZ, IL-7-Tg 7Z, IL-7-Tg 7SZ and wild-type (B6) mice (above), and expression of CCR7 and CD5 in mature TCRβ^hiCD8⁺ thymocytes (below). Numbers in outlined areas indicate frequency of cells in each gate. Dashed lines indicate control antibody staining. Summary of developmental implications of these results, Supplementary Figure 8. Data are representative of two experiments.