Notch1 signaling promotes the maturation of CD4 and CD8 SP thymocytes

Abstract

Notch proteins regulate many developmental processes. Notch1 is highly expressed on thymocytes, but its role in regulating their development is not known. We show that activation of Notch1 signaling in CD4+CD8+ double positive thymocytes promotes the maturation of both CD4+ and CD8+ single positive thymocytes and that this occurs in the absence of interactions between the T cell receptor and MHC molecules expressed on thymic epithelial cells. We have also identified several genes that are transcriptionally regulated by Notch1 in T cells and show that they are upregulated during maturation into both single positive lineages. These observations suggest that Notch1 signaling plays a role in promoting maturation into both the CD4 and CD8 T cell lineages.

Figure 1. Analysis of Transgene Expression in NotchIC Transgenic Mice

(A) Schematic representation of the NotchIC transgene. NotchIC corresponds to amino acids 1751–2444 of murine Notch1. Arrows correspond to the locations of primers used for RT-PCR. The bar corresponds to the region of Notch1 recognized by the antiserum used for Western blot analysis. (B) RT-PCR analysis of NotchIC transgene expression in thymocytes and peripheral lymphocytes. cDNA prepared from thymocytes and lymph node cells from NotchIC and littermate control mice was serially diluted 5-fold and amplified by PCR using transgene specific primers. The PCR products were analyzed by Southern blot using a Notch1 cDNA probe. (C) Western blot analysis of transgene expression and endogenous Notch1 expression in thymocytes. Thymocyte lysates from NotchIC or littermate control mice were analyzed by Western blot using an antiserum specific for amino acids 2426–2445 of Notch1. The arrow indicates the NotchIC transgene, and the arrowheads indicate the full-length and proteolytically processed forms of endogenous Notch1. (D) Total thymocyte numbers from NotchIC and littermate control mice. Total thymocyte cell numbers were determined for NotchIC and control mice from three representative litters between 5 and 12 weeks of age. Diamonds represent cell numbers from individual mice, and the numbers above the bars represent the average of these values.

Figure 2. Phenotypic Analysis of Thymocytes from NotchIC Mice

(A) FACS analysis of thymocytes from NotchIC and control mice. Thymocytes from two NotchIC mice and one littermate control were analyzed for CD4 and CD8 expression. Numbers indicate the percent of total thymocytes that fall within the indicated quadrants. (B) FACS analysis of maturation markers on CD4⁺ and CD8⁺ SP thymocytes from NotchIC and littermate control mice. CD4⁺ SP and CD8⁺ SP thymocytes from the mice shown in (A) were analyzed for expression of CD3, MHC class I, CD5, and HSA. Numbers above the bars represent the percent of total thymocytes that fall within the indicated gate. Numbers in the upper left corners of the histograms represent the mean florescence intensity of staining for the cells in this same gate. (C) Percent of DP, HSA^lo CD4⁺ SP, and HSA^lo CD8⁺ SP thymocytes in representative NotchIC mice. NotchIC mice and control mice from several litters were analyzed by FACS for CD4, CD8, and HSA expression. CD4⁺ SP and CD8⁺ SP thymocytes were electronically gated and analyzed for HSA expression as in (B). The percent of CD4⁺CD8⁺ (DP), HSA^lo CD4⁺ SP, and HSA^lo CD8⁺ SP thymocytes for individual NotchIC mice are graphed separately. The average value for five littermate control mice is shown for reference. The axis on the left gives the percent of total thymocytes that are DP, and the axis on the right gives the percent of total thymocytes that are HSA^lo CD4⁺ SP or HSA^lo CD8⁺ SP.

Figure 3. Decrease in Peripheral T Cell Numbers and Increase in Annexin V⁺ Thymocytes in NotchIC Mice

(A) Total numbers of CD4⁺ and CD8⁺ T cells in the peripheral lymph nodes of NotchIC and littermate control mice. Lymph nodes were collected from NotchIC and control mice from two representative litters. Total CD4⁺ and CD8⁺ T cell numbers were determined by multiplying total cell numbers by the percent CD4⁺ SP and CD8⁺ SP cells determined by FACS. Diamonds represent cell numbers from individual mice, and the numbers above the bars represent the average of these values. (B) FACS analysis of Annexin V⁺ thymocytes in NotchIC and littermate control mice. Thymocytes were analyzed for CD4 and CD8 expression and Annexin V binding. The upper panels show Annexin V staining for total thymocytes. The numbers represent the percent of total thymocytes that fall within the indicated region. Cells in this region were gated and analyzed for CD4 and CD8 expression in the lower panels.

Figure 4. Notch1 Signaling Promotes the Maturation of CD4⁺ and CD8⁺ SP Thymocytes in the Absence of MHC Expression on Thymic Epithelial Cells

(A) FACS analysis of CD4 and CD8 expression on thymocytes from one littermate control→MHC^−/− and two NotchIC→MHC^−/− bone marrow chimeric mice. Numbers indicate the percent of total thymocytes that fall within the indicated quadrants. (B) FACS analysis of HSA and MHC class I expression on CD4⁺ and CD8⁺ SP thymocytes from control→MHC^−/− and NotchIC→MHC^−/− bone marrow chimeric mice. CD4⁺ SP and CD8⁺ SP thymocytes from the mice shown in (A) were analyzed by FACS for expression of HSA and MHC class I. Numbers above the bars represent the percent of total thymocytes that fall within the indicated gate. (C) Percent of HSA^lo CD4⁺ SP and HSA^lo CD8⁺ SP thymocytes in NotchIC→MHC^−/− chimeric mice. CD4⁺ SP and CD8⁺ SP thymocytes from six NotchIC→MHC^−/− chimeric mice, two littermate control→MHC^−/−, and two littermate control→B6 chimeric mice were analyzed by FACS for HSA expression as in (B). The percent of HSA^lo CD4⁺ SP and HSA^lo CD8⁺ SP thymocytes for the NotchIC→MHC^−/− chimeras mice are graphed individually. The average values for the littermate control→MHC^−/− and littermate control→B6 chimeric mice are shown for reference. (D) FACS analysis of CD3 expression on CD4⁺ and CD8⁺ SP thymocytes from NotchIC→MHC^−/− and littermate control→B6 bone marrow chimeric mice.

Figure 5. FACS Analysis of CD25⁺ DP Thy-mocytes from NotchIC Mice

(A) FACS analysis of CD25 expression on DP thymocytes from NotchIC and littermate control mice. Numbers above the bars represent the percent of DP thymocytes in indicated region. (B) FACS analysis of CD3, CD5, and CD44 expression on CD25⁺ DP thymocytes from NotchIC mice. (C) FACS analysis of CD25 expression on AKR1 cells expressing NotchIC. The AKR1 DP thymoma cell line and a polyclonal population of AKR1 cells expressing NotchIC were analyzed for CD25 and CD44 expression. (D) FACS analysis of CD25⁺ DP thymocytes from B6 mice. CD25⁺ thymocytes were enriched by panning on antibody-coated plates and analyzed for expression of CD4, CD8, and CD25. The expression of CD25 on panned and unpanned DP thymocytes is shown in the histogram.

Figure 6. Expression Pattern of Notch1 Target Genes in T Cells

(A) RT-PCR analysis of the expression of Notch target genes in the AKR1 and AKR1010 thymomas and the 2B4.11 T cell hybridoma. Equivalent amounts of cDNA prepared from control and NotchIC expressing cells were serially diluted 5-fold and amplified using primers specific to the indicated genes. PCR products were analyzed by Southern blotting using specific probes. (B) RT-PCR analysis of the expression of Notch target genes in DP thymocytes from NotchIC and littermate control mice. DP thymocytes were sorted from NotchIC and littermate control mice. (C) RT-PCR analysis of the expression of Notch1 target genes in thymocyte subsets. Thymocyte subsets were sorted from B6 mice. (D) RT-PCR analysis of the expression of Notch1 target genes in peripheral T cells. CD4⁺ and CD8⁺ T cells were sorted from peripheral lymph nodes of B6 mice.