Fine-scale staging of T cell lineage commitment in adult mouse thymus

Abstract

T cell development is marked by the loss of alternative lineage choices accompanying specification and commitment to the T cell lineage. Commitment occurs between the CD4 and CD8 double-negative (DN) 2 and DN3 stages in mouse early T cells. To determine the gene regulatory changes that accompany commitment, we sought to distinguish and characterize the earliest committed wild-type DN adult thymocytes. A transitional cell population, defined by the first downregulation of surface c-Kit expression, was found to have lost the ability to differentiate into dendritic cells and NK cells when cultured without Notch-Delta signals. In the presence of Notch signaling, this subset generates T lineage descendants in an ordered precursor-product relationship between DN2, with the highest levels of surface c-Kit, and c-Kit-low DN3 cells. These earliest committed cells show only a few differences in regulatory gene expression, compared with uncommitted DN2 cells. They have not yet established the full expression of Notch-related and T cell differentiation genes characteristic of DN3 cells before beta selection. Instead, the downregulation of select stem cell and non-T lineage genes appears to be key to the extinction of alternative lineage choices.

Figure 1. Surface phenotypes of early thymic DN populations from adult B6 and NOD mice

FACs plots showing surface receptor expression and gates used for definition of thymic DN cell populations enriched by depletion of DP, SP, γδT, DC and NK cells and stained as described in Materials and Methods. (A) CD44 vs. CD25 and Kit vs. CD25 plots for B6 and NOD DN cells with quadrant lines indicating typical designations for “DN1-DN4”. (B) ETP, DN2a and DN2b populations gated first by CD44 and Kit and then by Kit and CD25, as indicated with lines and arrows, were used throughout this study. (C) Histograms showing surface expression of Kit and CD44 for the gated DN populations.

Figure 2. DN2b cells are a developmentally intermediate population between DN2a and DN3

Purified ETP, DN2a, DN2b, and DN3a precursors were plated and co-cultured with OP9-DL1 cells and analyzed for developmental progression. (A) Development of Bcl2 transgenic mouse DN cells after 4 days, showing changes in Kit vs. CD25 (top row) and CD44 vs. CD25 (bottom row). Histograms show direct comparisons of Kit and CD44 surfaces levels between the different cultured populations. (B) Kit vs. CD25 expression for purified DN populations from B6 (top row) and NOD (bottom row) mice, after 4 days of culture. (C) Plots showing development, after 13 days of culture, of NOD and B6 CD4⁺CD8⁺ DP cells (top panels), and CD45⁺ γδTCR⁺ T cells from the same cultures (bottom panels). Results are representative of 2–4 similar experiments.

Figure 3. DC and NK lineage potential is lost in the DN2a to DN2b transition

Sorted DN cells were co-cultured with OP9-Ctrl cells for 7 days and assayed for surface markers of DC and NK cells. (A) Left panels: CD11c and NK1.1 expression on CD45⁺ cells generated from ETP, DN2a, DN2b and DN3a cells. CD11c⁺ (DC, middle panels) and NK1.1⁺ (NK, right panels) gated subsets, further analyzed for MHC-II expression and side scatter. (B) DC and NK cell production from B6 and NOD.NK1.1 DN cells, showing phenotypes of CD45⁺ gated cells using CD11c with NK1.1 plus Dx5 (left), with MHC-II (middle), and with CD11b (right). Plots are representative of 12 independent wells of each cell type in the same experiment, as well as two additional independent experiments. (C) Graphs showing the numbers of DCs and NK cells produced per input cell from independent ETP, DN2a and DN2b cultures, starting with 100, 500 and 500 sorted input cells/well respectively, from B6, NOD and Bcl2 transgenic (Tg) mice. Lines indicate mean ± SD for each group and mean values are as shown.

Figure 4. DN2a and DN2b cell responses to attenuation of Notch signaling

Sorted B6 DN2a and DN2b cells were co-cultured with OP9-DL1 in the presence or absence of different doses of Notch inhibitor, GSI, for 3 days. (A) Plots showing CD25 and CD44 for two independent cultures at each GSI concentration as indicated. Red arrows show the CD44^hi CD25^lo non-T cells generated from DN2a cells, but not DN2b cells, in response to decreasing Notch signals. (B) Graphs showing the mean geometric MFI for CD25 and the mean height for FSC comparing responses of the DN2a and DN2b cells to the varying doses of GSI. Data shown are two separate wells of each GSI dose from one of two independent experiments giving similar results.

Figure 5. Proliferation and differentiation of DN subsets

Sorted ETP, DN2a, DN2b and DN3a cells from B6 mice were stained with CFSE and placed in co-cultures with OP9-DL1 cells. Dot plots of CD25 vs. CFSE after 2, 3 and 4 days, showing the downregulation of CD25 with cell division. Summary histograms are shown at the bottom comparing the CFSE levels through time for each DN population. The histogram at the right compares the forward scatter (FSC) values of CD25⁺ cells that have undergone only 1–2 cell divisions (upper right quadrant) vs. the CD25⁺ cells that have undergone >2 divisions (upper left quadrant) from the DN3a cell cultures on day 3. Results are representative of 3 similar experiments.

Figure 6. T lineage commitment precedes major changes in expression of genes related to Notch signaling and early T cell development

(A, C, D) Quantitative RT-PCR results showing relative expression levels of T and non-T cell genes in ETP, DN2a, DN2b, DN3a cells, plus DN3b (β-selected T cells), and γδT cells for comparison, sorted from B6 (black bars) and NOD (white bars) mice. Values for each sample were calculated relative to Actb controls and presented as mean ± SE, n=2–5, nd = not determined. Graphs show the relative expression of non-T cell genes (A), Notch and related genes (C), and T lineage identity and required transcription factor genes (D). NOD ETP populations analyzed here are likely to include a small percentage of more mature T cells, which is made apparent by the use of a log scale. (B) Intracellular PU.1 in ETP, DN2a, DN2b and DN3a cell populations from B6 and NOD mice. Large granular cells (SSC^hi) included for positive controls (gray dashed line). Data are representative of 3 independent experiments.