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Comparative Study
. 2011 Jan 15;186(2):826-37.
doi: 10.4049/jimmunol.1002630. Epub 2010 Dec 10.

Lineage divergence at the first TCR-dependent checkpoint: preferential γδ and impaired αβ T cell development in nonobese diabetic mice

Ni Feng  1 , Patricia Vegh, Ellen V Rothenberg, Mary A Yui
Affiliations
Comparative Study

Lineage divergence at the first TCR-dependent checkpoint: preferential γδ and impaired αβ T cell development in nonobese diabetic mice

Ni Feng et al. J Immunol. .

Abstract

The first TCR-dependent checkpoint in the thymus determines αβ versus γδ T lineage fate and sets the stage for later T cell differentiation decisions. We had previously shown that early T cells in NOD mice that are unable to rearrange a TCR exhibit a defect in checkpoint enforcement at this stage. To determine if T cell progenitors from wild-type NOD mice also exhibit cell-autonomous defects in development, we investigated their differentiation in the Notch-ligand-presenting OP9-DL1 coculture system, as well as by analysis of T cell development in vivo. Cultured CD4 and CD8 double-negative cells from NOD mice exhibited major defects in the generation of CD4 and CD8 double-positive αβ T cells, whereas γδ T cell development from bipotent precursors was enhanced. Limiting dilution and single-cell experiments show that the divergent effects on αβ and γδ T cell development did not spring from biased lineage choice but from increased proliferation of γδ T cells and impaired accumulation of αβ T lineage double-positive cells. In vivo, NOD early T cell subsets in the thymus also show characteristics indicative of defective β-selection, and peripheral αβ T cells are poorly established in mixed bone marrow chimeras, contrasting with strong γδ T as well as B cell repopulation. Thus, NOD T cell precursors reveal divergent, lineage-specific differentiation abnormalities in vitro and in vivo from the first TCR-dependent developmental choice point, which may have consequences for subsequent lineage decisions and effector functions.

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Figures

FIGURE 1
FIGURE 1. NOD DN cells generate and maintain few DP αβT cells while retaining γδT development in vitro
A. Flow cytometric analyses showing the development of DP and γδT cells from magnetic bead-purified DN cells from B6, NOD and BALB/c thymocytes, after 12 days of OP9-DL1 co-culture in 5 ng/ml IL-7 and Flt3L. Similar results were obtained from cultures harvested on day 8 and when IL-7 concentrations were dropped to 2 ng/ml for the last 5 days of culture or were maintained at 2 ng/ml throughout the culture period (data not shown). (B E) Development of FACs sorted DN1, DN2, DN3a, and DN3b (post-β-selection) cells from B6 and NOD thymuses cultured in 2.5 ng/ml IL-7 and 5 ng/ml Flt3L. B. Flow cytometric plots showing CD4 and CD8 DP cell development at days 7 and 14 from each progenitor population. C. Histograms showing levels of surface TCRβ expression in the same cultures for B6 and NOD cell populations at days 7 and 14 of culture. No data are shown for NOD DN3b cells on day 14 because very few cells are remaining. D. Flow cytometric plots of CD44 vs. CD25 showing differences in initial DN differentiation in the same cultures at day 7. E. γδT cell development in the same cultures at day 14. Percentages of cells in each gate are indicated. These results are representative of at least 3 independent experiments.
FIGURE 2
FIGURE 2. NOD bone marrow progenitor cells also show attenuated in vitro development of DP αβT cells and enhanced γδT cell development
Bone marrow cells enriched for progenitors were isolated from NOD and B6 mice and cultured on OP9-DL1 and DL4 cells as described in Materials and Methods. Flow cytometry plots show the generation of CD4/CD8 DP cells and γδT cells from B6 and NOD bone marrow cultures analyzed after 19 and 22 days (A) and two independent cultures established from mixing equal numbers of NOD and B6 bone marrow cells, analyzed after 20 days (B). Results are representative of at least 3 replicate wells from 1 of 2 independent experiments with similar results.
FIGURE 3
FIGURE 3. NOD and B6 DN2 cells generate more γδT and fewer DP cells in ten-cell and single cell OP9-DL1 co-cultures but retain bipotency
A. Plots showing the relative proportion of γδT cells in comparison with DP cells generated from each individual B6 and NOD 10-cell DN2 cell culture, calculated as %γδT/(γδT + DP). The absolute numbers of CD45+ total cells, γδT cells, and DP cells generated in each of those cultures are also shown. A horizontal bar indicates the median value of individual B6 or NOD cultures. B. Plot showing the relative proportions of γδT cells in comparison to DP (calculated as %γδT/(γδT + DP)) generated from single cell cultures of sorted DN2a, DN2b and DN3a cells (left). To determine the commitment of the cells to αβ vs. γδ lineages, the lineage choices of individual cells are shown as percentages of individual wells containing γδT cells only, both γδT and DP cells, or DP cells only (right). p-values given below plots were determined using Mann-Whitney U tests. Data are representative of two independent experiments.
FIGURE 4
FIGURE 4. B6 and NOD DN populations proliferate at similar rates in vitro, but NOD γδT cells proliferate more rapidly than B6 γδT cells
DN cell populations were purified by cell sorting and stained with CFSE before co-culture with OP9-DL1 cells. A. Total thymocytes (All) and purified DN1, DN2, DN3a, and DN3b cells were harvested for analysis of remaining CFSE levels on days 1, 2, 3 and 4. B. Sorted DN3a cells were cultured on OP9-DL1 cells, harvested after 4 days, and stained to distinguish TCRγδ+ cells. Histograms show CFSE levels for B6 (solid gray) and NOD (black line) cells. Data are representative of 3 independent experiments with similar results.
FIGURE 5
FIGURE 5. Expression of TCR-Vγ rearrangements and other genes in sorted thymic NOD and B6 γδT cells
QPCR results from TCRγδ+ cells sorted from NOD and B6 thymuses using primers to detect subsets of specific TCR-Vβ rearrangements and other T cell genes. Data are shown as the geometric mean +/− SD, n=2-5. For additional gene expression comparisons, see ref. 33.
FIGURE 6
FIGURE 6. NOD thymocytes show evidence of a partial block at β-selection and abnormalities in differentiation to DP cells in vivo
A. Histograms showing levels of activation-induced surface receptors CD27, CD28 and CD5, on freshly isolated DN thymocytes from NOD (black lines) and B6 mice (solid gray), using intracellular staining (ic) for TCRβ (top row) or TCRγδ (bottom row) to distinguish cells that have passed through β-selection or γδ-selection respectively. Data are representative of 3-4 independent experiments. B. Flow cytometric analysis showing CD44 vs. CD25 expression in DN subsets from freshly isolated NOD and B6 thymuses (left panels). Percentages of cells in each quadrant are as indicated. A histogram comparing CD25 surface expression levels between gated NOD and B6 CD44lo DN cells (DN3 and DN4 cells) from two mice of each strain. Data are representative of >10 mice of each strain. C. Flow cytometric analyses showing CD4 vs. CD8 plots for total thymocytes from NOD and B6 mice (left), CD8 and CD4 gated cells, stained to distinguish immature (ISP) cells from mature SP cells (middle panels), and histograms showing HSA levels for DN, DP, CD8+ and CD4+ cells (right) which distinguish less mature cells (HSAhi) from more mature cells (HSAlo). The red boxes indicate the intermediate HSA level for B6 CD8 ISP cells (top panel, arrow) with no corresponding major population in the NOD thymus (bottom panel, arrows). Data are representative of 3 independent experiments. D. Percentages of BrdU+ cells among DN2/3 (CD25+), DN4 (CD25−), and DP cell populations 1-3 days after in vivo BrdU pulse-labeling, showing a major difference in accumulation of BrdU+ DP cells on day 2 between B6 and NOD mice. Mean + SD values over time are plotted for B6 and NOD thymocytes.
FIGURE 7
FIGURE 7. NOD cells exhibit differences in establishment of γδT vs. αβT cells in mixed bone marrow chimeras
Bone marrow cells isolated from NOD and B6 mice and 1 × 106 cells from each strain were mixed and injected into irradiated (NOD × B6)F1.Rag1−/− recipients. Mice were sacrificed after 5-6 or 8-9 weeks and analyzed by flow cytometry for lymphocyte populations in thymus and spleen using CD45.1 (NOD) and CD45.2 (B6) to distinguish donor strains. A. Flow cytometric analysis of a representative chimeric mouse (#1) analyzed at 5 weeks post-injection. Plots on the left show thymocytes were stained for CD4 and CD8 (upper panels) and CD3ε and TCRγδ (lower panels). Splenocytes, shown on the right, were stained for αβTCR and CD19, to distinguish B cells (upper panels), and CD3ε and TCRγδ (lower panels). B, C. Summary of FACs staining results from six mixed bone marrow chimeric mice, three analyzed after 5-6 weeks (#1-3) and three after 8-9 weeks (#4-6). B. Graphs showing the ratio of TCRαβ+/TCRγδ+ cells in thymus (left) and spleen (right) generated from B6 (black bars) and NOD (white bars) donors. C. Graph showing of the relative percentages of B, αβT, and γδT lymphocytes derived from B6 and NOD donors in spleens from the same individual chimeric mice.
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