Massive apoptosis of thymocytes in T-cell-deficient Id1 transgenic mice - PubMed (original) (raw)

Massive apoptosis of thymocytes in T-cell-deficient Id1 transgenic mice

D Kim et al. Mol Cell Biol. 1999 Dec.

Abstract

Id1 is an inhibitor of a group of basic helix-loop-helix transcription factors, collectively called E proteins, which includes E12, E47, E2-2, and HEB. We have generated transgenic mice in which Id1 is specifically expressed in T cells. The total number of thymocytes in these mice is less than 4% of that in wild-type mice. The majority of the transgenic thymocytes are CD4 and CD8 double negative and bear the cell surface markers of multipotent progenitor cells. A small number of thymocytes, however, differentiate into CD4 or CD8 single-positive T cells, which also display different characteristics from their wild-type counterparts. More importantly, apoptotic cells constitute about 50% of the total thymocytes. These apoptotic thymocytes have rearranged their T-cell receptor genes, suggesting that they are differentiating T cells. This finding has raised the possibility that the T-cell deficiency in Id1 transgenic mice is the result of a massive apoptosis of differentiating T cells triggered by Id1 expression as opposed to a developmental block at the earliest progenitor stage. The progenitor cells accumulated in the transgenic mice might have survived because they are not susceptible to the apoptotic signals. Despite the massive cell death of the thymocytes at young ages, Id1 transgenic mice frequently develop T-cell lymphoma later in their life span, and lymphomagenesis appears to occur at different stages of T-cell development. Taken together, our data suggest that E proteins, being the targets of Id1, are essential regulators for normal T-cell differentiation and tumor suppression.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

(A) Reduced cellularity in the thymuses of Id-1 transgenic mice. Unpurified viable thymocytes were counted with a hemocytometer. The numbers of cells per thymus are the average for n wild-type (WT) or Id1-28 and Id1-29 transgenic mice at the indicated ages. (B). Hematoxylin and eosin stain of wild-type and Id1-28 transgenic thymus sections.

FIG. 2

FIG. 2

FACS analyses of cells from Id1 transgenic mice. (A) CD4 and CD8 profiles of the wild-type and transgenic littermates at the indicated ages. The percentage of each cell population is shown in each quadrant. (B) Further analysis of CD4+ and CD8+ single-positive thymocytes. Thymocytes from 5-week-old mice were stained with TC-CD4 and PE-CD8 together with FITC-conjugated antibodies as indicated. The CD4+ and CD8+ populations were defined as shown in panel A. The expression of each indicated cell surface marker is plotted as fluorescence intensity versus cell number (different scales were used for wild-type and transgenic cells). The profiles of the wild-type mice are shown as thick lines, and those of the Id1 transgenic mice are shown as thin lines. (C) Analyses of Ficoll-purified spleen cells from 5-week-old mice as described in panel B. The percentage of CD4+ and CD8+ single-positive cells are shown on top of the boxes.

FIG. 2

FIG. 2

FACS analyses of cells from Id1 transgenic mice. (A) CD4 and CD8 profiles of the wild-type and transgenic littermates at the indicated ages. The percentage of each cell population is shown in each quadrant. (B) Further analysis of CD4+ and CD8+ single-positive thymocytes. Thymocytes from 5-week-old mice were stained with TC-CD4 and PE-CD8 together with FITC-conjugated antibodies as indicated. The CD4+ and CD8+ populations were defined as shown in panel A. The expression of each indicated cell surface marker is plotted as fluorescence intensity versus cell number (different scales were used for wild-type and transgenic cells). The profiles of the wild-type mice are shown as thick lines, and those of the Id1 transgenic mice are shown as thin lines. (C) Analyses of Ficoll-purified spleen cells from 5-week-old mice as described in panel B. The percentage of CD4+ and CD8+ single-positive cells are shown on top of the boxes.

FIG. 3

FIG. 3

Further characterization of CD4 and CD8 DN cells. Thymocytes from wild-type (WT) and transgenic littermates at the indicated ages were stained with TC-CD4 and TC-CD8 together with PE- and FITC-conjugated antibodies as indicated. The TC-negative population was gated for these analyses.

FIG. 4

FIG. 4

TCR rearrangement. Total and sorted thymocytes (labeled at the top of each panel) from wild-type and transgenic littermates at the indicated ages were used to prepare DNA for PCR analyses. Thymocytes from wild-type and transgenic (lanes 1 and 3) mice and their respective 10-fold-diluted samples (lanes 2 and 4) were used in PCRs to detect TCR rearrangement events as indicated. The PCR product of the Id2 gene served as a control for the amount of DNA present in each sample. The PCR products were analyzed by Southern blotting.

FIG. 5

FIG. 5

Apoptosis in the Id1 transgenic thymus. (A) Forward- and side-scatter analysis of thymocytes with or without Ficoll purification as indicated. Live cells are circled. (B) Electrophoresis of DNA from wild-type and transgenic unpurified thymocytes (lanes 1 and 2), transgenic thymocytes in the pellet after centrifugation on Ficoll (lane 3), and transgenic thymocytes in the supernatant (lane 4). (C) TCR rearrangement in different fractions of thymocytes in 10-day-old transgenic mice. The fractionation procedure is diagrammed at the top. The different fractions are numbered 1 through 7, corresponding to the lane numbers. The PCR product representing the germ line configuration in Dβ-Jβ region is marked by an arrowhead.

FIG. 6

FIG. 6

T-cell lymphoma in Id1 transgenic mice. (A) The appearance of a thymic lymphoma in a 3.5-month-old mouse and histological examination of the tumor and its metastatic sites. (B) FACS analysis of the thymus and lymph nodes of four 4-month-old transgenic mice numbered 1 to 4. The size of each thymus was similar to that shown in panel A. Lymph nodes were enlarged to various degrees. Cells were stained with antibodies against CD4 and CD8 as labeled.

Similar articles

Cited by

References

    1. Amsen D, Kruisbeek A M. Thymocyte selection: not by TCR alone. Immunol Rev. 1998;165:209–229. - PubMed
    1. Anderson S J, Abraham K M, Nakayama T, Singer A, Perlmutter R M. Inhibition of T-cell receptor β-chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56lck. EMBO J. 1992;11:4877–4886. - PMC - PubMed
    1. Bain G, Robanus Maandag E C, Izon D J, Amsen D, Kruisbeek A M, Weintraub B, Krop I, Schlissel M S, Feeney A J, van Roon M, van der Valk M, te Riele H P J, Berns A, Murre C. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell. 1994;79:885–892. - PubMed
    1. Bain G, Robanus Maandag E C, te Riele H P, Feeney A J, Sheehy A, Schlissel M, Shinton S A, Hardy R R, Murre C. Both E12 and E47 allow commitment to the B cell lineage. Immunity. 1997;6:145–154. - PubMed
    1. Bain G, Engel I, Robanus Maandag E C, te Riele H P, Voland J R, Sharp L L, Chun J, Huey B, Pinkel D, Murre C. E2A deficiency leads to abnormalities in alphabeta T-cell development and to rapid development of T-cell lymphomas. Mol Cell Biol. 1997;17:4782–4791. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources