Disruption of T cell homeostasis in mice expressing a T cell-specific dominant negative transforming growth factor beta II receptor

Abstract

The immune system, despite its complexity, is maintained at a relative steady state. Mechanisms involved in maintaining lymphocyte homeostasis are poorly understood; however, recent availability of transgenic (Tg) and knockout mouse models with altered balance of lymphocyte cell populations suggest that cytokines play a major role in maintaining lymphocyte homeostasis. We show here that transforming growth factor (TGF)-beta plays a critical role in maintaining CD8(+) T cell homeostasis in a Tg mouse model that specifically overexpresses a dominant negative TGF-beta II receptor (DNRII) on T cells. DNRII T cell Tg mice develop a CD8(+) T cell lymphoproliferative disorder resulting in the massive expansion of the lymphoid organs. These CD8(+) T cells are phenotypically "naive" except for the upregulation of the cell surface molecule CD44, a molecule usually associated with memory T cells. Despite their dominance in the peripheral lymphoid organs, CD8(+) T cells appear to develop normally in the thymus, suggesting that TGF-beta exerts its homeostatic control in the peripheral immune system.

Figure 1

Construction and expression of the DNRII transgene. (A) Schematic representation of the expression vector with transgene. (B) Association of TGF-β1 with DNRII. Either splenocytes (SP) from C57BL/6 mice or TG1124 cells (TG) were affinity labeled with ¹²⁵I–TGF-β1 in the absence (−) or presence (+) of cold TGF-β1. Cell lysates were subjected to immunoprecipitation by either anti–TGF-β II receptor, specific for cytoplasmic tail of endogenous TGF-β II, or anti-HA tag, specific for DNRII. Immunoprecipitation of receptors was analyzed by SDS-PAGE and autoradiography. (C) Semiquantitative RT-PCR to establish tissue-specific expression of DNRII transgene. CSK, a ubiquitously expressed kinase, was used to establish relative expression of the transgene in T cell and non-T cell populations (number in parentheses). (D) Semiquantitative RT-PCR to establish expression within T cell subsets. Expression of G3PDH was used to calculate relative expression between CD4⁺ and CD8⁺ T cell subsets (number in parentheses).

Figure 2

Small lymphocyte infiltration in DNRII Tg founder T12H1. (A) Extensive perivascular infiltration of small lymphocytes into the lung with little inflammation. (B) Higher magnification showing lymphoid cells in alveolar walls. (C) Spleen containing large white pulp areas with disruption of normal architecture. (D) Higher magnification showing a uniform population of lymphoid cells. (E) Liver with extensive perivascular and periportal lymphoid infiltration, with many lymphoid cells present in the blood but little inflammation. (F) Higher magnification showing infiltration around the blood vessel and in the sinusoids. Magnifications: A, 75×; C and E, 50×; B, D, and F, 150×.

Figure 3

Flow cytometric analysis of B and T cells in DNRII Tg mice. Splenocytes from 12-wk-old founder T12H1 (right panels) or an aged-matched C57BL/6 mouse (left panels) were analyzed for expression of T cell markers, CD4 and CD8 (top panels), and B cell markers, B220 and MHC class II (CII) molecules (bottom panels). Absolute cell numbers ×10⁻⁶ shown in parentheses. At least 10,000 events were collected for each group.

Figure 4

T cell activation marker expression on surfaces of DNRII Tg CD8⁺ T cells. LN T cells from 27-wk-old non-Tg littermate mouse (left histograms) or DNRII Tg mouse I2A1 (right histograms) were stained with Abs specific for a panel of T cell activation/memory markers. At least 10,000 events were collected for each group.

Figure 5

Time course analysis of CD8⁺ peripheral T cells in DNRII Tg mice. (A) PBLs from founder line E1A3 (□) or non-Tg littermate mice (○) were analyzed by flow cytometry for T cell markers CD4 and CD8 (left; expressed as CD4/CD8 ratio) and CD8⁺CD44^hi surface marker (right) over time. (B) TCR V-β repertoire was measured by a panel of TCR V-β–specific mAbs. Percentage of CD8⁺ PBLs expressing a specific TCR V-β receptor was plotted over time. At least 10,000 events were collected per time point.

Figure 6

Thymic development and cell cycle analysis of DNRII Tg T cells. (A) LN cells (top panels) or thymocytes (bottom panels) from 27-wk-old DNRII Tg line I2A1 (right panels) or littermate control mice (left panels) were stained with anti-CD4 and -CD8 mAbs and were detected by flow cytometric analysis. (B) CD8⁺ LN T cells (four leftmost panels) or thymocytes (right panels) from 19-wk-old DNRII Tg line I2C2 (center panels) or littermate control mice (left and right panels) were stained with propidium iodide (PI). Percentage of cells in cycle (S/M/G2) is indicated as percentage of incorporation of PI as detected by flow cytometric analysis. Thymocytes (right panels) were used as a positive control for the detection of cycling cells. At least 10,000 events were collected for each group.