Korean J Physiol Pharmacol.  2012 Oct;16(5):349-353. 10.4196/kjpp.2012.16.5.349.

CTLA-4-Tg/CD-28-KO Mice Exhibit Reduced T Cell Proliferation in vivo Compared to CD-28-KO Mice in a Graft-versus-host Disease Model

• Yoo JS
• Lee YJ
• Yoon JW
• Hyung KE
• Hwang KW

• KMID: 1493969
• DOI: http://doi.org/10.4196/kjpp.2012.16.5.349

Abstract

Activated T cells express inhibitory receptors such as CTLA-4 that can downregulate immune responses. Blockade of or genetic deficiency in CTLA-4 can result in autoimmunity. Therefore, strategies to increase the inhibitory function of CTLA-4 may be attractive in settings of undesirable T cell responses such as autoimmunity or transplant rejection. We have tested the hypothesis that transgenic constitutive expression of CTLA-4 can further attenuate immune responses when compared with normal inducible expression. Our results indicate that transgenic expression of CTLA-4 in mouse T cells (CTLA-4-Tg T cells) results in reduced cell cycle progression and increased apoptosis of TCR-stimulated T cells. CTLA-4-Tg T cells display reduced T cell proliferation in an in vivo model of graft versus host disease (GVHD). These results further our understanding of how CTLA-4 can be manipulated to inhibit immune responses and may help development of new therapeutic strategies for clinical settings of autoimmunity and transplantation.

Keyword

Cell surface molecules; Cellular activation; T lymphocytes; Transgenic; Rodent

MeSH Terms

Animals
Apoptosis
Autoimmunity
Cell Cycle
Cell Proliferation
Graft Rejection
Graft vs Host Disease
Mice
Rodentia
T-Lymphocytes
Transplants

Figure

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  Fig. 1 Reduced proliferation by CTLA-4-Tg splenocytes. Splenocytes from WT and CTLA-4-Tg mice were incubated with soluble anti-CD3 mAb and incorporation of 3H-thymidine for the last 6 h of a 72 h culture was assessed. The data are expressed as the mean±SD. The result is representative of 3 independent experiments.

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  Fig. 2 TCR-stimulated CTLA-4-Tg T cells have reduced cell-cycle progression and increased apoptosis than WT T cells. CD4+ T cells were purified from WT and CTLA-4-Tg splenocytes and stimulated with anti-CD3 (1 µg/ml) in the presence of T-depleted irradiated syngeneic WT splenocytes. Cells were stained with anti-CD4-APC, fixed, permeabilized, stained with PI and analyzed by flow cytometry. Doublets were excluded based on FL2-Width. The histograms represent PI fluorescence intensity on CD4+-gated cells. The numbers in the upper right corner of the plot represent the percent of CD4+ cells in S/G2/M phases of the cell-cycle. The numbers in the upper left corner represent the percent of CD4+ cells with subdiploid DNA content. This result is representative of 3 independent experiments.

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  Fig. 3 Greater number of CTLA-4-Tg than WT T cells undergoes apoptosis following TCR stimulation. T cells were purified from WT and CTLA-4-Tg splenocytes and stimulated as for Fig. 2. Cells were stained with APC-conjugated antiCD4 or anti-CD8, FITC-coupled annexin V and PI and analyzed by flow cytometry. Dot plots represent annexin V versus PI fluorescence of cells following gating on CD4+ or CD8+ cells. Numbers in the plots represent percent CD4+ or CD8+ cells that stain positive for annexin V (lower right quadrant) or for annexin V and PI (upper right quadrant). This result is representative of 2 independent experiments.

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  Fig. 4 Reduced in vivo responses by CTLA-4-Tg/CD28-KO than CD28-KO T cells in a GVHD model. T cells were purified from CD28-KO and CTLA-4-Tg/CD28 KO splenocytes and labeled with CFSE. 4×106 T cells were injected intravenously into sub-lethally irradiated BALB/c recipients. Animals were sacrificed on day 2 and splenocytes were stained with PE-coupled anti-Kb and APC-coupled anti-Thy 1.2, CD4, or CD8 mAbs. Events were gated on Kb+/Thy l.2+, Kb+/CD4+, or Kb+/CD8+ cells and CFSE fluorescence are displayed as histograms. Results are representative of 2 independent experiments.

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