Cutting edge: The transmembrane E3 ligase GRAIL ubiquitinates the costimulatory molecule CD40 ligand during the induction of T cell anergy - PubMed (original) (raw)

Cutting edge: The transmembrane E3 ligase GRAIL ubiquitinates the costimulatory molecule CD40 ligand during the induction of T cell anergy

Neil B Lineberry et al. J Immunol. 2008.

Abstract

Activation of naive T lymphocytes is regulated through a series of discrete checkpoints that maintain unresponsiveness to self. During this multistep process, costimulatory interactions act as inducible signals that allow APCs to selectively mobilize T cells against foreign Ags. In this study, we provide evidence that the anergy-associated E3 ubiquitin ligase GRAIL (gene related to anergy in lymphocytes) regulates expression of the costimulatory molecule CD40L on CD4 T cells. Using its luminal protease-associated domain, GRAIL binds to the luminal/extracellular portion of CD40L and facilitates transfer of ubiquitin molecules from the intracellular GRAIL RING (really interesting new gene) finger to the small cytosolic portion of CD40L. Down-regulation of CD40L occurred following ectopic expression of GRAIL in naive T cells from CD40(-/-) mice, and expression of GRAIL in bone marrow chimeric mice was associated with diminished lymphoid follicle formation. These data provide a model for intrinsic T cell regulation of costimulatory molecules and a molecular framework for the initiation of clonal T cell anergy.

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Conflict of interest statement

Disclosures: The authors have no financial conflict of interest.

Figures

FIGURE 1

FIGURE 1

Ionomycin-up-regulated GRAIL expression is associated with reduced CD40L expression. A and B, MACS-purified CD4 T cells from BALB/c mice were anergized with a low (0.1 _μ_M), medium (0.5 _μ_M), or high (1.5 _μ_M) ionomycin dose with or without PMA (200 ng/ml) overnight. Cells were then harvested for QPCR (A) or Western blot analysis (B) of GRAIL expression. N.S., nonspecific band used as a loading control; IB, immunoblot; Norm., normalized. C, MACS-purified BALB/c CD4 T cells were anergized with 0 _μ_M (left) or 1.5 _μ_M (right) ionomycin overnight. Live cells were then washed, stimulated, and stained for cell surface CD40L. D, Histograms representing the percentage of cell surface CD40L expressed on gated CD4 T cells with varying conditions of ionomycin anergy treatment and recall stimulation. Black histogram represents CD4 T cells treated with 0 _μ_M ionomycin, dark gray histogram denotes CD4 T cells treated with 0.5 _μ_M ionomycin, and light gray histogram represents CD4 T cells treated with 1.5 _μ_M ionomycin.

FIGURE 2

FIGURE 2

GRAIL binds and ubiquitinates CD40L. A, Schematic display of the domains and orientation of GRAIL and CD40L. B, 293T cells were transfected with 3× FLAG-tagged human CD40L (hCD40L) and the indicated V5-tagged GRAIL constructs. Eluted proteins from anti-FLAG conjugated agarose were separated by SDS-PAGE and blotted with the indicated Abs. IB, Immunoblot; IP, immunoprecipitation. C, 293T cells were transfected with 3× FLAG-tagged hCD40L, V5-tagged GRAIL constructs, and HA-tagged ubiquitin. Eluted proteins from anti-FLAG-conjugated agarose were separated by SDS-PAGE and blotted with the indicated Abs. D, 293T cells were transfected with 0.2 μg of 3× FLAG-tagged hCD40L and V5-tagged GRAIL at 0.4, 0.8, 1.2, and 1.6 μg (empty V5 vector was added for a total of 2 μg of total V5 plasmid for each transfection). Cell lysates were separated by SDS-PAGE and blotted with the indicated Abs.

FIGURE 3

FIGURE 3

GRAIL expression down-regulates endogenous CD40L expression. A, Freshly MACS-purified CD4 T cells from C57BL/6 and CD40−/− mice were stained for CD40L. Gray histogram represents unstimulated wild-type CD4 T cells, thick line denotes live CD40−/− CD4 T cells, and dotted line represents intracellular staining of CD40−/− CD4 T cells. B, MACS purified CD4 T cells from CD40−/− mice were stimulated overnight in vitro and then infected with retrovirus expressing the indicated GRAIL construct. After 36 h, cells were stained for total CD40L and gated GFP+ cells are shown. Thick line represents vector-transduced CD4 T cells, gray line denotes GRAIL transduced CD4 T cells, and dotted line represents ΔPA-transduced CD4 T cells.

FIGURE 4

FIGURE 4

GRAIL expression in naive CD4 T cells results in diminished follicle formation in vivo. DO11.10 bone marrow chimeric mice were generated as described (6). Mice were sacrificed 28 days after injection of transduced hematopoietic cells and lymphoid tissue was processed for histological analysis. A, GFP vector control. B, Wild-type GRAIL. C, Dominant negative H2N2 GRAIL. D, Otubain-1 ARF-1 (epistatic protein stabilizer of GRAIL). Original magnification was × 10;✩ denotes follicular zone. One representative mouse is shown from two to three mice per group from three independent experiments.

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