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Competitive Homing Assays to Study Gut-tropic T Cell Migration
Published on: March 2, 2011
Bovine gamma delta T cells express high levels of functional peripheral lymph node homing receptor (L-selectin)
1Montana State University, Bozeman 59717.
Lymphocyte migration from the blood into specific tissues is directed by their expression of adhesion molecules referred to as homing receptors. The homing receptor L-selectin, for example, directs the migration of lymphocytes into peripheral lymph nodes (PLN). Since bovine gamma delta T cells, a major lymphocyte subset in peripheral blood (25-50%), represent only a minor subset in PLN, we examined whether these cells lack expression or function of L-selectin. We found that bovine gamma delta T cells expressed L-selectin at levels higher (2- to 5-fold) than alpha beta T cells and B cells. Furthermore, gamma delta T cells accumulated along the vascular wall of venules that support lymphocyte extravasation into PLN (MECA-79+ venules) in vivo and bound mouse PLN high endothelial cell venules in an ex vivo binding assay. In contrast to this primary adhesive event, we directly demonstrate that gamma delta T cells in vivo do not appreciably extravasate from the blood into the parenchyma of lymph nodes. Since the lack of functional L-selectin expression could not account for the inability of gamma delta T cells to enter PLN, we tested for other differences between gamma delta T cells and PLN homing lymphocytes related to the processes following primary adhesion; for instance, the down-regulation of L-selectin expression following short-term activation and the expression of accessory adhesion molecules necessary for transendothelial migration. We found that gamma delta and alpha beta T cells demonstrate differential down-regulation of L-selectin after PMA activation. Kinetic analysis revealed that, at all time points after PMA treatment, L-selectin expression remained significantly higher on gamma delta T cells and was down-regulated at a slower rate compared with alpha beta T cells. However, the expression levels of CD44 and CD18 on gamma delta and alpha beta T cells were found to be equivalent. This study is the first to demonstrate for lymphocytes that the expression of L-selectin alone does not predict a PLN homing capacity. Our results suggest that the gamma delta T cells' reduced ability to enter PLN may be due to inefficient down-regulation of L-selectin compared with non-gamma delta lymphocytes, thus potentially disrupting the dynamics of the extravasation event.
Lymphocyte migration from the blood into specific tissues is directed by their expression of adhesion molecules referred to as homing receptors. The homing receptor L-selectin, for example, directs the migration of lymphocytes into peripheral lymph nodes (PLN). Since bovine gamma delta T cells, a major lymphocyte subset in peripheral blood (25-50%), represent only a minor subset in PLN, we examined whether these cells lack expression or function of L-selectin. We found that bovine gamma delta T cells expressed L-selectin at levels higher (2- to 5-fold) than alpha beta T cells and B cells. Furthermore, gamma delta T cells accumulated along the vascular wall of venules that support lymphocyte extravasation into PLN (MECA-79+ venules) in vivo and bound mouse PLN high endothelial cell venules in an ex vivo binding assay. In contrast to this primary adhesive event, we directly demonstrate that gamma delta T cells in vivo do not appreciably extravasate from the blood into the parenchyma of lymph nodes. Since the lack of functional L-selectin expression could not account for the inability of gamma delta T cells to enter PLN, we tested for other differences between gamma delta T cells and PLN homing lymphocytes related to the processes following primary adhesion; for instance, the down-regulation of L-selectin expression following short-term activation and the expression of accessory adhesion molecules necessary for transendothelial migration. We found that gamma delta and alpha beta T cells demonstrate differential down-regulation of L-selectin after PMA activation. Kinetic analysis revealed that, at all time points after PMA treatment, L-selectin expression remained significantly higher on gamma delta T cells and was down-regulated at a slower rate compared with alpha beta T cells. However, the expression levels of CD44 and CD18 on gamma delta and alpha beta T cells were found to be equivalent. This study is the first to demonstrate for lymphocytes that the expression of L-selectin alone does not predict a PLN homing capacity. Our results suggest that the gamma delta T cells' reduced ability to enter PLN may be due to inefficient down-regulation of L-selectin compared with non-gamma delta lymphocytes, thus potentially disrupting the dynamics of the extravasation event.
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