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Robert Barry, David Withers, Philip Murray, Peter Lane, John Curnow; Investigating the response of naive T-cells to the three isoforms of TGFβ. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2031.
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In both human and experimental animal models of disease, uveitis is thought to be mediated by CD4+ T-cells that on entering the eye fall under the control of TGFβ. Of the three TGFβ isoforms, TGFβ2 is found at high levels in the aqueous humour of uninflamed eyes, whereas TGFβ1 predominates in episodes of inflammation. The presence of TGFβ3 has not been demonstrated in uveitic eyes. Given the apparent importance of differential TGFβ signalling in the ocular microenvironment we set out to investigate the response of purified naïve CD4+ T-cells to the three mammalian isoforms of TGFβ at a range of physiological dilutions.
Cells were extracted from the lymph nodes of C57Bl/6 mice and purified to naïve CD4+ T-cells (CD4+CD25-CD62L+CD44lo) using flow cytometry. CD25+ cells were removed to exclude all regulatory T-cells (<1% Fox-P3+) and the CD62L+CD44lo population selected to exclude central memory cells. Cells were labelled with a proliferation dye (eFluor450) and cultured in 96-well plates (200,000 cells in 200ul) in serum-free media with IL-2, stimulated with plate-bound anti-CD3 and soluble anti-CD28. TGFβ1, 2 or 3 was added at 0.05 - 5.0 ng/ml. Cells were harvested following 4 days of culture with proliferating and apoptotic cells identified from forward/side-scatter profile on flow cytometric analysis, and cell count and cycles of proliferations calculated.
Increasing concentrations of all three TGFβ isoforms resulted in increased cell proliferation and decreased apoptosis at the dilutions tested. At the highest concentrations of TGFβ (5.0ng/ml) cells underwent >4 cycles of proliferation. No statistically significant difference between TGFβ isoforms was observed (average % cells undergoing proliferation 2.52 at 0.05ng/ml, 14.22 at 0.5ng/ml, 45.38 at 5.0ng/ml and apoptosis 55.45, 49.95, 20.11 respectively).
Our data illustrates that all isoforms of TGFβ are equal in their ability to induce proliferation and inhibit apoptosis of a highly purified population of naïve T-cells, possibly through inhibition of activation-induced cell death. As TGFβ also determines immune activation through its ability to generate both immunosuppressive Treg and pathogenic Th17 cells further work is underway to investigate the effect of different isoforms of TGFβ on Treg / Th17 differentiation.
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