Hypoxia modulates early events in T cell receptor‐mediated activation in human T lymphocytes via Kv1.3 channels

Abstract

T lymphocytes are exposed to hypoxia during their development and when they migrate to hypoxic pathological sites. Although it has been shown that hypoxia inhibits Kv1.3 channels and proliferation in human T cells, the mechanisms by which hypoxia regulates T cell activation are not fully understood. Herein we test the hypothesis that hypoxic inhibition of Kv1.3 channels induces membrane depolarization, thus modulating the increase in cytoplasmic Ca 2+ that occurs during activation. Hypoxia causes membrane depolarization in human CD3 + T cells, as measured by fluorescence-activated cell sorting (FACS) with the voltage-sensitive dye DiBAC 4 (3). Similar depolarization is produced by the selective Kv1.3 channel blockers ShK-Dap 22 and margatoxin. Furthermore, pre-exposure to such blockers prevents any further depolarization by hypoxia. Since membrane depolarization is unfavourable to the influx of Ca 2+ through the CRAC channels (necessary to drive many events in T cell activation such as cytokine production and proliferation), the effect of hypoxia on T cell receptor-mediated increase in cytoplasmic Ca 2+was determined using fura-2. Hypoxia depresses the increase in Ca 2+ induced by anti-CD3/CD28 antibodies in ∼50% of lymphocytes. In the remaining cells, hypoxia either did not elicit any change or produced a small increase in cytoplasmic Ca 2+ . Similar effects were observed in resting and pre-activated CD3 + cells and were mimicked by ShK-Dap 22 . These effects appear to be mediated solely by Kv1.3 channels, as we find no influence of hypoxia on IKCa1 and CRAC channels. Our findings indicate that hypoxia modulates Ca 2+ homeostasis in T cells via Kv1.3 channel inhibition and membrane depolarization.

Hypoxia controls CD4+CD25+ regulatory T‐cell homeostasis via hypoxia‐inducible factor‐1α

“…Additionally, HIF-1a was reported to play an inhibitory role in the regulation of T-cell receptor signal transduction by controlling intracellular calcium balance [8]. Collectively, these observations suggest that HIF-1 drives a Th2 favored response by downregulated Th1 programs [9][10][11]. Naturally occurring CD4 1 CD25 1 regulatory T cells (Treg) comprise a relatively newly characterized population that has evolved to tune down cellular immune responses [12].…”

The Ca2+-activated K+ channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes

“…To achieve this, we used anti-CD3 and anti-CD28 antibodycoated beads to induce T cell activation. This is a wellvalidated system shown by us and others to induce Ca 2ϩ influx and molecular reorganization, both indicative of a functional T cell activation (23,30). Furthermore, upon T cell activation, extensive cytoskeletal reorganization takes place, resulting in F-actin accumulation at the contact point; as such, it can serve as a marker of IS formation (3,5).…”

“…Cells were stored at 37°C in the dark for up to 2 h before use. All cell-imaging experiments were performed on a Cyt-Im2 Ca 2ϩ imaging system (Intracellular Imaging, Cincinnati, OH), using a ratiometric method as previously described (14,23). Cells were imaged on a Nikon-inverted epifluorescence microscope equipped with a heated microscopy chamber, a ϫ20 objective, and a xenon arc lamp, which was used for the alternative excitation of fura 2 at 340 and 380 nm.…”

“…ϩ and CD4 ϩ lymphocytes were isolated from healthy donors by E-rosetting (StemCell Technology, Vancouver, Canada) and Ficoll-Paque density-gradient centrifugation (ICN Biomedicals, Aurora, OH) and maintained as previously described (23). Freshly isolated human T cells were preactivated with 4 g/ml phytohemmaglutinin (PHA; Sigma-Aldrich, St. Louis, MO) and transfected 18 -24 h later with YFP-KCa3.1 with the Amaxa Nucleofector technology (Amaxa Biosystems, Cologne, Germany) using 10 ϫ 10 6 cells, 5 g DNA, and program T20 according to the manufacturer's instructions.…”

Glut1-mediated glucose transport regulates HIV infection

“…Thus, although lymphocytes encounter fluctuations in O 2 levels in vivo, the physiological range of O 2 levels to which they are exposed is at least two to six times lower than the 20% O 2 levels maintained in standard incubators. Culturing at atmospheric O 2 levels has been shown to result in altered cell proliferation rates and other skewed T-cell responses (16,(19)(20)(21)(22)(23). Thus, because the goal of in vitro studies is generally to reveal information of in vivo significance, our studies here focus on findings with peripheral blood T cells cultured at physiological O 2 levels.…”