T cell activation responses are differentially regulated during clinorotation and in spaceflight.

Studies of T lymphocyte activation with mitogenic lectins during spaceflight have shown a dramatic inhibition of activation as measured by DNA synthesis at 72 h, but the mechanism of this inhibition is unknown. We have investigated the progression of cellular events during the first 24 h of activation using both spaceflight microgravity culture and a ground-based model system that relies on the low shear culture environment of a rotating clinostat (clinorotation). Stimulation of human peripheral blood mononuclear cells (PBMCs) with soluble anti-CD3 (Leu4) in clinorotation and in microgravity culture shows a dramatic reduction in surface expression of the receptor for IL-2 (CD25) and CD69. An absence of bulk RNA synthesis in clinorotation indicates that stimulation with soluble Leu4 does not induce transition of T cells from G0 to the G1 stage of the cell cycle. However, internalization of the TCR by T cells and normal levels of IL-1 synthesis by monocytes indicate that intercellular interactions that are required for activation occur during clinorotation. Complementation of TCR-mediated signaling by phorbol ester restores the ability of PBMCs to express CD25 in clinorotation, indicating that a PKC-associated pathway may be compromised under these conditions. Bypassing the TCR by direct activation of intracellular pathways with a combination of phorbol ester and calcium ionophore in clinorotation resulted in full expression of CD25; however, only partial expression of CD25 occurred in microgravity culture. Though stimulation of purified T cells with Bead-Leu4 in microgravity culture resulted in the engagement and internalization of the TCR, the cells still failed to express CD25. When T cells were stimulated with Bead-Leu4 in microgravity culture, they were able to partially express CD69, a receptor that is constitutively stored in intracellular pools and can be expressed in the absence of new gene expression. Our results suggest that the inhibition of T cell proliferative response in microgravity culture is a result of alterations in signaling events within the first few hours of activation, which are required for the expression of important regulatory molecules.     

Activation of human T lymphocytes under conditions similar to those that occur during exposure to microgravity: a proteomics study

A number of experiments, conducted under microgravity conditions, i.e. in space shuttle biolaboratories or in ground based systems simulating the conditions occurring in microgravity, show that in hypogravity, in vitro human lymphocyte activation is severely impaired. However, very early stimulation steps of T lymphocytes are not compromised, since CD69 receptor, the earliest membrane activation marker, is expressed by T cells at a level comparable to that observed on 1 g activated lymphocytes. Since CD69 engagement, together with submitogenic doses of phorbol esters, transduces an activation signal to T lymphocytes, we undertook a comparative study on the stimulation mediated through this receptor on human CD3+ cells cultured under conditions similar to those which occur during exposure to microgravity, i.e. in clinorotation, or at 1 g. During the early hours of activation, increased levels of intracellular calcium and increased mitochondrial membrane potential were detectable in clinorotating as well as in 1 g cells. However, after 48 hours clinorotation, interleukin 2 production by T lymphocytes was significantly reduced and cell proliferation was greatly decreased. By means of a differential proteomics approach on T cells activated in clinorotation or at 1 g for 48 hours, we were able to detect statistically significant quantitative protein alterations. Seven proteins with modified expression values were identified; they are involved in nucleic acids processing, proteasome regulation and cytoskeleton structure.