The effects of actual microgravity and vector-averaged gravity on the development of T cells

Abstract

It is well documented that long-term spaceflight adversely affects immune system function. Using fetal thymus organ culture (FTOC), we examined the effects of spaceflight and vector-averaged gravity on T cell development. In order to perform this work, we needed to design and validate a culture system that supported FTOC in a microgravity environment. The system we built, and which is described herein, served successfully for ground-based experimentation built on the principle of the clinostat. Moreover, results of testing this system at NASA's Ames Research Center demonstrated that it is optimal for future flight experimentation. This system can also be used for other cell/organ culture methodologies where three-dimensional growth and organotypic organization optimizes function for ground-based as well as spaceflight experiments. Under both conditions (spaceflight and vector-averaged gravity), the development of T cells was significantly attenuated. Exposure to spaceflight for 16 days resulted in a loss of precursors for CD4⁺, CD8⁺, and CD4⁺CD8⁺ T cells in a rat/mouse xenogeneic co-culture. A significant decrease in the same precursor cells, as well as a decrease in CD4⁻ CD8⁻ T cell precursors, was also observed in a murine C57BL/6 FTOC after rotation in a clinostat. The observed block in T cell development appeared to occur between the pre-T cell and CD4⁺CD8⁺ T cell stage. Furthermore, flow cytometric analysis clearly illustrated a reduction in the expression of IL-7Rα (CD127) in clinorotated FTOC as well as an increase in the presence of TNF-α after 4 days of culture. Levels of phosphorylated Lck were unchanged in clinorotated FTOC when compared to motional and stationary controls. These findings suggest that the full sequelae of pre-TCR signaling is dependent upon the presence of gravity. However, this alteration may be occurring downstream of Csk/Lck regulation. In support of this line of reasoning, T cell development was partially rescued in clinorotated FTOC treated with anti-CD3 monoclonal antibody. Anti-CD3 treatment presumably partially substitutes for a signal that is required for proper T cell development and is absent in the microgravity environment. These data therefore indicate that gravity indeed plays a decisive role in β-selection and in broader terms the development of T cells.