T Cells in Microgravity (micACTin): High resolution microscopy and deep gene expression profiling to find the T cell gravisensor

Space flights pose extreme challenges on the human body and impair the immune system with changes persisting long after return to normal gravity. Among the Apollo crew members, 50% reported bacterial or viral infection upon landing back on Earth.

The reduced effectiveness of the immune system is evidenced by increased infections and re-activation of latent viruses in astronauts.

With the planned long-term manned missions to Moon and Mars, it will be important to better understand the effects of space flight on the immune system.

Cells of the immune system have evolved to undergo rapid dynamics of the cell cytoskeleton during cell migration and for cell-cell interaction.

We hypothesize that the force and tension of the cell cytoskeleton underneath the cell membrane is a gravity sensor for immune cells.

At normal gravity, activation of the T cell receptor for 5 minutes stimulates large rearrangement of the actin cytoskeleton that can be measured by advanced microscopy techniques.

The T cell changes in shape from a round cell to a “fried egg” mimics the T cell immune synapse formation with other cells.

Termed T cell spreading, this is the first critical step to induce changes in gene expression to become T effector cells that can help B cells to produce antibodies or can kill virus-infected cells and tumor cells.We will in this research programme compare the T cell spreading response in micro-g versus 1 g conditions during a sounding rocket flight.

This is the ideal set up since we can examine rapid cytoskeletal changes in T cells on glass surfaces during 5 minutes of microgravity exposure.

T cells, wildtype or gene-edited to lack specific cytoskeletal proteins, will be loaded in fully-automated culture units capable of administrating cells that will be exposed to micro-g at 37ºC for 5 minutes and thereafter fixed. A setup as used in the BIM-2 experimental module in the Maser 12 sounding rocket will be employed.

In BIM-5, cassettes with cells are either placed on a static rack permissive of exposing cells to changes in gravity or on a centrifuge that maintain cells at 1 g throughout the experiment.

High content and high resolution microscopy to image T cells and deep RNA and DNA sequencing for gene expression profiling will be performed in our laboratory at Karolinska Institutet using our already established assays.

To understand how short term (minutes) and long term (hours-days) micro-g exposure affect T cell differentiation into effector cells, we will use the random positioning machine and analyse T cells by microscopy, transcriptomics, and flow cytometry for functionality.

We propose: Aim 1: To use high resolution imaging to qualitatively and quantitatively investigate early T cell receptor immune synapse formation and identify changes in the actin cytoskeleton dynamics and key proteins of the T cell response to microgravity.Aim 2: To perform global transcriptome and open chromatin analysis using RNA and DNA deep sequencing to identify, select and profile differentially expressed actin cytoskeleton network gene transcripts as potential biomarkers of T cell sensitivity and response to microgravity.Aim 3: To validate and extend analysis of actin regulators in T cells, wildtype and gene edited T cells will be exposed to microgravity from minutes to days in random positioning machine to explore T effector cell differentiation and function, and response in 3D printed environments.

We expect that our new and unique approach to use gene-edited T cells and combined microscopy and gene expression data will identify microgravity sensors and help in development of countermeasures for long space flights.