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STS107 T-cell activation at 1g vs. vg

ABSTRACT: Peripheral blood leukocytes of three human donors (Donors 2, 4, and 5) were isolated from blood bank buffy-coat preparations by Ficoll-Hypaque density gradient centrifugation, and T-cells were purified with high affinity negative selection human CD3+ T-cell enrichment columns. The cells were resuspended in RPMI-1640 with 10% FCS with approximately 3-8 millions cells/mL at room temperature overnight. T-cells were then either unactivated (1g 0 hr), activated (1g 4 hr) with a final concentration of 5 mg/mL Con A + 4 mg/mL anti-CD28 antibody and incubated for 4 hours at 37°C, or loaded onto a RPM rotating at 60°/s (vg). T-cells loaded on the RPM were cultured for 2 hours prior to collection or activation to allow equilibration of cells to the new environment. After incubation, 1 mL of 4 M guanidinium isothiocyanate in 750 mM sodium citrate buffer, pH 7, with N-lauroylsarcosine and beta-mercaptoethanol was added to lyse cells and stabilize RNA. Keywords: ordered

ORGANISM(S): Homo sapiens

PROVIDER: GSE1709 | GEO | 2005/08/25

SECONDARY ACCESSION(S): PRJNA114695

REPOSITORIES: GEO

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Project description:This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in microgravity (mg). Immunosuppression during spaceflight is a major barrier to safe long-term human space habitation and travel. The goals of these experiments were to prove that mg was the cause of impaired T cell activation during spaceflight as well as understand the mechanisms controlling early T cell activation. T cells from 4 human donors were stimulated with concanavalin A (ConA) and anti-CD28 onboard the International Space Station (ISS). An onboard centrifuge was used to generate a 1g simultaneous control to isolate the effects of mg from other variables of spaceflight. Microarray expression analysis after 1.5 hours of activation demonstrated that mg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly differentially down-regulated in mg. Importantly, several key immediate early genes were inhibited in mg. T cells were isolated from human volunteers. T cells from each donor were kept separate and loaded into individual chambers in separate cassettes for the following treatments: mg non-activated, mg activated, and 1g activated. Therefore, samples represent biological triplicates. Experimental units were launched into space and placed into the KUBIK facility onboard the International Space Station. The 1g units were placed in the central centrifuge positions and centrifuged with an applied 1g force. The mg units were place in the static positions for continued mg exposure. After 30 minutes of pre-incubation, mg non-activated units were fixed by addition of RNALater (QIAGEN, Valencia, CA), removed from the incubator, and stored in 4M-BM-0C. The mg and 1g activated units were injected with final concentration 10mg/ml Con A and 4mg/ml anti-CD28. These cassettes were replaced into KUBIK on either the centrifuge or static positions and activated for 1.5 hours. Activation was stopped with the addition of RNALater and the units were then moved to 4M-BM-0C storage. All units were returned to Earth for analysis.

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STS107 T-cell activation at 1g vs. vg

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