Molecular understanding of oxygen-tension and patient-variability effects on ex vivo expanded T cells.
Immunotherapy with ex vivo cultured T cells depends on a large supply of biologically active cells. Understanding the effects of culture parameters is essential for improving the proliferation and efficacy of the expanded cells. Low oxygen tension (5% pO(2)) was previously reported to improve T-cell expansion and alter cellular phenotypic characteristics compared to T cells cultured at 20% pO(2). Here we report the use of DNA-array based transcriptional analysis coupled with protein-level analysis to provide molecular insights into pO(2) and patient-variability effects on expanded primary human T cells. Analysis of seven blood samples showed that reduced pO(2) results in higher expression of genes important in lymphocyte biology, immune function, and cell-cycle progression. 20% pO(2) resulted in higher expression of genes involved in stress response, cell death, and cellular repair. Expression of granzyme A (gzmA) was found to be significantly regulated by oxygen tension with cells at 5% pO(2) having greater gzmA expression than at 20% pO(2). Protein-level analysis of gzmA was consistent with transcriptional analysis. Granzyme K (gzmK) was coexpressed with gzmA, whereas Granzyme B (gzmB) expression was found to precede the expression of both gzmA and gzmK in 15-day cultures. Temporal gene expression patterns for seven blood samples demonstrate that most genes are expressed by all patient samples in similar temporal patterns. However, several patient-specific gene clusters were identified, and one cluster was found to correlate well with cell proliferation and may potentially be used to predict patient-specific T-cell expansion.
Department of Chemical and Biological Engineering, Northwestern University, 2145 N. Sheridan Road, Room E136, Evanston, IL 60208, USA.
Hadar
H
Haddad
Dirk
D
Windgassen
Christopher G
CG
Ramsborg
Carlos J
CJ
Paredes
Eleftherios T
ET
Papoutsakis
D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000235:genetics*; D014644:Genetic Variation; D006432:Hemochromatosis / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010100:Oxygen / Q000378:metabolism*; D013601:T-Lymphocytes / Q000378:metabolism*
D010100:Oxygen