Molecular insights into the pleiotropic effects of plasma on ex vivo-expanded T cells using DNA-microarray analysis.
OBJECTIVE
Immunotherapy with ex vivo-expanded T cells depends on a large supply of biologically active cells. Understanding the effects of culture parameters is essential for improving cell expansion and efficacy. We used DNA-microarray and flow-cytometric analysis coupled with functional assays to investigate mechanistic aspects of plasma supplementation in ex vivo T-cell expansion.


METHODS
The effect of plasma supplementation on 18 primary T-cell cultures over a 15-day expansion was investigated. Transcriptional analysis of 5 samples was done with time points every 2 to 3 days throughout the 15-day expansion. Quantitative RT-PCR analysis was used to confirm selected microarray data. The expression of granzyme A and vimentin were analyzed using intracellular flow cytometry. T-cell functionality was assessed using a mixed leukocyte reaction (MLR).


RESULTS
We show that the increased expansion of plasma-supplemented cultures of primary human T cells is mostly due to increased cell survival. T cells from plasma-supplemented cultures show higher expression of immunoglobulin genes, integrins, and genes of cytotoxic granules, suggesting a possible enhanced immune function. This was confirmed using a mixed leukocyte reaction and intracellular granzyme-A measurements. A distinct gene expression pattern was correlated to viability differences between plasma-supplemented and serum-free cultures. Ontological analysis of genes in this pattern suggests that the decreased viability of serum-free cultures correlates with higher expression of actin-cytoskeleton and lipid-metabolism genes. Vimentin was found to be expressed higher in serum-free cultures.


CONCLUSIONS
These results indicate that the observed decreased cytotoxicity of T cells cultured in serum-free media may be due to increased oxidative stress and cytoskeleton degradation.
Ramsborg
Christopher G
CG
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA.
Windgassen
Dirk
D
Fallon
Jonathan K
JK
Paredes
Carlos J
CJ
Papoutsakis
Eleftherios T
ET
D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology*; D016895:Culture Media, Serum-Free; D020869:Gene Expression Profiling; D053804:Granzymes; D006801:Humans; D016130:Immunophenotyping; D007167:Immunotherapy / Q000379:methods; D007700:Kinetics; D007959:Lymphocyte Culture Test, Mixed; D010949:Plasma; D012697:Serine Endopeptidases / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D014746:Vimentin / Q000235:genetics
D003470:Culture Media; D016895:Culture Media, Serum-Free; D014746:Vimentin; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human