Global Transcriptional Analysis Details Prior Knowledge and Identifies New Genes and Processes Associated with T-Cell Activation in the Context of Ex Vivo Expansion.

Introduction. Ex vivo expanded T cells are increasingly employed in a variety of immunotherapy protocols. The success of such protocols requires large numbers of biologically active T cells. Several activation and culture protocols have been examined, but none in significant or molecular detail to allow more targeted protocol interventions. In this study, our aim was to better understand early T-cell activation using a comprehensive, microarray-based transcriptional analysis, in conjunction with targeted protein-level and functional analyses.

Methods. Three biological experiments were transcriptionally analyzed. T cells were activated with anti-CD3/anti-CD28 Mab, cultured for 96 hours, and samples collected at 0, 4, 10, 48 and 96 hours. mRNA levels at each time point were compared to that of 0 hour. Genes were selected as follows: a minimum 1.8-fold difference in at least 6 time points of the 12 (3x4) time points. Data were subject to Gene Ontology (GO) analysis, EASE, to discover enriched biological themes. Interferon-γ (IFNG) and CCL20 ELISA assays and NF-κB p65 (active complex) assay by flow cytometry were carried out to confirm select findings of the transcriptional analysis.

Results. 2340 genes passed the gene-selection criteria. GO analysis showed that the GO groups Immune Response, Regulation of Apoptosis, and Mitochondrion are among the most over-represented groups among differentially expressed genes during T-cell activation. The upregulated Immune Response group contained many genes coding cytokines typically produced by helper T cells. Highly upregulated genes in this category include IL2, CCL20, CXCL11, CXCL9, CXCL10, CD83, IFNG, CCL3, CSF2, IL23A, XCL2, TNF, CCL4, TNFSF6 and ICOS. Although several of these findings would have been expected, our data include a more extensive gene list and suggest potential use of these cytokines as T-cell ex vivo culture supplements. We singled out CCL20, sharing a very similar transcription pattern with IL2, and IFNG, for further analysis. ELISA assays of IFNG and CCL20 showed that the kinetics of secreted protein levels were in agreement with transcriptional data. In the GO group Regulation of Apoptosis, the downregulated gene cluster was enriched by pro-apoptotic genes, while the upregulated cluster was enriched by anti-apoptotic genes. Our data indicated that NF-κB complex activity changed upon T-cell activation. This was verified by NF-κB p65 Phosflow assay, showing that phosphorylated NF-κB p65 was suppressed up to 10 hours, and then increased until hour 96. Although it is known that mitochondrial hyperpolarization is important in T-cell activation, our transcriptional data suggest more profound changes in mitochondrial biology. Genes in this category include several having oxidoreductase activity, such as SOD2, SCO1, NDUFAB1, GLUD2, consistent with reported increases in intracellular ROS levels upon T-cell activation, and suggest the potential use of anti-oxidants in ex vivo T-cell expansion.

Conclusion. Our data and analysis provide a better understanding of T-cell activation and identify several potential targets that can be explored to benefit T-cell expansion protocols for immunotherapy applications. Although there is much known about T-cell activation, modern genomic tools provide an extraordinary opportunity to verify, extend and enrich prior knowledge, and, discover new players and processes not previously associated with T-cell activation.