title: Knockdown of T-bet expression in Mart-127-35 -specific T-cell-receptor-engineered human CD4(+)  CD25(-) and CD8(+) T cells attenuates effector function.
abstract: Gene transfer to create tumour epitope-specific cytolytic T cells for adoptive immunotherapy of cancer remains an area of active inquiry. When the Mart-127-35 -specific DMF5 T-cell receptor (TCR) is transferred into peripheral human CD4(+) T cells, the reprogrammed cells exhibit a T helper type 1 (Th1) phenotype with significant multifactorial effector capabilities. The T-bet transcription factor plays an important role in determination of the Th1 differentiation pathway. To gain a deeper understanding of how T-bet controls the outcome of human T-cell reprogramming by gene transfer, we developed a system for examining the effects of short hairpin RNA-mediated T-bet gene knockdown in sorted cell populations uniformly expressing the knockdown construct. In this system, using activated peripheral human CD4(+)  CD25(-) and CD8(+) T cells, T-bet knockdown led to attenuation of the interferon-γ response to both antigen-specific and non-specific TCR stimulation. The interleukin-2 (IL-2) antigen-specific response was not attenuated by T-bet knockdown. Also, in TCR-reprogrammed CD8(+) cells, the cytolytic effector response was attenuated by T-bet knockdown. T-bet knockdown did not cause redirection into a Th2 differentiation pathway, and no increased IL-4, IL-10, or IL-17 response was detected in this system. These results indicate that T-bet expression is required for maintenance of the CD4(+)  CD25(-) and CD8(+) effector phenotypes in TCR-reprogrammed human T cells. They also suggest that the activation protocol necessary for transduction with retrovectors and lentivectors may commit the reprogrammed cells to the Th1 phenotype, which cannot be altered by T-bet knockdown but that there is, nevertheless, a continuous requirement of T-bet expression for interferon-γ gene activation.
authors.affiliation: Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Sidharth S
authors.initials: SS
authors.lastname: Jha
authors.forename: Nitya G
authors.initials: NG
authors.lastname: Chakraborty
authors.forename: Prashant
authors.initials: P
authors.lastname: Singh
authors.forename: Bijay
authors.initials: B
authors.lastname: Mukherji
authors.forename: David I
authors.initials: DI
authors.lastname: Dorsky
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D065150:Cellular Reprogramming; D016207:Cytokines / Q000235:genetics / Q000276:immunology; D055785:Gene Knockdown Techniques; D006801:Humans; D015202:Protein Engineering; D011946:Receptors, Antigen / Q000235:genetics / Q000276:immunology; D020825:T-Box Domain Proteins / Q000235:genetics / Q000276:immunology; D018417:Th1 Cells / Q000276:immunology*; D000098815:T-bet Transcription Factor
chemical_list: D016207:Cytokines; D011946:Receptors, Antigen; D020825:T-Box Domain Proteins; D000098815:T-bet Transcription Factor
keywords: Mart1; T-bet; knockdown