Knockdown of T‐bet expression in Mart‐1_27–35‐specific T‐cell‐receptor‐engineered human CD4⁺ CD25⁻ and CD8⁺ T cells attenuates effector function

Abstract

SummaryGene transfer to create tumour epitope-specific cytolytic T cells for adoptive immunotherapy of cancer remains an area of active inquiry. When the Mart-1 27-35 -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 À and CD8 + T cells, T-bet knockdown led to attenuation of the interferon-c 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 À 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-c gene activation.

pTregs or iTregs are the Potent Tolerance Inducer for the growth and Metastasis of Cancer

“…In our previous reports we have shown that when CTL precursors with specificity for a self but tumor-associated epitope are optimally stimulated, tTreg are not much of an impediment but iTreg or pTreg are [9][10][11][12]. Our previous results also indicated that the freshly isolated Treg cells consist of a heterogeneous group of CD4+ T cells (both tTreg and pTreg) [13].…”

“…If available and permitted by the pathologists, surgically removed tumor tissues were also obtained. The details of the collection of samples and cell preparation have been described [9,10].…”

“…Dendritic cells (DC) used in this study were isolated from peripheral myeloid antigen presenting cells (APC) expanded in vitro as described earlier [8,9,11]. Briefly, monocyte/macrophages were isolated as adherent cells from Ficoll Hypaque gradient derived mononuclear cell populations from blood.…”

“…Cytokine, IFNγ, IL-4 and IL-10 were assayed in appropriate ELISA assays as per manufacturer's (DuoSet ELISA Development System, R&D System, Minneapolis, MN) protocol and described earlier, Intracellular cytokine assay was done as described in our previous report [9][10][11]. Briefly, the effector cells were stimulated with peptide-pulsed DC for a total period of 6 hr at 37 °C (responder to stimulator ratio = 10:1).…”

Noninvasive detection of tumor-infiltrating T cells by PET reporter imaging

“…CD8 cells were capable of IFN-γ production, and no difference in the quantity of cytokine-producing cells was observed between groups ( Figure 1C). CD4 cells engineered to express the MHC class Irestricted F5 TCR have been previously described to exhibit a Th1 phenotype with increased IFN-γ production (33). CD4 cells expressing either F5 or F5/hdCK3mut produced equivalent IFN-γ when stimulated by PMA/ionomycin ( Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI77326DS1).…”