Bet v 1-Specific T-Cell Receptor/Forkhead Box Protein 3 Transgenic T-Cells Suppress Bet v 1-Specific T-Cell Effector Function in An Activation-Dependent Manner

Background: T-cell function can be modified by over-expression of transgenes. Recently, we have shown that transduction of T-cell receptor (TCR) alpha/beta chains specific for allergen-derived T-cell epitopes transfers allergen-specificity to peripheral blood T-cells. Similarly, over-expression of Foxp3 has been reported to generate transgenic regulatory T-cells (Treg). We aimed to combine these two strategies to engineer allergen-specific Treg. 

Methods: cDNAs encoding the a- and b-chains of a Bet v 1_142-153-specific TCR (TRAV6/TRBV20) and human Foxp3 were linked via picornaviral 2A-sequences and expressed as a single translational unit from an internal ribosomal entry site-green fluorescence protein containing retroviral vector. Subsequently, stably transduced PB T-cells were tested for expression of transgenes, Treg phenotype and regulatory capacity towards function of allergen-specific effector T-cells in response to allergen-specific and polyclonal stimuli.

Results: Transduced T-cells showed clear-cut transgene expression and the typical Treg phenotype with up-regulation of CD25, CD39 and CTLA-4 and down-regulation of CD127. Similar to endogenous Treg cells, the transduced cells were hyporesponsive in cytokine production and secretion and did not proliferate following antigen-specific or CD3/CD28-mediated stimulation. However, proliferation was inducible in response to exogenous IL-2. In co-culture experiments, TRAV6⁺TRBV20⁺Foxp3⁺ transgenic (tg) T-cells, unlike Foxp3⁺ single tg T-cells or CD4⁺CD25⁺ naturally-occurring Treg, suppressed T-cell cytokine production and proliferation of corresponding allergen-specific effector T-cells in an allergen-specific and dose-dependent manner. Interestingly, effector T-cell cytokine production was inhibited already at the level of cytokine production, suggesting that cytokine depletion by such tg Treg is not the major mechanism of suppression.

Conclusions: We demonstrate here a transgenic approach to engineer human allergen-specific Treg that exert regulatory function in an activation-dependent manner. Such customized Treg might become useful for tolerance-induction therapies in patients suffering from allergic and other immune-mediated diseases. Further efforts will also focus on the elucidation of transcription factors that are involved in the induction of an induced Treg/Tr1-like phenotype.