T cell exhaustion and microRNAs

Functional exhaustion of antigen-specific T cells is a feature of many chronic viral infections and the role of miRNAs in this process are not well understood. A typical exhausted T cell is characterized by dysfunctional responses such as low proliferative potential and decreased ability of cytokine production such IL-2, TNF-α and IFN-γ. These defects have been associated with upregulation of inhibitory receptors such as programmed death-1 ( PD-1), a member of the B7:CD28 family, capable to induce T cell exhaustion following the interaction with its own ligands (PD-L1 expressed on all cells and/or PD-L2 particularly expressed on dendritic cells). In this project we studied the early phases of PD-1-dependent T cell exhaustion in healthy individuals and we correlated this phenomenon with the microRNAs profile in these cells. PD-1 positive and PD-1 negative PBL populations from six healthy donors were FACS-sorted and the functional features of CD4+ and CD8+ T cells/ expressing or not PD-1 were analyzed for the intracellular cytokine production in response to polyclonal stimuli. The exhaustion is a gradual process in which IL-2 production is the first function to be lost: our data demonstrated that two samples analyzed were characterized by PBLs/PD-1+ in terminal exhaustion because they produced less IL-2 and IFN-γ after antiCD3/CD28 stimulation than PD-1- T cells; the other four samples analyzed instead were characterized by T lymphocytes in early exhaustion, because PD-1+ T cells produced only less IL-2 when stimulated with antiCD3-CD28. This result demonstrates that PD-1 expression correlates with different degree of exhaustion. To validate both PD-1 and TIM-3 as exhaustion markers, we stimulated T cell clones with mitogenic stimuli in vitro and we analyzed the expression level of both PD-1 and TIM-3, as well as the cytokine production upon the appropriate stimuli. In these conditions, T cell clones upregulated both PD-1 and TIM-3 in relation with impaired cytokine production. Interestingly, upon repeated rounds of stimulation in vitro, T cell clones rescued the capacity of performing their functions in relation with a dramatic downregulation of both PD-1 and TIM-3, suggesting that the exhaustion phenomenon is reversible. This was confirmed by experiments showing rescued cytokine production by PD-1+ T cell clones that had been stimulated in the presence of a blocking anti-PD-L1 antibody. To identify candidate microRNAs in early phases of the exhaustion process, we analyzed the microRNAs expression profile by Taqman Low density array on PD-1+ T cells producing low amounts of IL-2, but normal IFN- The analyses showed that only 23 microRNAs are deregulated in PD-1+ PBLs compared to PD-1- PBLs and that miR-21 resulted the most upregulated. The upregulation of miR-21 were also validated by RT-PCR in functionally-exhausted CD4+ T cell clones coexpressing PD-1 and TIM-3. This dimostrated that miR-21 could be a candidate microRNA in the early phases of T cell exhaustion.