HCV-specific CD4 T cells show strong Tfh functionality after DAA-mediated viral clearance

Background:

During Hepatitis C virus (HCV) infection peripheral HCV-specific CD4 T cells acquire a T follicular helper (Tfh) phenotype but rapidly disappear from circulation and seem to accumulate in the liver tissue as the infection progresses to chronicity. Recently we revealed that HCV-specific Tfh cells with a memory-like phenotype reappear in the periphery after direct acting antiviral (DAA)-mediated viral clearance but their low frequency prevented us from further functional research on these cells.

Methods:

We established a clone-based system to evaluate CD4 T cell functionality in vitro. CD4 T cell clones were generated by single cell sorting and antigen non-specific expansion. Influenza- (Flu-) and HCV-specific CD4 T cell clones were established from healthy donors (HD) and DAA-cured HCV patients, respectively. Tfh (CXCR5+, PD-1+, CXCR3-) and Th1 (CXCR5-, CXCR3+, CCR6-) clones with unknown antigen-specificity derived from HD or patients. Phenotype and cytokine production were analyzed by flow cytometry. The B cell helper capacity was assessed by coculture of naïve allogeneic B cells with the T cell clones.

Results:

Differences in phenotype and functions of Tfh and Th1 clones demonstrated the maintenance of lineage-specific features in vitro and therefore validated our methodological approach. Unlike Tfh, Th1 and Flu clones, HCV clones showed less alteration of their ex vivo phenotype maintaining high PD-1 and CXCR3 expression. Furthermore, HCV clones were highly activated, indicated by high CD38 and OX40 expression but also characterized by elevated levels of the inhibitory markers TIGIT and BTLA. Despite their unique phenotype that was clearly different from Tfh clones, HCV clones showed high B cell helper capacity. Moreover, HCV clones produced high amounts of TNF, IL-21 and IFNg with a cytokine profile that more closely resembled that of Tfh clones.

Conclusion:

The highly functional potential of HCV-specific CD4 T cells after DAA-mediated elimination of persistent infection suggests that chronic antigen and interferon exposure of intrahepatic HCV-specific CD4 T cells in vivo might not have resulted in functional exhaustion but rather altered Tfh functionality to facilitate long-term viral control. Displaying such strong Tfh characteristics HCV-specific CD4 T cells after DAA therapy could be targeted by vaccination as they would support B cell-mediated humoral immunity.