T cell receptor signaling mediates enhanced IFNγ production by γδ intraepithelial lymphocytes in response to type I interferon

Abstract Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor (TCR) provide a rapid response to limit enteric pathogen invasion. Despite constant γδTCR triggering in vivo, these IELs remain immunologically quiescent until their activation threshold is surpassed. Activated γδ IELs limit viral replication by producing type I interferons (IFN), such as IFNα, yet the extent to which IFNα activates γδ IELs remains unknown. To this end, murine small intestinal γδ IELs were stimulated ex vivowith 1 ug/mL αCD3, 10 ng/mL IFNα or both for 5 h. We observed a 26%±10 increase in IFNγ +γδ IELs treated with αCD3 and IFNα compared to αCD3 treatment alone (p<0.0001) and a 2-fold increase in IFNγ MFI (p<0.0001). Using Nur77-GFP mice, we found that only Nur77 +γδ IELs produced IFNγ (p<0.0001), indicating that TCR signaling is necessary for IFNα-induced effector function. Suboptimal αCD3 stimulation (0.1 ug/mL) was sufficient to increase the frequency of IFNγ +γδ IELs following IFNα exposure relative to αCD3 alone (p<0.001). To interrogate the molecular mechanism involved in IFNα co-stimulation, γδ IELs were treated with PMA and/or ionomycin in the presence or absence of IFNα. Ionomycin and IFNα increased the frequency of IFNγ +γδ IELs by 16%±7 compared to ionomycin alone (p<0.0001), whereas PMA had no effect. Pharmacological inhibition of NFAT signaling (10 uM INCA6) abrogated both TCR- or IFNα-induced γδ IEL IFNγ production (p<0.01 or p<0.0001, respectively). Lastly, we observed that the co-stimulatory effect of IFNα was lost in STAT4-deficient γδ IELs compared to WT (p<0.0001). Together, these data indicate that low level TCR signaling through NFAT is required for IFNα to rapidly enhance γδ IEL IFNγ production in a STAT4-dependent manner. This work was supported by the National Institutes of Health R01 DK119349, the New Jersey Commission on Cancer Research, Busch Biomedical Research grant and the RBHS Chancellor Scholar Award (KLE).