Plasmacytoid Dendritic Cells Prevent T Regulatory Cell (Tregs) Re-programming to Effector Ex-Tregs in Herpes Simplex Keratitis

Abstract

Purpose : Despite our recent understating on the role of pDCs in limiting herpes simplex virus (HSV)-1 keratitis, their significance in regulating Treg survival remains to be elucidated.

Methods : Bone marrow chimeric mice were generated by irradiating and reconstituting wildtype C57BL/6 mice by adoptive transfer of bone marrow cells from BDCA2-DTR (pDCs can be ablated by administration of diphtheria toxin [DT]) and Foxp3-eGFP/cre×Rosa-tdTomato mice (Foxp3⁺ Tregs are GFP⁺ tdTomato⁺ and Foxp3^neg ex-Tregs are converted to eGFP^neg tdTomato⁺) enabling concurrent depletion of pDCs and assessing Treg fate. pDC depletion was performed by subconjunctival (sconj.) injection of 30ng DT prior to HSV-1 inoculation and was repeated every 48 hours. Sconj. injection of PBS served as control. HSV-1 keratitis was induced by inoculation of 2×10⁶ PFU of HSV-1 strain McKrae on scarified corneas, 4 weeks after generation of chimeric mice. Corneas and draining lymph nodes (dLNs) were evaluated by confocal microscopy and flow cytometry, respectively. Co-cultures of pDCs and Tregs under treatment with 10⁵ PFU UV-inactivated HSV-1 were performed. T test was used to compare the groups. P<0.05 was considered significant.

Results : Confocal microscopy of whole-mounted corneas showed that pDC depletion resulted in a higher density of infiltrating eGFP^neg Tdtomato⁺ ex-Tregs compared to sham depletion in both the peripheral (89.6±20.4 cells/mm² vs 17.3±6.6; p<0.001) and central (20.8±9.6 vs. 3.3±1.9; p<0.001) corneas on day 7 following HSV-1 inoculation. Similarly, pDC depletion lead to 2.5-fold higher density of eGFP^negTdtomato⁺ ex-Tregs in the dLNs, suggesting that pDC depletion facilitates re-programming of Foxp3⁺Tregs to Foxp3^neg ex-Tregs. Further phenotyping of Foxp3^neg ex-Tregs in the dLNs showed that in both control and pDC-depleted mice, the majority of Foxp3^neg ex-Tregs expressed IFN-γ, suggesting their re-programming to effector T cells. Co-culture of splenic Tregs with different densities of splenic pDCs under stimulation of UV-irradiated HSV-1 showed that while in the presence of UV-irradiated HSV-1, Tregs converted to ex-Tregs, the presence of 1:10 and 1:5 densities of pDCs prevented their re-programming to effector ex-Tregs by 2.6-folds and 3.4-folds, respectively.

Conclusions : pDCs prevent re-programing of Tregs to effector ex-Tregs in the dLNs and promote Treg survival during HSV keratitis.

This is a 2020 ARVO Annual Meeting abstract.