Abstract
Purpose :
Plasmacytoid dendritic cells (pDCs), a highly functional subset of immune cells, play a key role in anti-viral responses and link innate and adaptive immune responses through (Toll Like Receptor) TLR 7/9 activation. Here we investigate the presence of activated pDCs in draining lymph nodes (dLNs) and their role in modulating T cell responses in the acute herpes simplex virus (HSV)-1 keratitis
Methods :
Corneal infection was induced by inoculation of HSV-1 after scarification. Submandibular dLNs were excised and quantified for pDCs by flow cytometry or immunofluorescence staining of cryosections for pDC markers (CD11c, PDCA-1), T cells (CD3) and activation markers (CD86 and MHC-II). Intravital multiphoton microscopy (IV-MPM) was performed in dLNs in Tred mice (T cells are red) after adoptive transfer of pDC-GFP (expression green fluorescence protein) in steady state and after corneal inoculation with TLR9 agonist and HSV-1 to determine the kinetics of pDCs and their interactions with T cells
Results :
We observed an increased number of pDCs in dLNs (4-folds, P<0.05) after corneal HSV-1 infection, with an increased percentage of activated pDCs (MHCII+CD86+) (72% vs 42%, P<0.05) and in total T cells (CD45+ CD3+) (2.5 fold, P<0.05) compared to naïve mice. In dLN, pDCs are mainly distributed in the parafollicular cortex (2.9-fold) compared to the follicular, and (1.6-fold) to the subcapsular areas respectively (P<0.05) after corneal HSV-1 infection; similarly in steady state, the distribution is 3.8 fold in the parafollicular area compared to the follicular area (P<0.05). Further, IV-MPM imaging in dLNs from Tred mice showed an increased motility of adoptively transferred pDC after corneal inoculation of TLR9 agonist and HSV-1 with an increased velocity and track length (P<0.001) compared to steady state pDCs (corneal inoculation with PBS). Moreover, around 40% of actively motile pDCs established contact with T cells with durations of >5 minutes demonstrating direct interaction
Conclusions :
In dLNs, pDCs are increased during HSK with an increased number of activated pDCs. Also, pDCs are located mainly in the parafollicular cortex where they directly interact with T cells and mediate anti-viral immune responses. This study, for the first time, demonstrates in vivo imaging of submandibular LNs, allowing the study of pDCs and their interaction with T cells in context
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.