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245
Probing Novel HBV Therapies Using Fine Needle
Aspirates to Sample Compartmentalised Liver-
Resident Immunity and Hepatocytes
Upkar Singh Gill1, Laura J. Pallett2, Niclas Thomas2, Alice R.
Burton2, Kerstin A. Stegmann2, Patrick T. F. Kennedy3 and
Mala K. Maini2, (1)Barts Liver Centre, Blizard Institute, Barts
and the London SMD, Queen Mary University of London, (2)
Division of Infection & Immunity, University College London,
(3)Barts Liver Centre, Barts and the London School of
Medicine and Dentistry
Background: In order to optimally refine the multiple new
therapeutic strategies in the pipeline for HBV cure, evaluation
of virological and immunological changes at the site of
infection is required. This has been underscored by our recent
demonstration that specialised subsets of tissue-resident
CD8 T (Pallett et al., JEM 2017) and NK cells (Stegmann et
al., Sci Rep 2016), adapted to provide frontline defense, are
compartmentalised within the liver and consequently cannot
be sampled in the blood. We therefore investigated if fine
needle aspirates (FNA), well-suited for repeated sampling,
could be used to study the local immune landscape in parallel
with HBV-infected hepatocytes. Methods: Patients with HBV
(n=19) and those without viral infection (n=8) undergoing
percutaneous liver biopsy for diagnostic purposes were
included. Matched samples for blood, liver biopsy and FNA from
the same patients were analysed in parallel. 16-colour multiparameter
flow cytometry was used to characterise a range
of immune cells including circulating, liver-infiltrating (T, B, NK
& MAIT cells) and liver-resident populations (T and NK cells)
as well as hepatocytes. Results: Comparable populations of
CD4 T, CD8 T, B, NK and MAIT cells were identified by FNA to
those seen in material from liver biopsy. Populations of tissueresident
memory CD8 T cells (CD69+CD103+; CD8 TRM) with a
comparable PD-1hiCD39hi phenotype, were identified by both
FNA and liver biopsy, and their frequency by these sampling
techniques strongly correlated with each other (PD-1+ CD8
TRM r=0.91, p=<0.0001; CD39+ CD8 TRM r=0.93, p=0.002).
This cell population was not seen in the blood. Importantly,
HBV-specific T cells (analysed by HLA-A2/HBV-dextramer or
intracellular cytokine staining following overnight overlapping
peptide stimulation) could be identified by FNA at similar
frequencies to those from biopsies and significantly enriched
compared to blood. The large subset of liver-resident NK cells
(CXCR6+ T-betloEomeshi), seen on biopsy were also identified
by FNA and not seen in the blood. Moreover, by FNA we could
simultaneously identify populations of live hepatocytes, which
expressed SRB-1, PDL-1 and HBsAg, whereas these were
not viable after the processing required for liver biopsies.
Conclusion: We demonstrate for the first time that FNA
identifies a range of immune cells including local specialised
sentinel HBV-specific T cells and NK cells, together with HBVinfected
hepatocytes. The broad sampling achieved by this
rapid, painless and safe technique makes it an attractive
method for longitudinal sampling of the liver, important in
optimising new therapies for HBV. |
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发表于 2018-10-8 17:26