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PS-049
Modifications of virus-specific and total B cell populations by
hepatitis B virus infection
L. Salimzadeh1,2, N.L. Bert2, U.S. Gill1,3, P.T. Kennedy3, D. Samuel4,
N. Novikov4, S. Pflanz5, C.R. Frey5, A. Bertoletti1,2. 1Emerging Infectious
Diseases Program, Duke-NUS Medical School; 2Singapore Institute for
Clinical Sciences, A*STAR, Singapore, Singapore; 3Hepatology Unit,
Centre for Immunobiology, Blizard Institute, Barts and the London School
of Medicine & Dentistry, QMUL, London, United Kingdom; 4Departments
of Biology Core Support; 5Departments of Immunology/Inflammation,
Gilead Sciences, Foster City, United States
E-mail: [email protected]
Background and Aims: B cell biology during hepatitis B virus (HBV)
infection has been poorly characterized. To determine the impact of
chronic HBV infection on the B cell repertoire and function, we
developed a protocol for the direct ex vivo analysis of HBsAg-specific
and total B cells. We used this novel assay to characterize B cell
frequency and phenotype in healthy HBV vaccinated subjects as well
as patients with resolved or chronic HBV infection.
Methods: Phenotypic analysis of total and HBsAg-binding B cellswas
performed by flow cytometry using conjugated antibodies identifying
different markers of B cell function and maturation. Recombinant
HBsAg was fluorescently conjugated with either DyLight 550 or
DyLight 650 and used to doubly stain peripheral blood cells of
HBV vaccinated subjects (n = 17), patients with resolved (n = 13) and
chronic infection. Patients were categorized as follows: HBV-DNA
107–1010 IU/ml, ALT < 40 IU/L (n = 16); eAg+/−, HBV-DNA 102–108 IU/
ml, ALT 11–367 IU/L (n = 43); HBV-DNA 101–103 IU/ml, ALT < 40 IU/L
(n = 17). HBsAg was also quantified in all patients.
Results: CD19 + CD27+ cells from vaccinated individuals that bound
fluorochrome-conjugated HBsAg were sorted, cultured in vitro and
analyzed for anti-HBs production. Anti-HBs antibodies were only
detected in the supernatant of CD19 + CD27+, HBsAg-DyLight 550+
and HBsAg-DyLight 650+ cells, demonstrating that the staining
protocol identifies bona-fide anti-HBs specific B cells. The frequency
and phenotype of anti-HBs specific B cells were then analyzed in
patients. Surprisingly, the frequency of anti-HBs memory B cells
(∼0.3% of total memory B cells) were comparable in patients with
resolved or chronic infection, as well as in vaccinated individuals.
In contrast, B cell phenotype was different in patients with high
HBsAg/HBV-DNA and no inflammation (normal ALT) relative to
patients in other phases of disease and healthy vaccinated subjects.
Specifically, total and anti-HBs specific B cells in patients with high
HBsAg/HBV-DNA and normal ALT had higher expression of CD39 (a
regulation marker) and lower expression of CD38 (an activation
marker). Moreover, there was an increased frequency of atypical
memory B cells (CD21-CD27-) with exhaustion properties in these
patients.
Conclusions: We developed a protocol for the direct ex vivo
visualization of anti-HBs producing B cells. Using this new method
we demonstrate that HBsAg/HBV-DNA quantity affects the phenotype
of total and HBsAg-specific B cells, which may improve our
understanding of the natural history of CHB.
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发表于 2017-4-9 10:08