To the editor:
A unique cross talk between the innate and adaptive cells underlie
asthma pathobiology. Within the adaptive domain, T lymphocytes have a
clear pathogenic role, while that of B lymphocytes remain lesser known.
B-cells support IgE-dependant allergic inflammation, evident from
IgE+ B-cells in asthmatic airways, post-aeroallergen
challenge1. Moreover, the presence of anti-eosinophil
peroxidase (EPX) and anti-nuclear/extra-nuclear antibodies (ANAs)
detectable in sputa and not in the circulation, indicate a B-cell
dominant mechanism capable of in situ autoantibody
generation2. We therefore aimed to detect and
characterize B-cell subsets in severe eosinophilic asthma (SEA) and
further probe into a possible association with airway autoimmunity.
We recruited stable moderate and severe eosinophilic asthmatics despite
adherence to inhaled/oral corticosteroids (ICS/OCS), corresponding to
Global Initiative for Asthma (GINA) treatment steps 4-5, and a
comparator healthy control (HC) population. All subjects provided
written informed consent. Induced sputum and peripheral blood
mononuclear cells (PBMC) were collected for immunophenotyping (cohort 1,
n=36) and subsequent B-cell characterization (cohort 2, n=34) (refer toTable E1 and online supplementary for methodology).
Flow cytometry analysis (gating strategy Fig 1A, Table
E2)3 showed that out of CD45+ sputa
cells, only the CD3-CD19+ subset
(identified as B-cells) were increased in asthma vs. HC
(Fig.1B ), especially in SEA who remained eosinophilic despite
ICS/OCS (Fig.1C ). B-cells enumerated from PBMCs remain
unremarkable between the subgroups (Fig.E1 ). The B-cell numbers
correlated with indices of asthma severity viz., sputum
eosinophils (r=0.5, P =0.01), reduced lung function (r=-0.4,P =0.01), and cytokines that support B-cell recruitment,
activation, differentiation, and survival, such as IL-6 (r=-0.3,P =0.05) and B-cells activating factor (BAFF, r=-0.3,P =0.04)) (Table E3 )4.
In Cohort 2 (gating strategy Fig.1E, Fig.E1E, Table E2)compared to matched PBMC population, naïve B-cells
(CD3-CD19+CD27-IgD+),
classic memory cells
(CD19+CD27+IgD-CD38dim/-)
and a unique isotype-switched
CD3-CD19+IgM-CD27-IgD-or double-negative (DN) subset were significantly higher in sputa(Fig.1F). Within the sputa, DN B-cells constitute
~80% of the total B-cell population, and the numbers
are significantly elevated in SEA (Fig.1G, H ). These cells
exhibit both antigen-activated
(HLA-DR+CD40+) and immune-exhaustion
surface markers (CD11c+CD21-)
(Fig.1E , depicts subtyping of the DN B-cells), indicating
functional heterogeneity.
DN B-cells are typically extrafollicular and reported in several
autoimmune diseases associated with the pathology4, 5.
These cells are isotype-switched and has the capability of acting as
antibody-secreting cells (ASCs) in situ , with/without
antigen-stimulation6. Given, sputum autoantibodies
(localized to the airways) have been reported in a similar SEA
population2, we investigated (i) sputum ANAs and
anti-EPX IgG (as described2), and (ii) evidence ofin situ Immunoglobulin (Ig) isotypes and autoantibody secretion
by isolated sputum cells with increased DN B subsets (method online).
Both ANAs and anti-EPX IgGs were detectable, and the former was
significantly higher in the SEA subset, in particular those who were
OCS-dependent (Fig.2A, B ). ANAs correlated significantly with
the anti-EPX IgG (Fig2.C ) confirming our previous
observation2, but more importantly with the percentage
(r=0.3, P =0.03) and the number of B-cells (r=0.4, P =0.009)
in the sputa (Fig.2D,E ). Sputum B-cells showed an increased
trend in autoimmune airways (Fig.2F ). Finally, ELISpot (cohort
2) confirmed that sputum cells that comprise of high percentage of DN
B-cells, derived from moderate-severe eosinophilic asthmatics are
capable of producing the key mucosal Ig subtype IgA, as well as IgGex vivo , in addition to specific Igs reactive to the coated
autoantigen, EPX (Fig.2G-I ).
We acknowledge that the study is limited by its small sample size; HC
are not age-matched, mainly due to increasing co-morbidities with age
that precludes recruitment, and also inadequate sputum production.
Detecting mechanisms of local class-switch of IgA to IgG autoantibodies
were also beyond the immediate scope of the study. Nevertheless, we
report an increased B-cells with a unique population of DN B-cells in
SEA airways at steady state (i.e., no exacerbation), that can
spontaneously secrete Igs, including autoreactive-Igs, without antigen
stimulation. This warrants a longitudinal study to examine the role of
DN B-cells in asthma, their functional relevance at exacerbations,in situ autoantibody generation and development/maintenance of
airway autoimmunity over time.
Ethical approval of the project was obtained from the National
Healthcare Group Domain Specific Review Board (Ref:2018/00910). All
subjects provided written informed consent.
Author contributions: H.F.L. and V.A. conceived the idea and
takes responsibility for overall guarantee of the paper. N.S.T and M.M.
developed the experimental design. S.Y.L. and C.H.T. were involved in
flow cytometry and protein assay analysis. A.R. and K.F. were involved
in development of ELISpot. Y.Y.H. and A.L. were involved in the planning
and acquisition of multiparametric flow cytometry panel. W.S.D.T., W.P.
and W.S.F.W. performed multiplex assay and PBMC processing. N.S.T.,
M.F.L. and H.F.L. were involved in patient care, recruitment and
coordination. M.M., P.N., D.Y.W. were involved in scientific advice.
H.F.L., V.A. and N.S.T. analyzed data. All authors have read and
consented to current manuscript draft.
Funding: The study was supported by grants from the National
Research Foundation, under its Campus for Research Excellence and
Technological Enterprise (CREATE) and National Medical Research Council
(Project ID: MOH-000146-01).