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\begin{document}

\title{Early expansion of allergen-responsive LAP + B regulatory cells in
allergic rhinitis but not in allergic asthma subjects during allergen
immunotherapy}



\author[1]{Hermelijn Smits}%
\author[1]{Astrid L. Voskamp}%
\author[2]{Nicolette w Jong}%
\author[1]{Simon P. Jochems}%
\author[1]{Arifa Ozir-Fazalalikhan}%
\author[1]{Luciën E.P.M. van der Vlugt}%
\author[1]{Koen A. Stam}%
\author[3]{Gerrit Braunstahl}%
\author[4]{Gertrude M. Möller}%
\author[2]{Roy Gerth van Wijk}%
\affil[1]{Leids Universitair Medisch Centrum Center for Infectious Diseases}%
\affil[2]{Erasmus MC Afdeling Inwendige Geneeskunde}%
\affil[3]{Franciscus Gasthuis en Vlietland}%
\affil[4]{Netherlands Center of Clinical Occupational Medicine}%


\vspace{-1em}



  
  \date{June 12, 2024}


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\textbf{Early expansion of allergen-responsive LAP\textsuperscript{+} B
regulatory cells in allergic rhinitis but not in allergic asthma
subjects during allergen immunotherapy}

Astrid L. Voskamp\textsuperscript{1}, Nicolette W. de
Jong\textsuperscript{2}, Simon P. Jochems\textsuperscript{1}, Arifa
Ozir-Fazalalikhan\textsuperscript{1}, Luci\selectlanguage{ngerman}ën E.P.M. van der
Vlugt\textsuperscript{1}, Koen A. Stam\textsuperscript{1}, Gert-Jan
Braunstahl\textsuperscript{3,4}, Gertrude M. Möller\textsuperscript{5},
Roy Gerth van Wijk\textsuperscript{2}, Hermelijn H.
Smits\textsuperscript{1}

\textsuperscript{1}Department of Parasitology, Leiden University Center
for Infectious Diseases (LU-CID), Leiden University Medical Center;
Leiden, The Netherlands.

\textsuperscript{2}Dept of Internal Medicine, Section Allergology and
Clinical Immunology, Erasmus University Medical Center; Rotterdam, The
Netherlands.

\textsuperscript{3}Department of Pulmonology, Franciscus Gasthuis and
Vlietland, Rotterdam, 3045 PM, the Netherlands.

\textsuperscript{4}Department of Pulmonology, Erasmus Medical Center,
Rotterdam, 3015 GD, the Netherlands.

\textsuperscript{5}Netherlands Center of Clinical Occupational Medicine,
The Netherlands.

\textbf{Correspondence to:}

Prof dr. H.H. Smits, ORCID-ID: 0000-0001-9279-2890

Dept of Parasitology, Leiden University Center of Infectious Disease
(LU-CID)

Leiden University Medical Center (LUMC)

Albinusdreef 2, 2333 ZA Leiden, The Netherlands

h.h.smits@lumc.nl

ORCID ID co-authors:

A.L. Voskamp: 0000-0001-9389-5138

N.W. de Jong: 0000-0002-7216-0906

S.P. Jochems: 0000-0002-4835-1032

A. Ozir-Fazalalikhan: 0000-0002-0613-8372

E.P.M. van der Vlugt: 0009-0008-7180-6534

K.A. Stam: 0000-0003-0982-4439

G.J. Braunstahl: 0000-0001-7671-3742

G.M. Möller: 0009-0004-5666-6079

R. Gerth van Wijk: 0000-0002-9608-8742

GJB reports grants from Dutch Lung Foundation, Stichting BeterKeten and
Research Grants from GSK, AstraZeneca and Sanofi; HHS reports grants
from Dutch Lung Foundation, National Science Council; SPJ reports a
grant from European Commission Horizon 2020 Framework Programme; all
others report no conflict of interest in relation to this work.

To the Editor,

Allergen immunotherapy (AIT) is used successfully for treatment of
allergic rhinitis (AR). There is also strong evidence of AIT being
effective in allergic asthma (AA), when used as an add-on treatment to
pharmacotherapy such as oral corticosteroids\textsuperscript{1}. There
is limited data available on immunological effects of AIT in AA compared
to AR and the impact of pharmacotherapy on mechanisms of AIT. Induction
of B-regulatory (Bregs) and T-regulatory (Tregs) cells are key
components in tolerance induction to allergens in AIT. Bregs produce
immunosuppressive cytokines IL-10 and TGF-\selectlanguage{greek}β, \selectlanguage{english}which not only suppress Th2
cell responses, but also mediate induction of Tregs\textsuperscript{2}.
In this study, allergen-responsive B-cells were monitored in 7 AA and 8
AR subjects prior to and after 4, 9 and 24 months of subcutaneous
allergen immunotherapy (\textbf{figure 1A} ). Allergen-responsiveness
was determined through \emph{in vitro} proliferative cell responses,
indicated by loss of CFSE, to HDM allergen at day 5. In total, 10
clusters of allergen-responsive B-cells were identified (\textbf{figure
1B} ). Four clusters expressed the memory marker CD27 and six did not,
mostly representing na\selectlanguage{ngerman}ïve/transitional B-cell populations. Within those
B-cell populations, a
CD71\textsuperscript{+}CD73\textsuperscript{-}CD25\textsuperscript{+}LAP\textsuperscript{+}cluster
was identified, consistent with a Breg phenotype. In AR subjects, the
frequency of LAP\textsuperscript{+} Bregs within allergen-responsive
B-cells increased at 24 months of AIT with nominal significance and was
already significantly increased at 4 months (with fdr-correction)
(\textbf{figure 1C} ). This increase was however not observed in AA
subjects. LAP is `latency associated peptide' and serves as a marker for
TGF-\selectlanguage{greek}β \selectlanguage{english}expression. TGF-\selectlanguage{greek}β \selectlanguage{english}is vital for induction of
Tregs\textsuperscript{2} and has important roles in Breg-induced control
of autoimmunity and allergy\textsuperscript{3,4}. The increased
percentage of LAP\textsuperscript{+} Bregs in AR subjects at 4 months
was accompanied with a (nominal) significant decrease in
CD25\textsuperscript{+}CD71\textsuperscript{-}CD27\textsuperscript{+}memory
B-cell clusters (Cluster 1 and 5; \textbf{figure 1D} ). Again, this
change was not observed in AA subjects. The frequency of
LAP\textsuperscript{+} B-cells was markedly lower in non-HDM responsive
B-cells compared to HDM-responsive B-cells and was not increased in AR
or AA at 24 months of AIT (\textbf{Supplementary figure 1} ).

Changes also occurred within the transcriptome of allergen-specific
memory B-cells (\textbf{figure 2A} ). In AR subjects, there was a change
in gene expression at 4 months of AIT, which did not occur in AA
patients (\textbf{figure 2B} ). The gene ontology pathways included
small GTPases-mediated signal transduction, important in cellular
processes (signal transduction, cell adhesion, chemotaxis and motility,
cell growth and division), along with plasma cell, immunoglobulin (Ig)
and MHC-TLR7-TLR8 pathways, necessary for germinal center forming and Ig
production (\textbf{figure 2C} ). This was further reflected in reduced
Ig heavy chain transcripts in AR subjects at 4 and 9 months of AIT, but
not in AA patients (\textbf{figure 2D} ). Of interest, TLR7/8 pathways
have been implicated in Breg function\textsuperscript{5}.

In summary, AR subjects displayed early changes in B-cell populations
during the first 4 months of AIT consisting of increased HDM-responsive
LAP\textsuperscript{+} B-cells and reduced Ig-related transcripts of
allergen-responsive memory B-cells, which do not occur in allergic
asthma subjects. Asthma and quality of life scores showed improvement at
24 months of AIT (\textbf{Supplementary figure 2} ); while allergen
challenges post-immunotherapy confirming clinical efficacy were lacking.
However as other studies showed variations in clinical efficacy between
AR and AA\textsuperscript{6}, the differences observed in B-cells at 4
months of AIT between these groups may shed light on immunological
mechanisms involved in achieving clinical efficacy and how it may differ
between diseases. Additionally, use of steroid-based medication may
prevent changes that would otherwise occur during AIT. Indeed, steroids
were shown to suppress Breg cell(s) (development) in patients with
myasthenia gravis\textsuperscript{7}. Further investigation should
determine consequences of immunological discrepancies between AR and AA
on clinical outcome of AIT.

\textbf{Acknowledgements:} The study was funded by research grants from
Longfonds (\textbf{3.2.10.072} , \textbf{5.1.15.015)} and NWO
(ZonMW-vidi: 91714352).

\textbf{References}

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\textbf{Figure legends}

\textbf{Figure 1. Allergen-responsive B-cell populations in allergic
rhinitis (AR) and allergic asthma (AA) subjects during allergen
immunotherapy (AIT).} (A) Allergen immunotherapy study set-up and dosing
schedule. (B) Allergen-responsive B-cell identification and
clustering-based phenotyping. (C) Cluster density plots and cluster 4
(LAP\textsuperscript{+} B cells) frequency in AR and AA groups during
AIT. (D) Cluster density plots and cluster 1\&5
(CD27\textsuperscript{+}memory B cells) frequency in AR and AA groups
during AIT. * fdr \textless{} 0.05

\textbf{Figure 2. Allergen-responsive memory B-cell populations in
allergic rhinitis (AR) and allergic asthma (AA) subjects during allergen
immunotherapy (AIT).} (A) Allergen-responsive memory B-cells selected
for FACS isolation for RNA-sequencing. (B) Molecular degree of
perturbation quantifying the heterogeneity between samples to indicate
overall changes in gene expression. (C) Gene set enrichment analysis
indicating increased (red dots) or decreased (blue dots) expression of
gene pathways compared to time point 0. Dot size indicated fold change.
(D) IgH mapping of B cell receptors.

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