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\begin{document}

\title{Functional CTLA-4 variants associate to both allergic asthma and
rhinitis potentially by modulating na\selectlanguage{ngerman}ïve regulatory T cells\selectlanguage{english}}



\author[1]{Anand Kumar Andiappan}%
\author[1]{Kia Joo PUAN}%
\author[2]{Yang Yie Sio}%
\author[1]{Fauziah ALLY}%
\author[1]{Bernett LEE}%
\author[2]{Sri Anusha MATTA}%
\author[1]{Nurhashikin YUSOF}%
\author[1]{Anis LARBI}%
\author[3]{De Yun Wang}%
\author[2]{Fook Chew}%
\author[1]{Olaf Rotzschke}%
\affil[1]{Singapore Immunology Network}%
\affil[2]{National University of Singapore Department of Biological Sciences}%
\affil[3]{National University of Singapore}%


\vspace{-1em}



  
  \date{May 27, 2024}


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Article type: Letter to the Editor

\selectlanguage{ngerman}\textbf{Functional \emph{CTLA-4} variants associate to both allergic
asthma and rhinitis potentially by modulating naïve regulatory T cells}

To the editor,

A genome-wide linkage study previously indicated the chromosome 2q
region containing the cytotoxic T-lymphocyte protein 4 (\emph{CTLA-4} )
gene might be a candidate asthma locus (\emph{1} ). However, prior
reports of associations between \emph{CTLA-4} genetic variants and
asthma are conflicting and inconclusive (\emph{2-4} ). This study aimed
to determine the role of \emph{CTLA-4} single nucleotide polymorphisms
(SNPs) on allergy risk by carrying out genetic association and
functional analyses.

We identified 5 tag-SNPs (rs733618, rs4553808, rs16840252, rs231775, and
rs3087243) in \emph{CTLA-4} from the Hapmap Chinese Han population (CHB,
Figure S1). These tag-SNPs were genotyped in a cohort of 1703 Singapore
Chinese adults (age: 22.2 ± 5.6, 42\% male, table S1). Of these
5\emph{CTLA-4} tag-SNPs genotyped, rs3087243 has the highest
significance level of associations with AR without asthma (\emph{p} =
4.69 x 10\textsuperscript{-3}, OR = 1.38), asthma without AR (\emph{p} =
3.49 x 10\textsuperscript{-4}, OR = 1.58), and AR with asthma (\emph{p}
= 6.67 x 10\textsuperscript{-4}, OR = 1.52, \textbf{Table 1} ).

Next, we determined if rs3087243 has a functional effect on\emph{CTLA-4}
. We extracted \emph{CTLA-4} mRNA expression data from 31,300 whole
blood samples constituting 36 different cohorts collected by the eQTLgen
consortium. In these cohorts, meta-analysis showed a strong correlation
between allele ``A'' of rs3087243 and increasing\emph{CTLA-4} mRNA
expression (meta \emph{p} -value = 2.67E-69, combined\emph{Z} -score =
17.60, \textbf{Figure 1A} ). From the HapMap CHB data, tag-SNP rs3087243
is in a strong linkage disequilibrium (r\textsuperscript{2} = 1) with
rs11571316 that is located \textasciitilde{}1.4 kb upstream of the
\emph{CTLA-4} gene (\textbf{Figure S1} ). We cloned -1686 bp to +110 bp
of the \emph{CTLA-4}gene to conduct an \emph{in vitro} luciferase assay
and showed a significantly higher \emph{CTLA-4} promoter activity
associated with allele ``A'', as compared to allele ``G'' of rs3087243
(\emph{p}\textless{} 0.01, \textbf{Figure 1B and 1C} ). This suggests
rs11571316 might represent a functional SNP that influences the
\emph{CTLA-4}promoter activity and causes the apparent genetic
association between the tag-SNP rs3087243 and allergy.

Lastly, when analysing the \emph{CTLA-4} surface expression of T cells
by flow cytometry we demonstrated a functional effect of rs11571316 on
protein levels in T regulatory (Treg) cells. On peripheral blood
mononuclear cell (PBMC) samples from a Singapore Chinese cohort
(\emph{n} = 201, age: 21.0 ± 2.3, 58\% male) we observed robust CTLA-4
expression on Treg cells (gated for CD4+ CD25+ Foxp3+), while only lower
amounts of the protein were detected on CD25-Foxp3- T effector (Teff)
cells (\textbf{Figure S2} ). Compared to memory-type Treg (CD45RA-) the
basal levels in naïve-type Treg cells (CD45RA+) were also lower.
However, in this subset we observed a dosage effect of
increasing\emph{CTLA-4} protein levels from the ``CC'' to ``TT''
genotype of rs11571316 (ANOVA \emph{p} = 8.5 x
10\textsuperscript{-10},\textbf{Figure 1D} ). Besides this strong effect
on naïve Treg, nominal association between rs11571316 genotype and
\emph{CTLA-4} protein levels was only observed in T effector (Teff)
cells (ANOVA \emph{p} = 0.011,\textbf{Figure S3} ). This suggests that
rs11571316 might regulate\emph{CTLA-4} protein levels mainly in naïve
Treg cells and contribute to the development of allergy. In agreement
with this, prior reports have also demonstrated an important role of
Treg cells in the control of airway inflammation in asthmatic patients
(\emph{5} ). Also, a separate linkage block in 2q was previously
reported to modulate the mRNA expression of \emph{ICOS} among Treg and
Teff subsets and associated with the risk of atopy and asthma (\emph{6}
). Therefore, we speculated an interplay between \emph{ICOS} and
\emph{CTLA-4} in these T cell subsets might contribute to the
pro-inflammatory condition that underlies allergy manifestation
(\textbf{Figure S4} ).

In conclusion, the present study identified a functional genetic signal
that was associated with allergy via modulating \emph{CTLA-4} expression
in Treg cell subsets. Our data highlight a possible mechanism for how a
targeted immune dysregulation can affect the risk for multiple disease
phenotypes. The naïve Treg phenotype warrants further investigation as a
potential target for therapeutic intervention in affected patients by
harnessing strategies to attenuate \emph{CTLA-4} expression on these
cells.



\textbf{References}

1. M. Wjst \emph{et al.} , A genome-wide search for linkage to asthma.
German Asthma Genetics Group. \emph{Genomics} \textbf{58} , 1-8 (1999).

2. M. C. Munthe-Kaas \emph{et al.} , CTLA-4 polymorphisms in allergy and
asthma and the TH1/ TH2 paradigm. \emph{J Allergy Clin
Immunol}\textbf{114} , 280-287 (2004).

3. T. D. Howard \emph{et al.} , Fine mapping of an IgE-controlling gene
on chromosome 2q: Analysis of CTLA4 and CD28. \emph{The Journal of
allergy and clinical immunology} \textbf{110} , 743-751 (2002).

4. N. Hizawa \emph{et al.} , Increased total serum IgE levels in
patients with asthma and promoter polymorphisms at CTLA4 and FCER1B.
\emph{J Allergy Clin Immunol} \textbf{108} , 74-79 (2001).

5. K. Jansen \emph{et al.} , T regulatory cells from atopic asthmatic
individuals show a Th2-like phenotype. \emph{Allergy} , (2021).

6. A. K. Andiappan \emph{et al.} , Genetic variants of inducible
costimulator are associated with allergic asthma susceptibility. \emph{J
Allergy Clin Immunol} \textbf{135} , 556-558 (2015).

\textbf{Author names:} Anand Kumar ANDIAPPAN\textsuperscript{1} PhD, Kia
Joo PUAN\textsuperscript{1} PhD, Yang Yie SIO\textsuperscript{2} PhD,
Fauziah ALLY\textsuperscript{1}, Bernett LEE\textsuperscript{1} PhD, Sri
Anusha MATTA\textsuperscript{2} PhD, Nurhashikin
YUSOF\textsuperscript{1}, Anis LARBI\textsuperscript{1} PhD, De Yun
WANG\textsuperscript{3} MD PhD, Fook Tim CHEW\textsuperscript{2*} PhD,
Olaf ROTZSCHKE\textsuperscript{1,*}
PhD\textsuperscript{}\textbf{Affiliations:} \textsuperscript{1}
Singapore Immunology Network (SIgN), \#04-06, 8A Biomedical Grove,
Singapore.

\textsuperscript{2} Department of Biological Sciences, National
University of Singapore, Singapore

\textsuperscript{3}Department of Otolaryngology, National University of
Singapore, Singapore

\textbf{*Correspondence} : Olaf ROTZSCHKE :
Olaf\_Rotzschke@immunol.a-star.edu.sg; or Fook Tim CHEW :
dbscft@nus.edu.sg

\textbf{Acknowledgements:} The authors would like to thank the
volunteers for participating in this study, Dr. Ramani Anantharaman and
Parate Pallavi Nilkanth for help with sample collection.

\textbf{Funding:} Olaf ROTZSCHKE and Fook Tim CHEW have received
research support from the Singapore Ministry of Education Academic
Research Fund, Singapore Immunology Network (SIgN), National Medical
Research Council (NMRC) (Singapore), Biomedical Research Council (BMRC)
(Singapore), and the Agency for Science Technology and Research (A*STAR)
(Singapore); Grant Numbers: N-154-000-038-001, R-154-000-191-112,
R-154-000-404-112, R-154-000-553-112, R-154-000-565-112,
R-154-000-630-112, R-154-000-A08-592, R-154-000-A27-597,
R-154-000-A91-592, R-154-000-A95-592, R-154-000-B99-114,
BMRC/01/1/21/18/077, BMRC/04/1/21/19/315, BMRC/APG2013/108, SIgN-06-006,
SIgN-08-020, NMRC/1150/2008, and H17/01/a0/008. Fook Tim CHEW has
received consulting fees from Sime Darby Technology Centre; First
Resources Ltd; Genting Plantation, and Olam International, outside the
submitted work. Data generated by the SIgN Immunomonitoring platform was
supported by a BMRC IAF 311006 grant and BMRC transition funds
\#H16/99/b0/011. Dr. Anand Kumar Andiappan was supported by NMRC YIRG
grant ``OFYIRG17nov065'' and A*STAR CDA (Career Development Award) grant
(202D800012) and fellowships from the EAACI (European Academy of Allergy
and Clinical Immunology) and ERS (European Respiratory Society). Y.Y.S.
has received research support from the NUS Resilience \& Growth
Postdoctoral Fellowships with grant number: R-141-000-036-281. All
funding agencies had no role in the study design, data collection and
analysis, decision to publish, or preparation of the manuscript.

\textbf{Table 1: Genetic association of CTLA-4 tag SNPs to allergic
rhinitis and asthma in Singapore Chinese population}

Logistic regression test was performed adjusting for age and gender. CI:
Confidence Interval; Logistic P: adjusted logistic regression p-value;
OR: adjusted odds ratio, using minor allele as the reference category.
*Logistic P \textless{} 0.05 is considered significant association\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\textbf{Reference/Phenotype} & \textbf{Reference/Phenotype} & \textbf{AR
without Asthma (N=455) vs Non-atopic Non-allergic controls (N=525)} &
\textbf{AR without Asthma (N=455) vs Non-atopic Non-allergic controls
(N=525)} & \textbf{AR with Asthma (N=338) vs Non-atopic Non-allergic
controls (N=525)} & \textbf{AR with Asthma (N=338) vs Non-atopic
Non-allergic controls (N=525)} & \textbf{Asthma without AR (N=379) vs
Non-atopic Non-allergic controls (N=525)} & \textbf{Asthma without AR
(N=379) vs Non-atopic Non-allergic controls (N=525)}\tabularnewline
\textbf{SNP} & \textbf{Allele} & \textbf{Logistic P} & \textbf{OR} &
\textbf{Logistic P} & \textbf{OR} & \textbf{Logistic P} &
\textbf{OR}\tabularnewline
\textbf{rs733618} & G/A & \textbf{3.04E-03*} & \textbf{0.76(0.63-0.91)}
& 5.64E-02 & 0.82(0.66-1.01) & \textbf{4.27E-02*} &
\textbf{0.81(0.67-0.99)}\tabularnewline
rs4553808 & G/A & 9.08E-01 & 0.98(0.74-1.31) & 2.33E-01 &
0.82(0.58-1.14) & 1.92E-01 & 0.81(0.60-1.11)\tabularnewline
rs16840252 & T/C & 7.96E-01 & 0.96(0.73-1.28) & 2.34E-01 &
0.82(0.59-1.14) & 2.00E-01 & 0.82(0.60-1.11)\tabularnewline
\textbf{rs231775} & A/G & \textbf{9.24E-03*} & \textbf{1.30(1.07-.157)}
& \textbf{2.86E-02*} & \textbf{1.29(1.03-1.61)} & \textbf{4.91E-02*} &
\textbf{1.24(1.00-1.54)}\tabularnewline
\textbf{rs3087243} & A/G & \textbf{4.69E-03*} & \textbf{1.38(1.10-1.73)}
& \textbf{3.49E-04*} & \textbf{1.58(1.23-2.02)} & \textbf{6.67E-04*} &
\textbf{1.52(1.19-1.93)}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure Legend}

\textbf{Figure 1.} \textbf{Functional association between 2q variants
and \emph{CTLA-4} expression.} \textbf{(A)} Meta-analysis of whole blood
eQTL association between \emph{CTLA-4} mRNA expression and rs3087243
genotypes, using 31,300 samples constituting 36 different cohorts from
the eQTLgen consortium. \textbf{(B)} Schematic illustration of
the\emph{CTLA-4} gene region with the relative location of SNPs. The
cloned region and haplotypes used for the \emph{in vitro} luciferase
assay were also indicated. \textbf{(C)} Relative luciferase units (RLUs)
of the\emph{CTLA-4} promoter construct were compared across haplotypes.
Promoter-less luciferase plasmid pGL4.10 (empty vector, EV) was used as
a background control. Lipo: Lipofectamine transfection
reagent.\textbf{(D)} Protein expression of \emph{CTLA-4} in na\selectlanguage{ngerman}ïve-type
CD45RA+ T regulatory (Treg) cells was compared across rs11571316
genotypes. An independent t-test was conducted to determine statistical
significance in \textbf{(C)} and \textbf{(D)} that was defined as
\emph{p} -value \textless{} 0.05.\selectlanguage{english}
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