Functional 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 (CTLA-4 )
gene might be a candidate asthma locus (1 ). However, prior
reports of associations between CTLA-4 genetic variants and
asthma are conflicting and inconclusive (2-4 ). This study aimed
to determine the role of 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 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 5CTLA-4 tag-SNPs genotyped, rs3087243 has the highest significance
level of associations with AR without asthma (p = 4.69 x
10-3, OR = 1.38), asthma without AR (p = 3.49 x
10-4, OR = 1.58), and AR with asthma (p = 6.67
x 10-4, OR = 1.52, Table 1 ).
Next, we determined if rs3087243 has a functional effect onCTLA-4 . We extracted 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 increasingCTLA-4 mRNA expression (meta p -value = 2.67E-69, combinedZ -score = 17.60, Figure 1A ). From the HapMap CHB data,
tag-SNP rs3087243 is in a strong linkage disequilibrium
(r2 = 1) with rs11571316 that is located
~1.4 kb upstream of the CTLA-4 gene
(Figure S1 ). We cloned -1686 bp to +110 bp of the CTLA-4gene to conduct an in vitro luciferase assay and showed a
significantly higher CTLA-4 promoter activity associated with
allele “A”, as compared to allele “G” of rs3087243 (p< 0.01, Figure 1B and 1C ). This suggests rs11571316
might represent a functional SNP that influences the CTLA-4promoter activity and causes the apparent genetic association between
the tag-SNP rs3087243 and allergy.
Lastly, when analysing the 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
(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 (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 increasingCTLA-4 protein levels from the “CC” to “TT” genotype of
rs11571316 (ANOVA p = 8.5 x 10-10,Figure 1D ). Besides this strong effect on naïve Treg, nominal
association between rs11571316 genotype and CTLA-4 protein levels
was only observed in T effector (Teff) cells (ANOVA p = 0.011,Figure S3 ). This suggests that rs11571316 might regulateCTLA-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 (5 ). Also, a separate
linkage block in 2q was previously reported to modulate the mRNA
expression of ICOS among Treg and Teff subsets and associated
with the risk of atopy and asthma (6 ). Therefore, we speculated
an interplay between ICOS and CTLA-4 in these T cell
subsets might contribute to the pro-inflammatory condition that
underlies allergy manifestation (Figure S4 ).
In conclusion, the present study identified a functional genetic signal
that was associated with allergy via modulating 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 CTLA-4 expression on these
cells.