To the Editor,
Allergic asthma in childhood is characterized by a dominance of type 2
immunity driven by CD4+ T helper 2 (Th2) cells
expressing the transcription factor (TF) GATA3 and inefficient
counter-regulation by Tregs among other mechanisms.1However, a detailed analysis of T-cells associated with paediatric AA is
still needed.
To explore T-cell phenotypes associating with paediatric AA, we applied
a 42-antibody mass cytometry panel in combination with unsupervised
computational analyses in a cohort of well-characterized 14
treatment-naïve AA and 9 healthy children (HC) from the CLARA/CLAUS
study (Table S1,S2 Figure S1A).2 Integrating
information from 12 lineage-markers we identified seven major T- and
NK-cell populations within peripheral blood mononuclear cells (PBMC)
(Figure S1B,C) of which CD8+ T-cell abundance was
reduced with underrepresented memory compartment in AA vs HC (Figure
1A,S1D,E).
Accordingly, the CD4+/CD8+ T-cell
ratio was elevated in AA vs HC and correlated with blood eosinophil
frequencies, a determinant of AA severity3, in AA but
not in HC (Figure 1B,C,S1F). To address potential disease-associated
changes within the CD4+ T-cell compartment, we
selected 30 markers for subsequent clustering using FlowSOM algorithm.
Two clusters, cluster_c6 and cluster_c30, were expanded in AA vs HC
(Figure 1D,E,S1G,H). Cluster CD4_c6 represented Th2 cells (Figure 1F),
since it expressed the Th2-specific TF GATA3 and chemokine receptors
CRTH2 and CCR4.4 It uniquely co-expressed TIGIT and
ICOS, which was not reported in AA before and could be specific for
childhood, since TIGIT hypomethylation has been described only in
paediatric AA.5 Manual gating confirmed higher
abundance of ICOS+TIGIT+Th2-cells in
AA vs HC (Figure S2A-C) and revealed increased CD161 expression
characterising Th2-cells restricted to atopic adults,6thereby underpinning their pro-allergic nature. The frequency of the
TIGIT+ICOS+Th2_cluster, correlated
with eosinophilia in AA with allergic comorbidities and also with
CD4/CD8 T-cell ratio in asthmatics having intermittent disease symptoms,
while inversely in stable disease (Figure 1G,H), suggesting its
association with more symptomatic disease and with allergic comorbidity
linked to eosinophilia.
Cluster CD4_c30 expressing markers characterizing naïve/resting Tregs
(Figure S2D) matched the previously described
CD45RA+FOXP3low Treg
fraction(Fr)-I,7 while effector (e)Tregs represented
Fr-II (CD45RA-FOXP3high) and Fr-III
(CD45RA-FOXP3low) (Figure 1I).
Accordingly, the frequencies of Fr-I were enriched in AA vs HC, while
Fr-II and Fr-III were similar (Figure S2E). Cluster CD4_c30 abundance
tended to correlate inversely with lung function, and significantly with
memory CD8+ T-cell frequencies (Figure 1J,S2F),
indicating its partial connection to lung function and
CD8+ T-cell alterations. The abundances of Fr-I vs
Fr-II correlated inversely (Figure 1K), consistent with the linear
developmental model7, suggesting a
differentiation-block from Fr-I towards Fr-II, and a possible altered
eTreg compartment.
Therefore, we next analyzed manually gated eTregs (Figure S1B), by
FlowSOM clustering, which revealed an underrepresented cluster_c2 and
partially cluster_c10 in AA (Figure 2A,B,S2G). Considering a possible
eTregs impairment and an overrepresentation of
TIGIT+ICOS+Th2-cells (CD4_c6), we
asked if the two phenomena are connected. Indeed, the
TIGIT+ICOS+ Th2/eTregs ratio tended
to be higher in children with AA versus HC, but failed to associate with
eosinophilia in AA and in HC, whereby eosinophilia was markedly lower in
HC vs AA (Figure 2C,D,S2H). In contrast,
TIGIT+ICOS+ Th2-cell ratio to
eTreg_c2 and eTreg_c10 correlated significantly in AA but not in HC,
suggesting a specific relation between eTreg_c2 and eTreg_c10
underrepresentation and
TIGIT+ICOS+Th2-associated
eosinophilia in paediatric AA. Thus, eTregs (eTreg_c2 and eTreg_c10)
and naïve/resting Tregs (CD4_c30) are linked to two different
pathological features of AA, to the eosinophilia and in part to the lung
function, respectively.
Next, we performed principal component analysis (PCA) based on
significantly changed ratio and subset-frequencies, which separated AA
from HC children at the first PC, indicating that the detected
dysbalanced T-cell composition allows a discrimination between these two
groups (Figure 2E). Additionally, ROC analyses revealed a relation of
resting/naïve Tregs (CD4_c30), eTreg_c2 and eTreg_c10 to the
paediatric AA phenotype (sensitivity, true positive rate) (Figure 2F),
further supporting the relevant involvement of the Treg dysbalance in
childhood AA.
Summarizing, our approach identifies a unique T-cell signature of
childhood AA and provides insights for pathophysiological involvement of
dysbalanced Tregs, TIGIT+ICOS+ Th2
and memory CD8+ T-cells. This can be useful for
immunomonitoring, immunomodulation and for confirmatory larger studies
in childhood AA.