Mass cytometry‐based identification of a unique T‐cell signature in childhood allergic asthma
Authors: Hartmann Raifer, Axel R. Schulz, Johanna Theodorou, Addi J. Romero‐Olmedo, Andreas Böck, Wilhelm Bertrams, Bernd T. Schmeck, Ho‐Ryun Chung, Michael Lohoff, Hyun‐Dong Chang, Bianca Schaub, Henrik E. Mei, Magdalena Huber
Journal: Allergy (2021)

To the Editor, Allergic asthma (AA) 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.1 However, a detailed analysis of T-cells associated with pediatric AA is still needed. To explore T-cell phenotypes associating with pediatric 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 positively correlated with blood eosinophil frequencies, a determinant of AA severity,3 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, CD4_c6 and CD4_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 pediatric AA.5 Manual gating confirmed higher abundance of ICOS+TIGIT+Th2-cells in AA vs HC (Figure S2A-C). This cell population was also characterized by CD161 expression, as described for Th2-cells restricted to atopic adults,6 thereby underpinning its probable pro-allergic nature. The frequency of the CD4_c6 cluster positively 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 in relation to the CD4/CD8 T-cell ratio and allergic comorbidity in connection with 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 model,7 suggesting a differentiation block from Fr-I toward Fr-II, and a possible altered eTreg compartment. Therefore, we next analyzed manually gated eTregs (Figure S1B), by FlowSOM clustering, which revealed an underrepresented eTreg_c2 and partially eTreg_c10 in AA (Figure 2A,B, Figure 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 CD4_c6/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, Figure S2H). In contrast, CD4_c6 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-cell (CD4_c6)-associated eosinophilia in pediatric AA. Thus, eTregs (eTreg_c2 and eTreg_c10) and naïve/resting Tregs (CD4_c30) are involved in two different pathological features of AA, in the eosinophilia and in part in 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 pediatric AA phenotype (sensitivity, true positive rate) (Figure 2F), further supporting the relevant involvement of the Treg dysbalance in childhood AA. Summarizing, despite an explorative character without multiple testing adjustment, our study 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. This work was generously supported by the Else Kröner-Fresenius-Stiftung (Research group: “Functional, therapy-aimed clustering of T-cell sub-phenotypes across plasticity”). MH is also supported by the Deutsche Forschungsgemeinschaft (DFG) (HU-1824/9-1, HU-1824/7-1, HU-1824/5-1). BS is supported by grants from BMBF 01GL1742A, DFG (DFG-SCHA 997/3-1, DFG-SCHA 997/7-1, DFG-SCHA 997/8-1), from SFB TR22, grants Comprehensive Pneumology Center. JT is supported by grants from the DFG (DFG-SCHA 997/7-1). AB is supported by grants from BMBF (01GL1742A). HR, ARS, JT, AJRO, AB, WB, BTS, HRC, ML, HDC, BS, HEM, and MH have nothing to disclose. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.