Altered Phenotype and Gene Expression of Regulatory T cells (Tregs) in Children with Autism, and the Relationship with Comorbid Gastrointestinal Symptoms

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by social deficits and stereotypic behaviors. Increased numbers of inflammatory cells and their mediators have been found in peripheral blood, brain, and gastrointestinal (GI) tissues of individuals with ASD. Regulatory T cells (Tregs) play a crucial role in suppressing inflammatory processes that, if disrupted, can result in inflammation and development of a variety of immunological conditions. In this study, we sought to characterize Tregs populations using flow cytometry and gene transcriptomic approaches in children with ASD (n = 36) and typically developing (TD) children (n = 18) enrolled in the Childhood Autism Risk from Genetics and Environment (CHARGE) study. We also examined differences in the frequencies of activated Tregs in ASD groups when stratified by co-occurring GI status. The frequency of gut homing α4b7 ⁺ Tregs positive for inhibitory receptors GITR, LAP, and/or GARP were altered based on the presence of GI symptoms. Analysis of mRNA isolated from CD4 ⁺ CD25 ⁺ Tregs, showed 213 differentially expressed genes (DEGs) between the ASD and TD children. Upregulated DEGs were enriched in Gene Ontology (GO) Biological Processes involved in epigenetic regulation including 'chromatin organization', whereas Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were involved in metabolism, including 'lipid and atherosclerosis’ and 'pantothenate and CoA biosynthesis'. Upregulation of immune signaling genes (MAPK3, JAK2, and CASP3) was also noted in the ASD Tregs. Downregulated DEGs consisted of genes enriched in immune terms including the GO term 'leukocyte differentiation' and KEGG pathways ‘Parkinson disease’ and 'protein processing in endoplasmic reticulum’. Correlation analysis revealed a relationship between inappropriate speech scores and lower frequencies of Tregs in ASD children. Overall, these data support the hypothesis of altered Tregs cell biology in ASD, including their lower frequencies and altered gene expression. Furthermore, altered metabolic and immune signaling processes may contribute to changes in frequencies of Tregs in ASD based on co-morbidities, ultimately driving the immune status in ASD from a balanced to a dysregulated/inflammatory state.