Altered T Cell Subsets in Children with Autism Spectrum Disorders and Co-Morbid Gastrointestinal Symptoms

Background: The existence of gastrointestinal (GI) symptoms in children with autism spectrum disorders (ASD) has been estimated to occur 6 to 8 times more often than in typically developing children. The most commonly reported symptoms include: diarrhea, constipation, alternating bowel habits, gassiness or bloating, vomiting, and abdominal pain. While intestinal inflammation, altered microbiome profiles, and impaired intestinal permeability have been observed in children with ASD who experience GI symptoms, mechanisms underlying GI symptoms in ASD and relationship with behavioral phenotypes or other comorbid symptoms is still poorly understood.

Objectives: To characterize and assess T cell populations in children with ASD with and without GI co-morbidities, and the association with behavioral symptoms.

Methods: Children were recruited from the CHildhood Autism Risk from Genetics and the Environment (CHARGE) study and placed into one of four study groups based on responses from the GI symptom survey (adapted from standardized Rome III Diagnostic Questionnaire for the Pediatric Functional GI Disorders): children with ASD who experience GI symptoms of irregular bowel habits (ASD^GI) and children with ASD who do not have a history of GI symptoms (ASD^NoGI) compared to typically developing children with GI symptoms (TD^GI) and typically developing children without a history of GI symptoms (TD^NoGI). Peripheral blood mononuclear cells (PBMC) were isolated from participant’s blood, stimulated in-vitro for 4 hrs and stained for surface markers and intracellular cytokines for flow cytometry; including markers for T cell subsets and for β7, part of the α4β7 integrin that is upregulated on lymphocytes homing to the GI tissue. Behavioral assessments and diagnosis were performed by trained clinicians and included Autism Diagnostic Observation Schedule (ADOS), Autism Diagnostic Interview – Revised (ADI-R) and Aberrant Behaviors Checklist (ABC) scores.

Results: Flow cytometric analysis revealed several differences between groups. Children in the ASD^GI group had significantly decreased regulatory T cells (T_regs), identified as CD25⁺Foxp3⁺ T cells, compared to ASD^NoGI, furthermore, ASD^GI children were found to have a lower frequencyof gut homing T_regs (B7⁺CD25^Hi cells) compared to TD^NoGI, whereas both ASD groups were found to have reduced number of IL-10⁺ T cells compared to TD^NoGI. However, ASD^GI children had the highest percentage of IL-17⁺ CD4 T cells compared to all other groups, while ASD^NoGI displayed the highest percentage of IL-13⁺ CD4 T cells. In addition to changes in T cell populations, children in the ASD^GI group displayed increased aberrant behaviors, as indicated by increased scores on the ABC subscales for: Irritability, Hyperactivity and Social Withdrawal.

Conclusions: Children with ASD displayed deficits in regulatory T cell populations compared to typically developing controls, regardless of the presence of GI symptoms, however, the ASD^GI group had the lowest T_reg population numbers. Interestingly, the two ASD groups differed in T helper subset populations with ASD^GI children displaying more of a T_H17 phenotype (elevated IL-17⁺ T cells), whereas ASD^NoGI exhibited more of a T_H2 phenotype (elevated IL-13⁺ T cells), suggesting that children with ASD may have deficits in immune regulation that may lead to elevated inflammatory T cell subsets.