Cytotoxic T Lymphocytes Are Significantly Higher in Colonic Tissue from GI-Symptomatic Children with Autism Compared to Controls

Background: Gastrointestinal (GI) problems are more common in children with autism spectrum disorder (ASD) than in typically developing (TD) children. Moreover, many children with ASD present with GI symptoms suggestive of an inflammatory bowel disease-like (IBD-like) condition however, following investigative ileocolonoscopy with biopsy, conventional histology with hematoxylin and eosin (H&E) staining often does not reveal any marked abnormalities. Using immunohistochemistry, an earlier study reported an increase in cytotoxic T lymphocyte (CD8+) cell density and intraepithelial lymphocyte numbers in children with ASD and GI symptoms that was disproportionate to the inflammation seen on routine histologic evaluation, indicating a distinct lymphocytic colitis.

Objectives: The goal of this study was to investigate this finding further by comparing levels of CD8, a cytotoxic T cell and gut inflammation marker, in colonic biopsy samples from GI-symptomatic children with ASD compared to TD children in a completely separate and unrelated case/control cohort.

Methods: Biopsies from the right colon were obtained via colonoscopy from children with ASD and TD children who presented with GI symptoms suggestive of an IBD-like disease. These tissues were embedded in paraffin blocks, and we first prepared slides and performed conventional H&E staining. This was followed by immunohistochemistry with a primary CD8 antibody from a mouse host (dilution 1:750) and a secondary goat anti-mouse antibody (1:1000) with an emission wavelength of 594 nm. Tissues were permeabilized with 0.2% Triton-X100 in PBS and antigen retrieval was performed with 0.01 M citrate buffer at pH 6. Dako protein block and TrueBlack Autofluorescence Quencher were used to reduce nonspecific staining and auto fluorescence. Tissues stained with secondary antibody only served as the negative control and the positive control was a human spleen sample (rich in CD8+ cells). Density of cells with CD8, average intensity of CD8 fluorescence per area, and total intensity of CD8 fluorescence per area was assessed semi-quantitatively following a one second exposure with fluorescent microscopy.

Results: As predicted, H&E staining showed unremarkable colonic mucosa with no diagnostic abnormalities in both the ASD and TD group. Immunohistochemical analysis using a CD8-specific antibody resulted in a mean total intensity of fluorescence (cells expressing CD8) that was significantly greater in ASD than in TD samples (p=0.003), and the total intensity per area was also significantly greater in samples from ASD versus TD children (p=0.0007).

Conclusions: These findings revealed that, compared to GI-symptomatic TD children, children with ASD and GI symptoms displayed a marked increase in CD8 fluorescence intensity, suggesting a cytotoxic T lymphocyte infiltration that was not apparent on routine histologic examination. Further analysis of CD8 reactivity is required to determine the significance of the observed CD8 infiltration. Other inflammatory markers such as CD3 and CD4 should also be examined via immunohistochemical analysis.