Hirschsprung disease (HSCR) is a congenital enteric neuropathy where incomplete migration and population of neural crest cells in the colon during development result in a pseudo-obstruction in the distal colon. There are 2 distinct regions of the HSCR bowel, the ganglionic region where the enteric innervation is seemingly “normal” and the aganglionic region where there is no enteric innervation. Up to 50% of individuals with HSCR experience potentially life-threatening Hirschsprung Associated Enterocolitis (HAEC), an inflammatory condition of the bowel mucosa that can lead to overwhelming sepsis and death (Gosain and Brinkman, 2015). Interestingly, HAEC remains an ongoing complication post-surgical removal of the aganglionic bowel, suggesting that distal bowel obstruction is not the only causative factor and likely to be an interplay between enteric nervous system dysfunction, abnormal mucin secretion and altered immune cell presence and function (Jiao et al., 2016). To understand the immune phenotype in the bowel mucosa of HSCR broad-spectrum flow-cytometry analysis was conducted. This demonstrated a decrease in γδT cell populations in both the ganglionic and aganglionic bowel regions of these patients compared with age matched controls. We further assessed these changes, with in-depth flow cytometry analysis of the γδT cell population. Additionally, we have developed a novel 3-dimensional imaging technique to visualise the complete mucosal region of the colon using optical clearing techniques and digital light sheet imaging. Using this imaging strategy with colon tissue from patients, we will be able to map the topographical spatial localisation of γδT cells in situ. In combination with the relative frequencies of these cells and their subtypes this data provides novel insight into the immunological compartment of HSCR.