Intraepithelial lymphocytes (IELs) are very unique in the intestinal immune system. They include gamma delta T cells and CD4(-)CD8(-)TCR alpha beta+T cells (double negative: DNT), both of which are specific for the intestine, in addition to CD4(+) and CD8(+) T cells. IELs exist within the monolayer of the intestinal epithelial cells and dynamically move between lamina propria (LP) and intraepithelial (IE) region. The localization and movement patterns of IEL subsets and the regulatory factors have been unknown. Here, we developed a novel in vitro live imaging system and quantified the motility and morphological changes among subsets of IELs. We identified CD8 alpha alpha as the key regulatory factor. IELs, especially gamma delta and DNT cells, showed amoeboid shape and frequent morphological change, while most T cells in MLN or SP showed round shape in vitro. TCR signal, IL-15, gut microbes, CCL25, and integrin alpha E beta 7 expression were non-essential for IEL movement in vitro. CD8 alpha alpha(+) cells showed higher motility and larger morphological changes than CD8 alpha alpha(- )cells. Adoptive transferred CD8 alpha alpha(+) CD4-IELs localized to IE region of recipient NSG mice, while CD8aa CD4-IELs localized to the LP. Our results showed that the CD8 alpha alpha/TL signal is essential for the localization of IELs to IE region in vivo. CD8 alpha alpha/TL may be an effective target to increase the number of IELs, which protects against intestinal infection, allergy, tumorigenesis or inflammation. (C) 2019 Elsevier Inc. All rights reserved.