Flow cytometric analysis of intraepithelial lymphocytes from human small intestinal biopsies reveals populations of CD4+CD8+ and CD8αα+ cells

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To seek evidence of extrathymic maturation in the human small intestine using flow cytometric analysis of intraepithelial lymphocytes (IEL) subpopulations.


Single cell suspensions were prepared from the epithelial layer of endoscopically obtained small intestinal tissue from 11 healthy adults. These preparations were composed of approximately 70% epithelial cells and 30% lymphocytes, which were viable and suitable for flow cytometric studies.


Using two-colour analysis, 71–89% of the CD3+ lymphocytes were CD8+, while 2–20% were CD4+. The majority of IELs expressed the αβ T-cell receptor (TCR; mean 89%, range 79–99%), while the remainder (mean 6%, range 1–12.5%) expressed the γδ TCR. Less than 1% of the gated IEL population was CD22+, indicating the absence of B cells and thus suggesting minimal lamina propria contamination. Moreover, histological analysis of post-treatment tissue confirmed that the integrity of the basement membrane was maintained. A significant population of CD3+ lymphocytes co-expressed CD4 and CD8 (mean 8%, range 3–13%). Significantly, γδ+ cells were exclusively CD8+, indicating that the double positive population was αβ TCR+. Previously, double positive lymphocytes were thought to be an immature population found only in the thymus.


The presence of double positive lymphocytes in the gastrointestinal epithelium supports the hypothesis that the gut is a site of extrathymic differentiation. The demonstration that a significant percentage of the CD8+ population was homodimeric for the a chain (mean 20%, range 12–27%) is additional supportive evidence for this theory.

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