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

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Abstract

Objective:

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

Methods:

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.

Results:

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.

Conclusion:

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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