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Updated: Aug 8, 2026
Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes
Published on: May 8, 2016
Pitfalls in the characterization of small intestinal lymphocytes
1UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08903-0019.
Insights
Human intestinal lymphocytes (IEL and LPL) are similar in size and granularity to peripheral blood mononuclear cells (PBMC). Discrepancies in lymphocyte characterization arise from isolation methods and epithelial cell contamination.
Area of Science:
- Immunology
- Gastroenterology
- Cell Biology
Background:
- Morphological and phenotypic characteristics of human intestinal lymphocytes can differ when analyzed in situ versus ex vivo.
- Previous studies reported discrepancies in lymphocyte activation markers, macrophage function, and B cell counts in the intestine.
Purpose of the Study:
- To investigate the causes of discrepancies in the characterization of human intestinal lymphocytes.
- To clarify the morphology and function of intestinal lymphocytes, macrophages, and B cells.
- To identify factors that may artifactually alter experimental results.
Main Methods:
- Comparative analysis of intraepithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), and peripheral blood mononuclear cells (PBMC) using flow cytometry and immunohistochemistry.
- Assessment of epithelial cell (EC) contamination in lymphocyte preparations.
- Evaluation of macrophage-like properties and B cell marker expression in intestinal cell isolates.
Main Results:
- Most IEL and LPL are similar in size and granularity to PBMC, challenging the notion that IEL are predominantly large granular lymphocytes.
- Low-density surface antigen expression on LPL, detectable by flow cytometry but not immunohistochemistry, explains discrepancies in activation marker studies.
- Contamination with EC, which can exhibit macrophage-like features and non-specific staining with anti-immunoglobulin antibodies, accounts for variations in reported intestinal macrophage and B cell numbers and functions.
Conclusions:
- Artefactual alterations during sample processing, particularly EC contamination, significantly impact the accurate characterization of intestinal immune cells.
- Researchers must account for these factors to obtain reliable data on intestinal lymphocyte populations and their functions.
- Understanding these discrepancies is crucial for advancing research in gut immunology and related diseases.
Abstract:
The morphological characteristics of human intestinal lymphocytes may vary depending upon whether they are examined in tissue sections or after isolation. This study uncovered some causes of these discrepancies. The majority of intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) are of the same size and granularity as peripheral blood mononuclear cells (PBMC), negating the idea that most IEL are large granular lymphocytes. LPL were previously shown to express markers of early activation by flow cytometry but not by immunohistochemistry. This study shows that it is the low density of surface antigen expression that explains this discrepancy: only the highly sensitive flow cytometric analysis can detect such low-density expression. Previous reports of intestinal macrophage and B cell numbers and functions vary markedly. This may be due to variable amounts of contamination with epithelial cells (EC) that lose their characteristic columnar appearance and cytoplasmic mucin upon isolation. They share many features of macrophages (such as morphology and binding to plastic and latex beads) but lack phagocytic ability and superoxide generation, explaining, perhaps, the wide variation in the reported functions of presumed intestinal macrophages. In addition, a large fraction of EC non-specifically strain with anti-immunoglobulin antibodies. Their contamination of intestinal lymphocyte preparations accounts for the discrepancy in B cell numbers identified by surface immunoglobulin expression versus B cell markers. Intestinal lymphocytes do indeed differ in many ways from circulating lymphocytes, but investigators must be attentive to factors that may artifactually alter their results.
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