Background

Intestinal intraepithelial lymphocytes (IELs) are located between the epithelial cells of the digestive tract and can be divided into two main subpopulations: natural IELs and induced IELs. Natural IELs are mainly composed of γδT lymphocytes that develop and reside in the intestinal epithelium. In contrast, induced IELs are conventional CD4 and CD8 peripheral αβ T cells that migrate into the epithelium in response to antigens. Furthermore, small numbers of lymphocytes that do not express the T-cell receptor (TCR), such as intracytoplasmic (i)CD8 and iCD3 cells and type 1 innate lymphoid cells, are present in the epithelium.

IELs serve as the first line of defense against enteric pathogens and are vital for maintaining intestinal homeostasis [1]. Once activated, IELs are able to eliminate pathogens through the secretion of pro-inflammatory cytokines [e.g., interferon gamma (IFNγ)] and cytotoxic molecules (e.g., granzyme and perforin). These cells also have a regulatory role in the epithelium through the secretion of anti-inflammatory cytokines like interleukin (IL)-10 and transforming growth factor beta (TGF-β).

An imbalance in IEL inflammatory responses can significantly affect intestinal mucosa integrity. In celiac disease, for example, the unchecked expansion of IELs leads to the destruction of intestinal epithelial cells (IECs) and the atrophy of villi [2]. In patients with colorectal cancer, a greater number of TCRγδ IELs with a strong cytotoxic profile in the area around the tumor is associated with a lower risk of metastasis [3]. Although the specific role of this population in tumor cell regulation and elimination is not fully understood, one can hypothesize that deficiencies in this population substantially affect the patient’s response to treatments in general and to immunotherapy more specifically. Furthermore, many studies have documented anomalies in the total IEL count in various digestive pathologies [4].

Thus, an accurate assessment of the absolute IEL count is of great importance. A widely used approach for counting IELs involves analyzing histological sections. This multi-step process, including tissue fixation, paraffin embedding, block sectioning, slide mounting, and antibody labeling, extends experiment time compared to cytometric methods. Additionally, cell counting requires independent double readings by trained operators, similar to conventional hemocytometer counting of cell suspensions obtained after the epithelium has been detached from the rest of the mucosa, which also demands multiple readings by experienced personnel. So, a standardized procedure for counting IELs is lacking. Some experts express the IEL count with regard to the weight of the tissue, while others normalize it against the IEC count in the same sample. This lack of methodological consensus might explain the great disparities observed from one publication to another.

Recently, Lockhart et al. [5] employed flow cytometry to phenotype IELs and used BD TruCount^TM beads to yield the absolute IEL count in murine intestinal mucosa. The high-precision BD TruCount^TM procedure described below goes a step further by counting not only IELs but also IECs as an internal control in each sample, to ensure the assay’s reproducibility. Indeed, our flow cytometry technique is the first to have used BD TruCount^TM beads to provide the absolute IEL account and the IEL/IEC ratio. The IEL population is phenotypically identified by the expression of CD3 and CD103, while the epithelial population is characterized by the expression of CD326/EpCAM+. Moreover, unlike cells of hematopoietic origin such as IELs, IECs do not express the CD45 marker. During the development of this protocol, we observed that protein-based viability probes, such as Zombie dye, resulted in an overestimation of the percentage of dead cells compared to nuclear viability probes like DAPI. Indeed, mucus that remains adherent to epithelial cells nonspecifically catches protein-based viability probes and is therefore responsible for off-target binding. This overestimation subsequently biased the IEL count when compared to conventional microscopic counting. This finding underscores the importance of carefully selecting the viability probe when using this type of assay. We believe that our assay protocol will help to advance research in this field by providing a new tool for studying IEL density in the intestinal epithelium and, potentially, in other mucosal tissues. Through its use of commercially available reagents and antibodies, this flow cytometry–based protocol offers the advantages of (i) standardization, ensuring reproducibility and, therefore, the reliability of results, (ii) time efficiency, as it reduces the overall duration of the process, (iii) reduced workforce requirements, as it minimizes the need for multiple operators, and (iv) improved practicality, as it simplifies implementation while maintaining accuracy. This method is applicable to all rodent models used in the laboratory, with the main limitation being the availability of suitable antibodies for cytometry, which may not exist for all species. The protocol can also be adapted for human samples, though adjustments in reagent quantities will be necessary due to variations in specimen size.