Abstract
Mucosal tissues are critical immune effector sites containing complex populations of leukocytes in a tissue microenvironment that remains incompletely understood. We identify and quantify in human distal colorectal tissue absolute mucosal CD3+ lymphocytes, including CD4+ and CD8+ subsets, by direct visualization using immunohistochemistry (IHC), immunofluorescence (IF), and an automated counting protocol (r2=0.90). Sigmoid and rectal mucosal tissues are both densely packed with T lymphocytes in the mucosal compartment. Both compartments had similar densities of CD3+ T lymphocytes with 37,400 ± 2,801 cells/mm3 and 33,700 ± 4,324 cell/mm3, respectively. Sigmoid mucosa contained 57% CD3+CD4+ and 40% CD3+CD8+ T lymphocytes which calculates to 21,300 ± 1,476/mm3 and 15,000 ± 275/mm3 T lymphocytes, respectively. Rectal mucosa had 57% CD3+CD4+ and 42% CD3+CD8+ or 21,577 ± 332, and 17,090 ± 1,206 cells/mm3, respectively. By comparison, sigmoid mucosal biopsies subjected to conventional collagenase digestion, mononuclear cell (MMC) isolation and staining for flow cytometry yielded 4,549 ± 381/mm3 and 2,708 ± 245/mm3 CD4+ and CD8+ T lymphocytes. These data suggest only ~20.7% recovery compared to IHC results for these markers. Further studies will determine if this reflects a selective bias in only CD3+, CD4+ and CD8+ T cells or can be generalized to all flow-analyzed cells from mucosal tissues for phenotyping and functional testing.
Highlights
The mucosal immune compartment is the largest lymphoid reservoir of the body, a critical compartment in immune-protection such as in the setting of various infectious diseases and cancer
Given improved automated approaches to quantifying cells in situ, we sought to investigate the numbers and distributions of mucosal CD3+, CD4+ and CD8+ T lymphocyte populations in healthy human distal colon and rectum. The numbers of these cells in tissue have been debated and under-described in the scientific literature [4,5,6,7]. To accomplish this comparative but descriptive study with minimal additional resources, we utilized numbers of mucosal CD3+, CD4+ and CD8+ T lymphocyte populations quantified by flow cytometry from healthy controls in other NIH-funded studies, leveraging that data to compare with counts via IHC using newly recruited subjects through the UCLA Center for AIDS Research (CFAR)’s Mucosal Immunology Laboratory Core
It is believed that the gastrointestinal tract and gut-associated lymphoid tissue (GALT) contain the majority of total body lymphocytes [16]
Summary
The mucosal immune compartment is the largest lymphoid reservoir of the body, a critical compartment in immune-protection such as in the setting of various infectious diseases and cancer. Given improved automated approaches to quantifying cells in situ, we sought to investigate the numbers and distributions of mucosal CD3+, CD4+ and CD8+ T lymphocyte populations in healthy human distal colon and rectum. The numbers of these cells in tissue have been debated and under-described in the scientific literature [4,5,6,7]. To accomplish this comparative but descriptive study with minimal additional resources, we utilized numbers of mucosal CD3+, CD4+ and CD8+ T lymphocyte populations quantified by flow cytometry from healthy controls in other NIH-funded studies, leveraging that data to compare with counts via IHC using newly recruited subjects through the UCLA CFAR’s Mucosal Immunology Laboratory Core
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