T lymphocyte density and distribution in human colorectal mucosa, and inefficiency of current cell isolation protocols

• Published in: PloS one 2015-04, Vol.10 (4), p.e0122723-e0122723
• Main Authors: Preza, Gloria Cuevas, Yang, Otto O, Elliott, Julie, Anton, Peter A, Ochoa, Maria T
• Format: Article
• Language: English
• Subjects: Acquired immune deficiency syndrome; AIDS; CD3 antigen; CD4 antigen; CD8 antigen; Cell Count; Cell Separation - methods; Collagen; Collagenase; Colon; Compartments; Cytometry; Data recovery; Flow Cytometry; Fluorescent Antibody Technique; Functional testing; HIV; Human immunodeficiency virus; Humans; Immunofluorescence; Immunohistochemistry; Intestinal Mucosa - immunology; Intestine, Large - immunology; Leukocytes; Lymphocytes; Lymphocytes T; Mucosal immunity; Phenotyping; Practice guidelines (Medicine); Rectum; T-Lymphocyte Subsets - immunology; Tissues
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0122723

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.