title: T lymphocyte density and distribution in human colorectal mucosa, and inefficiency of current cell isolation protocols.
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.
authors: Department of Dermatology, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States of America.
authors: Gloria Cuevas
authors: GC
authors: Preza
authors: Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, United States of America; Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, United States of America; UCLA AIDS Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, United States of America; AIDS Healthcare Foundation, Los Angeles, CA, United States of America.
authors: Otto O
authors: OO
authors: Yang
authors: Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, United States of America; UCLA AIDS Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, United States of America.
authors: Julie
authors: J
authors: Elliott
authors: Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, United States of America; UCLA AIDS Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, United States of America.
authors: Peter A
authors: PA
authors: Anton
authors: Department of Dermatology, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States of America.
authors: Maria T
authors: MT
authors: Ochoa
mesh_terms: D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000276:immunology*; D007420:Intestine, Large / Q000276:immunology*; D016176:T-Lymphocyte Subsets / Q000276:immunology*