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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 (r 2 =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/mm 3  and 33,700 ± 4,324 cell/mm 3 , respectively. Sigmoid mucosa contained 57% CD3 + CD4 +  and 40% CD3 + CD8 +  T lymphocytes which calculates to 21,300 ± 1,476/mm 3  and 15,000 ± 275/mm 3  T lymphocytes, respectively. Rectal mucosa had 57% CD3 + CD4 +  and 42% CD3 + CD8 +  or 21,577 ± 332, and 17,090 ± 1,206 cells/mm 3 , respectively. By comparison, sigmoid mucosal biopsies subjected to conventional collagenase digestion, mononuclear cell (MMC) isolation and staining for flow cytometry yielded 4,549 ± 381/mm 3  and 2,708 ± 245/mm 3  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.

T Lymphocyte Density and Distribution in Human Colorectal Mucosa, and Inefficiency of Current Cell Isolation Protocols