Application of an automated algorithm to quantitate T lymphocytes in immunostained sigmoid colonic and rectal mucosa.

<p>A membrane detection algorithm was applied to CD3-immunostained sigmoid colonic and rectal mucosal biopsies from 5 healthy donors (5 sigmoid colon, 5 rectum). (A) Digital images with AEC chromogen staining for CD3 and hematoxylin counterstaining. (B) Mark-up images with red, orange, and yellow pixels depicting immunopositive cells (strong, moderate, and weak intensity, respectively), and black pixels depicting nuclei for immunonegative cells. Bar = 100 μm. Arrowheads denote manual enumeration. (C) Tissue concentrations of T lymphocytes obtained using both methods and analyzed by paired t test. (D) Correlation between counts obtained by manual and automated enumeration of the same regions. R² value = 0.90 (E) Tissue concentrations of T lymphocytes in epidermis (Epi), lamina propria (Lp), and lymphoid aggregates (La; when present) of sigmoid colon and rectum. (F) Average T lymphocyte concentrations obtained from averaging the various compartments from the sigmoid colon and rectum.</p

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

<div><p>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.</p></div

CD3⁺ lymphocytes are similar in normal human sigmoid colonic and rectal mucosa.

<p>Sigmoid colonic and rectal mucosal sections immunostained for CD3. The membrane red/brown colors denote positive staining (red/brown, AEC chromogen). Specimens were counterstained with hematoxylin. (A)Upper panel original magnification 200x (B) lower panel 400x (C) lower magnification showing distribution of lymphoid aggregates in both sigmoid and rectum (100x). Arrows represent lymphoid aggregates.</p

Distribution and number of CD4⁺ and CD8⁺ T lymphocytes in sigmoid mucosal and rectal mucosa.

<p>(A) Immunoperoxidase for CD4 and CD8 with hematoxylin counterstaining. (B) Distribution of CD4⁺ and CD8⁺ cells in lymphoid aggregates. (C) Confocal microscopy of CD3⁺CD4⁺ and CD3⁺CD8⁺ subsets of T lymphocytes. Bar = 50μm. (D) Densities of CD4⁺ and CD8⁺ T lymphocytes compared to densities obtained from flow analysis. GL = gland; Lu = lumen</p

Comparison of automated counts of <i>in situ</i> sigmoid colonic and rectal mucosal T lymphocytes versus isolation and flow cytometric counting.

<p>Mucosal CD3⁺ T lymphocytes were isolated by mechanical tissue disruption and collagenase digestion. (A) Gating strategy for flow cytometric analysis of isolated lymphocytes. (B) Comparison of the mean numbers of sigmoid colonic mucosal CD3⁺ lymphocytes isolated from 30 sigmoid biopsies from similar persons versus automated (n = 48) and manual counting of 5 sigmoid biopsies and 5 rectal biopsies from 5 subjects. Dots represent the means of 3–5 regions analyzed with bars representing standard error of the means. (Mann-Whitney Rank Sum test p<0.001).</p

HIV-1 Nef Sequence and Functional Compartmentalization in the Gut Is Not Due to Differential Cytotoxic T Lymphocyte Selective Pressure

<div><p>The gut is the largest lymphoid organ in the body and a site of active HIV-1 replication and immune surveillance. The gut is a reservoir of persistent infection in some individuals with fully suppressed plasma viremia on combination antiretroviral therapy (cART) although the cause of this persistence is unknown. The HIV-1 accessory protein Nef contributes to persistence through multiple functions including immune evasion and increasing infectivity. Previous studies showed that Nef’s function is shaped by cytotoxic T lymphocyte (CTL) responses and that there are distinct populations of Nef within tissue compartments. We asked whether Nef’s sequence and/or function are compartmentalized in the gut and how compartmentalization relates to local CTL immune responses. Primary <i>nef</i> quasispecies from paired plasma and sigmoid colon biopsies from chronically infected subjects not on therapy were sequenced and cloned into Env⁻ Vpu⁻ pseudotyped reporter viruses. CTL responses were mapped by IFN-γ ELISpot using expanded CD8+ cells from blood and gut with pools of overlapping peptides covering the entire HIV proteome. CD4 and MHC Class I Nef-mediated downregulation was measured by flow cytometry. Multiple tests indicated compartmentalization of <i>nef</i> sequences in 5 of 8 subjects. There was also compartmentalization of function with MHC Class I downregulation relatively well preserved, but significant loss of CD4 downregulation specifically by gut quasispecies in 5 of 7 subjects. There was no compartmentalization of CTL responses in 6 of 8 subjects, and the selective pressure on quasispecies correlated with the magnitude CTL response regardless of location. These results demonstrate that Nef adapts via diverse pathways to local selective pressures within gut mucosa, which may be predominated by factors other than CTL responses such as target cell availability. The finding of a functionally distinct population within gut mucosa offers some insight into how HIV-1 may persist in the gut despite fully suppressed plasma viremia on cART.</p></div

Selected PBMC data for CD4+, CD8+, CD4+CCR5+, CD8+CCR5+, CD4+CD38+DR+, and CD8+CD38DR+ T cells (boxplots showing the median, quartiles, and range for each cell population).

<p>Selected PBMC data for CD4+, CD8+, CD4+CCR5+, CD8+CCR5+, CD4+CD38+DR+, and CD8+CD38DR+ T cells (boxplots showing the median, quartiles, and range for each cell population).</p

Amount of selective pressure correlates with the CTL response regardless of location.

<p>Regression analysis shows a statistically significant correlation between the total mean number of SFC and the estimated global dN/dS from all samples, both blood and gut, for each subject (A). The correlation between CTL and dN/dS from all samples is more significant if only Nef-specific SFCs are considered (B). Finally, there is a significant correlation between the total mean number of SFCs and the number of positive peptide pools (C).</p

PBMC Comparisons.

<p>PBMC Comparisons.</p

MMC Comparisons.

<p>MMC Comparisons.</p