IL-17 producing cell function is higher in colorectum than blood in uninfected RMs.

<p><b>(A)</b> Comparison between cytokine profiles of Th17, Th22, and Th17/Th22 cells in PBMC and RB in uninfected RMs (d. -20 p.i.). Cytokine profiles were generated for each cell population by SPICE program v. 5.33, and were calculated by Flowjo Boolean gating. <b>(B)</b> Functional scores were compared for all three subsets between PBMC and RB. Functional scores represent average number of cytokines produced per individual cell (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005412#sec011" target="_blank">Methods</a>). Averaged data are presented as means ± SEM.</p

Loss of Th17, Th22, and Th17/Th22 cell function and levels are correlates and predictors of SIV persistence.

<p>Correlations between colorectal SIV-DNA content at late-ART (d. 256 p.i.) and IL-17 and IL-22 producing cell function and levels at both ART (<b>A-C</b>) and pre-ART infection (d. 58 p.i). <b>(D,E)</b>. Levels and function of the three subsets at pre-ART and after ART interruption additionally correlated with levels of blood DNA levels after ART interruption (d. 440 p.i.) <b>(F-I)</b>. Pearson product-moment correlation coefficients were measured for all plots except for 8E, which required Spearman’s rank correlation coefficient.</p

Loss of Th17, Th22, and Th17/Th22 cell function correlates with colorectal immune activation, soluble markers of inflammation, and levels of CD4⁺ T-cells.

<p>Correlations between Th17, Th22, and Th17/Th22 cell function and plasma viral loads (copies viral RNA per mL of plasma) <b>(A,B)</b>, absolute number of CD4⁺ T-cells <b>(C)</b>, levels of activated colorectal (HLA-DR⁺CD38⁺) CD4⁺ T-cells <b>(D,E)</b>, the level of proliferating (Ki-67⁺) RB CD4⁺ T-cells <b>(F)</b>, as well as soluble markers of inflammation sCD14 and sCD163 <b>(G, H)</b>. Pearson product-moment correlation coefficients were measured for all plots except for 7A, which required Spearman’s rank correlation coefficient.</p

Levels of Th17, Th22, and Th17/Th22 are more drastically depleted by SIV infection in RB than PBMC.

<p>Comparison of frequencies (measured as percentages of total CD4⁺ T-cell populations) of circulating <b>(A)</b> and colorectal <b>(B)</b> Th17 (red), Th22 (blue), and Th17/Th22 (green) cells before (d.-20 p.i.) and after (d. 58 p.i.) SIV infection. Averaged data are presented as means ± SEM.</p

Study timeline and representative cytokine panel staining.

<p><b>(A)</b> Complete study timeline of longitudinal tissue collections, SIVmac239 i.v. infection (d.0 p.i.), ART treatment (d. 58 to d.256 p.i.), and off-ART time period. <b>(B)</b> Representative staining for IL-17, IL-22, IFNγ, TNFα, and IL-2 within intestinal CD4⁺ T-cells in a representative uninfected RM.</p

SIV infection severely ablates intestinal IL-17 and IL-22 producing cell function and levels.

<p><b>(A)</b> Comparison between PBMC and RB Th17, Th22, and Th17/Th22 cell cytokine profiles at pre-infection (d.-20 p.i.) vs SIV infection (d. 58 p.i.). While all RB cytokine profiles significantly changed after SIV infection, the blood cytokine profiles remained unchanged. <b>(B)</b> No changes in functional score in PBMC after SIV infection. <b>(C)</b> In all three subsets, colorectal functional scores drastically decreased after SIV infection. Th17 cells marked as red, Th22 cells marked as blue, and Th17/Th22 cells marked as green. Averaged data are presented as means ± SEM.</p

ART treatment is insufficient for full restoration of cell subset levels and function.

<p><b>(A-C)</b> Longitudinal functional scores of Th17, Th22, and Th17/Th22 cells in RB at pre-infection (d. -20 p.i.), pre-ART (d. 58 p.i.), and throughout ART (d. 84 p.i. through d. 256 p.i.). Data are presented as box and whisker plots, with the median functional score plotted in between the 25% and 75% quartiles. Dotted line marks time of SIV infection and shaded gray box represents time of ART treatment. ART significantly increased all three subsets’ functional scores, but did not bring Th17 cell function back to pre-infection level. <b>(D)</b> Levels of subsets (as percentages of total CD4⁺ T-cell populations) in RB during ART remain significantly lower than pre-infection. <b>(E)</b> Longitudinal cumulative subset scores, calculated by multiplying cell frequencies and functional scores, showed the inability of ART to fully restore the levels and function of Th17, Th22, and Th17/Th22 cells. Th17 cells marked as red, Th22 cells marked as blue, and Th17/Th22 cells marked as green. Averaged data are presented as means ± SEM.</p

Differentially regulated genes between SIV+ CD4-low and SIV+ CD4-healthy mangabeys.

<p>Differentially regulated genes between SIV+ CD4-low and SIV+ CD4-healthy mangabeys.</p

Spectratyping analysis of DN T cells from SIV infected and uninfected mangabeys.

<p>+ indicates detected Vβ, — indicates undetected Vβ,</p>a<p>indicates specific Vβ had single clonally expanded peak.</p

Transcriptome analysis utilizing microarray of TCR stimulated DN T cells from mangabeys depicts upregulation of transcription factors and immunomodulatory genes.

<p>Microarray analysis comparing TCR-stimulated and unstimulated DN T cells. A) DN T cells from two SIV+CD4-healthy and two SIV+CD4-low mangabeys were stimulated with anti-CD3/anti-CD28 and comparative mRNA expression of differentially regulated genes following stimulation of is depicted with less than 4 fold differential regulation depicted in grey, while those with greater than 4 fold differential expression depicted in color (n = 568). In blue are genes regulated to the same extent in DN T cells from both SIV+CD4-low and SIV+CD4-healthy mangabeys, while genes specific to DN T cells from SIV+CD4-low animals are depicted in purple and those specific to DN T cells from SIV+CD4-healthy animals are in green. B) Enrichment data of functional clusters in gene families that are differentially regulated upon stimulation with number of genes shown as columns (dark blue) and extent of enrichment of functional gene clusters (enrichment score) depicted as lines (light blue). Genes involved in immune response and transcription were highly enriched. C) Evaluation of immunomodulatory genes with subsets representing adaptive immunity, innate immunity, apoptosis, signaling and transcription genes are presented as a heat map. Differentially regulated genes from SIV+ CD4-healthy, SIV+ CD4-low and uninfected mangabeys with ≥4 fold differential regulation following TCR stimulation depicting upregulated (red) and downregulated (green) gene expression.</p