Levels of gp120 and p27 in the Plasma, Lymph Node and Spleen of RM 12 weeks post-inoculation with R5-SHIV.

<p>LN: Lymph Node.</p

T regulatory cells migrate toward R5 HIV gp120.

<p>CD4+CD25+T regulatory cells were purified from naive human PBMC and exposed to various concentrations of gp120 in a Boyden chamber migration assay. A) Normalized transmigration index of Tregs that migrate towards various gp120 concentrations (500 pg/ml, 5 ng/ml, 500 ng/ml) in presence of CCR5 antagonist, TAK-779, or pretreated with pertussis toxin (Ptx 100 ng/ml) (*p<0.05 vs TAK-779). B) Normalized transmigration index of Tregs that migrate away from gp120 concentrations (500 pg/ml, 5 ng/ml, 500 ng/ml) in presence of CCR5 antagonist, TAK-779 (40 nM), or pretreated with pertussis toxin (Ptx, 100 ng/ml).</p

Reduced antigen-specific CD4 and CD8 T cell responses in LN RM during early R5-SHIV infection.

<p>RM were inoculated intravaginally (n = 4) or intravenously (n = 2) with SHIV-1157ipd3N4 (R5-SHIV). Blood samples were drawn for viral load analysis at 0, 1, 2, 3, 8 and 12 weeks post infection (A). Paired peripheral blood samples (PB) and lymph nodes (LN) from RM during early infection were sampled at 5 and 12 weeks post-inoculation (B-G). PB and LN lymphocytes were stimulated with overlapping clade C gp120 peptide pools at 5 and 12 weeks post inoculation and IFN-γ⁺ gp120-specific CD4 (B,C) and CD8 (D,E) T cell were assayed. F & G) PB and LN derived cells were stimulated with overlapping SIVmac239 Gag peptide pools at 5 and 12 weeks post inoculation and IFN-γ⁺ Gag-specific CD4 (F) and CD8 (G) T cell responses were assayed. Data representative of frequency of total (C,E-G) or frequency of parent (B,D) are shown. Error bars ±SEM, *p<0.05, ** p<0.01.</p

Increased numbers of CD4+CD25+CD127low T cells in the LN of R5-SHIV infected RM.

<p>RM were sampled at 5 and 12 weeks post-inoculation. PB and LN samples were examined by multi-colour flow cytometry for the proportion of Treg cells present. A) Representative dotplots from PB and LN gated on size and granularity as well as CD3 and CD4, and subsequently on CD25 vs CD127 as shown (% frequency of parent). B) Gating strategy demonstrating the fluorescent minus one scheme utilized as above and CD25 vs CD127 as shown. C & D) Individual RM Treg cell frequencies of parental gate at 5 weeks post-infection *p<0.05 LN compared to PB (C), and 12 weeks <u>(non-significant)</u> (D) post-infection. E) Time course of Treg accumulation at 5 and 12 weeks post infection in PB and LN, mean ±SEM. F) LN samples depleted of CD4+CD25+ T cells and stimulated with overlapping SIVmac239 Gag peptide pools. Data depicted as relative change of Gag- and gp120 specific CD8 responses in CD4+CD25+ depleted compared to non-depleted samples, (p = 0.06 vs change in gp120 specific CD8 T cells.) G) Correlation of Tregs with gp120 in the LN of intravaginally challenged RM (r² = 0.7, two-tailed p = 0.035).</p

Enhanced basal T cell apoptosis from PB, but not LN of R5-SHIV-infected RM.

<p>Naïve and R5-SHIV challenged RM were sampled at 5 and 12 weeks post-inoculation. PB and LN samples from either naïve or 5 and 12 weeks post-inoculation samples were either non-stimulated (NS) or stimulated with plate-bound anti-CD3 (CD3) for 24 hours. A) Percent of apoptotic CD4 T cells from naïve RM or infected RM at 5 or 12 weeks post inoculation comparing non-stimulated to CD3 stimulated is shown (naïve PBMC non-stimulated vs naïve PBMC stimulated p<0.005; naïve PBMC non-stimulated vs 5 weeks infected PBMC non-stimulated p<0.005; naïve PBMC non-stimulated vs 12 weeks infected non-stimulated p<0.05). B) Percent of apoptotic T cells from naïve RM or infected RM at 5 or 12 weeks post inoculation comparing non-stimulated to CD3 stimulated, (naïve PBMC non-stimulated vs naïve PBMC stimulated p<0.005; naïve PBMC, non-stimulated vs 5 weeks infected PBMC, non-stimulated p<0.05).</p

Enhanced expression of PD-1 on LN-derived CD4 and CD8 T cells.

<p>Naïve and intravaginally challenged RM were sampled at 5 and 12 weeks post-inoculation. Samples from PB and LN derived lymphocytes were examined by flow cytometry for the amount of PD-1 that was expressed at 5 and 12 weeks post-inoculation. Mean Fluorescent Intensity (MFI) for CD4 (A) and CD8 (B) of samples from infected and naïve RM are shown. Significant increases in PD-1 expression by LN derived CD4 and CD8 T cells were seen at 5 weeks post inoculation compared to naïve animals.</p