Comparison of populations observed via in situ tetramer staining to flow cytometry.

<p>The percentage of tetramer⁺ cells that were CD8⁺, CD8^low, and CD8⁻ within lymph nodes (A) and vagina submucosa tissues (B) using in situ tetramer staining and flow cytometry. For in situ tetramer staining, all lymph nodes were axillary with the exception of animal #R80072 in which axillary and mesenteric lymph nodes were stained together. For flow cytometry, all lymph nodes were axillary except #27338 which was inguinal.</p

Localization of SIV-specific CD8^low/− T cells in the genital tract.

<p>In each set of panels, the left panels show tetramer staining (red), the middle panels show CD8 antibody stain (green), and the right panels are merged images of the left and middle images. Mamu-A*01 Gag CM9 staining is shown in (A, D and G) and negative control Mamu-A*01 FLP is shown in (J). All panels are representative images of vagina from animal #27338 at 28 day post-infection, with panels (D) to (F) showing a higher magnification of the area indicated by the white box in (C). White arrow heads in (F) indicate tetramer⁺ CD8^low T cells. White arrows indicate tetramer⁺CD8^low/− cells. The outer edge of the genital tract epithelium is indicated with a white line. All images are confocal Z-scans collected using a 10× (A to C), 20× (G to L) and 60× objective (D to F). Bars: (A to C) 100 microns, (G to L) 50 microns and, (D to F) 20 microns.</p

Localization of SIV-specific T cells in B cell follicle.

<p>In each set of panels, the left images (A and D) show Mamu-A*01 Gag CM9 tetramer staining (red), the middle images (B and E) show CD20 antibody staining (green), and right images (C and F) are merged image of the left and middle images. The bottom panels shows a higher magnification image taken from within the large B cell follicle shown in the top panels. The white arrows point to a tetramer+CD20⁻ cell within the B cell follicle. Representative images are from an axillary lymph node from animal #24225 at 28 day post-infection. Confocal images were collected using a 20× (A to C) and 60× objective (D to F). Bars: (C) 50 microns, (F) 20 microns.</p

Protection of CD4-IgG2-treated rhesus macaques in a high-dose SIVmac239 challenge experiment.

<p>To maintain serum concentrations, CD4-IgG2 (or control human polyclonal IgG) was administered subcutaneously over a two-week period by an ALZET osmotic pump. Animals were challenged intrarectally with a single high dose inoculum (3–5×10³ TCID₅₀) of SIVmac239 3-days after initiation of CD4-IgG2 administration. (<b>A</b>) Viral loads for animals treated with 200 mg of control polyclonal human IgG as a function of time following SIVmac239 challenge. All control animals became infected. (<b>B</b>) Viral loads for animals administered 20 mg CD4-IgG2 as a function of time following SIVmac239 challenge. Three out of 6 animals were fully protected and one infected animal showed delayed primary viremia. Due to a technical problem with the ALZET osmotic pump, one of the protected animals (98045) did not receive the full dose of 20 mg but this animal did not become infected. (<b>C</b>) Viral loads for animals administered 200 mg CD4-IgG2 as a function of time following SIVmac239 challenge. Five out of 7 animals were protected and showed no sign of infection at any time point. The minimum detection level was 125 SIV RNA copies/ml with a 95% confidence level. Open symbol indicates protected animal, closed symbol indicates infected animal.</p

Localization of SIV-specific T cells and γδ TCR+ cells in lymph node and vagina.

<p>In each set of panels, the left images (A and D) are Mamu-A*01 Gag CM9 tetramer stain (red), the middle image (B and E) are γδ TCR antibody stain (green), and right images (C and F) are merged images of the left and middle images. Panels A to F are representative images from animal #27572 that show staining in inguinal lymph node and vagina at 27 day post-infection. Images are all confocal Z-scans collected using a 20× objective. Bar = 50 microns.</p

Plasma concentration of CD4-IgG2 in treated animals.

<p>Animals administered 200 mg of CD4-IgG2 showed plasma concentrations in the range of 500 to 1400 ng/ml at the time of challenge. No apparent correlation between plasma concentration and protection was observed. The CD4-IgG2 concentration at the time of challenge in animals administered 20 mg was 100 ng/ml for 1 animal and below the limit of detection (8 ng/ml) for 2 animals. Serum samples from the remaining 3 animals administered 20 mg were unavailable for this analysis.</p

Localization of SIV-specific CD8^low/− T cells in lymph nodes.

<p>In each set of panels, the left panels show tetramer staining (red), the middle panels show CD8 staining (green), and the right panels merged images of the red and green stain (A) and (D) show Mamu-A*01 Tat SL8 staining and (F) shows staining from the negative control Mamu-A*01 FLP tetramer. Panels (D) and (E) are derived from (A) and (B), but show only the tetramer stained in order to more easily note the lack of CD8 staining on the cells in the cluster of cells in the upper right-hand quadrant of the image delineated by the purple line. These are representative images from axillary lymph nodes from SIV-infected rhesus macaque #27357 at 20 days post-infection. All images are confocal Z-scans collected using a 20× objective. Scale Bar = 50 microns.</p

Presence of tetramer+CD8^low/− cells in tissues from SIV-infected rhesus macaques.

<p>N/A = not available. We could not distinguish the genital epithelium from lamina propria in sections from animals 27572 and 80025.</p

Tetramer⁺CD3⁺CD8^low/− stained cells from disaggregated lymph nodes and vagina analyzed by flow cytometry.

<p>Disaggregated cells from SIV-infected macaques were stained with Mamu-A*01 Gag CM9, Mamu-A*01 Tat SL8, and negative control Mamu-A*01 FLP tetramers; counterstained with CD3 and CD8 antibodies and analyzed by flow cytometry. Populations of SIV-specific tetramer⁺CD3⁺CD8^low/− lymphocytes were not detected above negative control staining. However, populations of tetramer⁺CD3⁺CD8^low cells were detected above background levels. Representative data from inguinal lymph node from animal #R80072, and vaginal submucosa from #27338 is shown. Note negative control staining FLP was not done with the vaginal submucosal cells.</p

Anti-human CD4 response in animals treated with CD4-IgG2.

<p>Animal sera were tested in a human CD4-specific ELISA to detect macaque antibody responses against CD4-IgG2. Serum samples were tested up to 23 days post-viral challenge and no responses were detected before day 15, indicating that the animal protection outcome was independent of a response against human CD4. Serum samples from 3 animals administered 20 mg CD4-IgG2 were unavailable for this analysis.</p