Staining Patterns of Gag CM9. A) Fluorescence image of a lymphocyte from an SIV infected Mamu-A*01 positive RM stained with Gag CM9 Qdot 655 multimer (red) and CD8 (green). B) Fluorescence image of a lymphocyte from an SIV infected Mamu-A*01 positive RM stained with Gag CM9 APC Tetramer (red) and CD8 (green). C) Confocal fluorescence image of a colon tissue section from an SIV infected Mamu-A*01 positive RM stained with Gag CM9 Qdot 655 multimer (Red), CD8 (green) and Dapi (blue). Scale bar = 10 μm. D) A magnified view of the region indicated in panel C. Cells stained with Qdot 655 multimer or APC tetramer show a punctate Gag CM9 staining pattern. All images were acquired with a 100×/1.4 oil immersion objective and further deconvolved. E-H) Volocity (Improvision) software was used to generate a surface model of the CD8⁺Gag CM9⁺ cells shown in panel A (E, F) and panel D (G,H). E, G: CD8 (green), Gag CM9 Qdot 655 multimer (red); F, H: overlap (yellow) of CD8 and Gag CM9 Qdot 655 multimer staining.

Gag CM9 T cells are located in the extra follicular area of lymphoid tissue. Fluorescence images showing sections from an SIV infected Mamu-A*01 positive RM. A-C) Images of a solitary lymphoid follicle in a colon section. A) Gag CM9 positive cells are located in the extra follicular region. Gag CM9 Qdot 655 multimer (red), CD20 (green). B) No CD8⁺ cells were located in the extra follicular region. CD8 (red), CD20 (green). C) All CM9 positive cells were CD8⁺. Gag CM9 Qdot 655 multimer (red), CD8 (green). D) Image of a mesenteric lymph node section showing that the majority of the Gag CM9 positive cells are located in the extra follicular region, but some are in the junction of the extra follicular and follicular area. Gag CM9 Qdot 655 multimer (red), CD20 (green) E) A magnified view of the region indicated in panel D showing the Gag CM9 positive cells the junction of the extra follicular and follicular area. All sections were stained with DAPI (blue). Images were collected with a 20×/0.75 objective. Scale bar = 50 μm.

Gag CM9 Qdot 655 multimer validation. A) Flow cytometry analysis of Gag CM9 specific CD8⁺ T cell clones showed that > 99% of the cells bound to the Gag CM9 Qdot 655 multimer or to the Gag CM9 APC tetramer. The multimer and the tetramer are coupled with the same Gag CM9 monomers. ≤ 0.13% of Gag CM9 specific cells bound to the negative control FLP Qdot 655 multimer. B) Flow cytometry analysis of PBMCs and single cell suspension of lymph nodes demonstrated that a distinct population of CD3⁺CD8⁺ cells, 1.74% in blood and 3.82% in lymph node single cell suspension, from SIV infected Mamu-A*01 positive RM bound the Gag CM9 Qdot 655 multimer. ≤ 0.17% of CD3⁺CD8⁺ cells from Mamu-A*01 positive RM that were not SIV infected or cells from Mamu-A*01 negative RM that were either SIV infected or uninfected bound the Gag CM9 Qdot 655 multimer. ≤ 0.31% of CD3⁺CD8⁺ cells bound the FLP Qdot 655 multimer. The gating strategy was as described in Methods. ND; not done due to lack of material.

In situ staining of CD8⁺Gag CM9⁺ cells in frozen tissue. A) Fluorescence images of a spleen tissue section from an SIV infected Mamu-A*01 positive RM stained with CD8 (green), Gag CM9 or FLP Qdot 655 multimer (red), and DAPI (blue) demonstrating that the Gag CM9 positive cells are CD8⁺. Fluorescence images of B) spleen sections, C) mesenteric lymph node sections, D) ileum sections with peyer's patch, E) solitary lymph node in colon and F) images of lamina propria in colon tissue from SIV infected Mamu-A*01 positive RM. Left-most column, second column, and right most column show Gag CM9 Qdot 655 multimer (red). The second column is a magnification of the left column images. Third column; FLP Qdot 655 multimer (red). Right-most column, similar sections from a non-infected Mamu-A*01 negative RM. Images were collected with a 20×/0.75 objective. Scale bar = 50 μm.

Intensity comparison between Gag CM9 Qdot 655 multimer and Gag CM9 APC-tetramer. A) Single cell suspension cells of a lymph node from an SIV infected Mamu-A*01 positive RM were stained with Gag CM9 Qdot 655 multimer or with Gag CM9 APC tetramer and the staining intensity was measured. Z stack average intensity projections for six cells was used and the mean average intensity was calculated by using Image J Software. A ten-fold higher mean average intensity was found for Gag CM9 Qdot 655 multimer as compared to the Gag CM9 APC tetramer, even though the same monomers are used in each case. B-D) Fluorescence images of tissue sections from an SIV infected Mamu-A*01 positive RM stained with CD8 in green, Gag CM9 Qdot 655 multimer in red (left column) or Gag CM9 APC Tetramer in red (right column) and dapi in blue. B) Images of spleen sections stained with Gag CM9 Qdot 655 multimer demonstrated abundant Gag CM9 specific cells (left column) whereas the consecutive section stained with Gag CM9 APC Tetramer showed no Gag CM9 positive cells (right column). C) Gag CM9 positive cells were detected in a solitary lymphoid follicle within the colon section (right column) when Gag CM9 Qdot 655 multimer was used, whereas when the consecutive slide was stained with Gag CM9 APC Tetramer no Gag CM9 positive cells could be detected (left column). D) Gag CM9 positive cells were detected both in the lamina propria and in a small solitary lymph node in the colon (right column) when stained with Gag CM9 Qdot 655 multimer but when the consecutive slide was stained with Gag CM9 APC tetramer no Gag CM9 positive cells were detected (left column). Images were collected with a 20×/0.75 objective. Scale bar = 50 μm.