2W:I-A^b tetramer staining via flow cytometry and <i>in situ</i> staining.

<p>2W:I-A^b-ExAv-FITC tetramer identified an antigen-specific CD4⁺ T cell population that had expanded in response to intranasal GAS-2W infection. A) Flow cytometric analysis of 2W:I-A^b tetramer+ cells from disaggregated NALT showed populations of tetramer+CD4+CD44+ cells in GAS-2W but not GAS mice. Cells were gated on total CD4⁺ cells. <i>B</i>) 2W:I-A^b tetramer <i>in situ</i> staining of NALT from littermates showed tetramer+ cells in GAS-2W infected mice that were above background levels of staining detected in similar regions of NALT in the negative control GAS infected mice. For these tissue sections, <i>in situ</i> tetramer staining was done with TSA amplification.</p

Population of 2W:I-A^b tetramer+ cells detected <i>in situ</i> in NALT co-stained with CD3 but not IgG antibodies.

<p>(<i>A</i>) Representative image of a whole NALT section from a GAS-2W infected mouse stained with 2W:I-A^b tetramers (red) with one round of TSA amplification, CD3 antibodies (blue), and IgG antibodies (green). For this image, several confocal z-scan fields were collected using a 20X objective and reconstructed as a montage. The enlargement in (<i>B</i>) shows a 2W:I-A^b tetramer-binding T cell that is co-stained with CD3 antibodies but not IgG antibodies. The enlargement in (<i>C</i>) shows 2W:I-A^b tetramer-binding cells that are not co-stained with CD3 or IgG antibodies. (<i>B</i>) and (<i>C</i>) are confocal z-scans collected using the same parameters with a 60X objective and zoom of 3. 2W:I-A^b tetramer⁺CD3⁺IgG^- cells are indicated with arrowheads, and 2W:I-A^b tetramer⁺CD3^-IgG^- cells are indicated with arrows.</p

Percentage of 2W:I-A^b tetramer binding cells that counterstained with CD3 and CD4 antibodies.

<p>(<i>A</i>) Flow cytometry plots from the NALT of two GAS-2W infected mice showing the percentage of 2W:I-A^b tetramer-binding cells that were co-stained with CD4 antibodies. Cells were gated on all cells stained with 2W:I-A^b-ExtrAvidin−FITC tetramers. (<i>B</i>) Shows the average percentage of 2W:I-A^b binding cells from four mice that were co-stained with CD4, CD3, and IgG antibodies <i>in situ</i>, with standard deviations indicated.</p

Populations of 2W:I-A^b tetramer+ cells detected <i>in situ</i> in NALT co-stained with CD4 antibodies.

<p>(<i>A</i>) Representative image of a NALT section stained with 2W:I-A^b tetramers with no TSA amplification (red) and CD4 antibodies (blue). 2W:I-A^b tetramer⁺ cells that were co-labeled with CD4 are indicated by arrowheads. In this field, all of the tetramer⁺ cells are CD4⁺. For this image, several confocal z-scan fields were collected using a 20X objective and reconstructed as a montage. (<i>B</i>) Shows two 2W:I-A^b tetramer-binding CD4⁺ T cells (indicated by arrowheads). (<i>C</i>) Shows a 2W:I-A^b tetramer-binding cell (red) from another field that is not co-stained with CD4 antibodies. (<i>B</i>) and (<i>C</i>) are confocal z-scans collected using the same parameters with a 60X objective.</p

<i>In Situ</i> Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4⁺ T Cells in Tissues

<div><p>The invention of peptide-MHC-tetramer technology to label antigen-specific T cells has led to an enhanced understanding of T lymphocyte biology. Here we describe the development of an <i>in situ</i> pMHC-II tetramer staining method to visualize antigen-specific CD4⁺ T cells in tissues. This method complements other methods developed that similarly use MHC class II reagents to stain antigen-specific CD4⁺ T cells <i>in situ</i>. In this study, we used group A streptococcus (GAS) expressing a surrogate peptide (2W) to inoculate C57BL/6 mice, and used fresh nasal-associated lymphoid tissues (NALT) in optimizing the <i>in situ</i> staining of 2W:I-A^b specific CD4⁺ T cells. The results showed 2W:I-A^b tetramer-binding CD4⁺ T cells in GAS-2W but not GAS infected mice. This method holds promise to be broadly applicable to study the localization, abundance, and phenotype of antigen-specific CD4⁺ T cells in undisrupted tissues.</p></div

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 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

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

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

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