Limits...
In Situ Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4+ T Cells in Tissues.

Dileepan T, Kim HO, Cleary PP, Skinner PJ - PLoS ONE (2015)

Bottom Line: This method complements other methods developed that similarly use MHC class II reagents to stain antigen-specific CD4+ T cells in situ.The results showed 2W:I-Ab 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Minnesota, Minneapolis, MN, United States of America.

ABSTRACT
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 in situ 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 in situ. 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 in situ staining of 2W:I-Ab specific CD4+ T cells. The results showed 2W:I-Ab 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.

No MeSH data available.


Related in: MedlinePlus

Population of 2W:I-Ab tetramer+ cells detected in situ in NALT co-stained with CD3 but not IgG antibodies.(A) Representative image of a whole NALT section from a GAS-2W infected mouse stained with 2W:I-Ab 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 (B) shows a 2W:I-Ab tetramer-binding T cell that is co-stained with CD3 antibodies but not IgG antibodies. The enlargement in (C) shows 2W:I-Ab tetramer-binding cells that are not co-stained with CD3 or IgG antibodies. (B) and (C) are confocal z-scans collected using the same parameters with a 60X objective and zoom of 3. 2W:I-Ab tetramer+CD3+IgG- cells are indicated with arrowheads, and 2W:I-Ab tetramer+CD3-IgG- cells are indicated with arrows.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4465905&req=5

pone.0128862.g003: Population of 2W:I-Ab tetramer+ cells detected in situ in NALT co-stained with CD3 but not IgG antibodies.(A) Representative image of a whole NALT section from a GAS-2W infected mouse stained with 2W:I-Ab 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 (B) shows a 2W:I-Ab tetramer-binding T cell that is co-stained with CD3 antibodies but not IgG antibodies. The enlargement in (C) shows 2W:I-Ab tetramer-binding cells that are not co-stained with CD3 or IgG antibodies. (B) and (C) are confocal z-scans collected using the same parameters with a 60X objective and zoom of 3. 2W:I-Ab tetramer+CD3+IgG- cells are indicated with arrowheads, and 2W:I-Ab tetramer+CD3-IgG- cells are indicated with arrows.

Mentions: In order to differentiate tetramer+ antigen-specific CD4+ T cells from other cells that bind tetramers, we co-stained tissue sections with anti-CD4 or anti-CD3 antibodies to label T cells and anti-IgG antibodies to label IgG expressing B cells and plasma cells. 2W:I-Ab tetramer staining combined with anti-CD4 staining revealed populations of 2W:I-Ab tetramer+ cells co-stained with anti-CD4 antibodies distributed amongst other CD4+ cells in NALT tissues from GAS-2W infected mice (Fig 2). In addition, many non-CD4 cells were also 2W:I-Ab tetramer+ but these were easily distinguishable from antigen-specific tetramer+CD4+ T cells through CD4 co-staining (Fig 2 panels B and C). Similar 2W:I-Ab tetramer+ CD4- cells were also detected in negative control animals (S1 Fig). We have historically found that CD3 antibody staining is generally superior in quality and more reliable than CD4 staining of T cells in tissue sections. Given this, we also stained sections with CD3 antibodies. Since class II tetramers do not bind CD8 T cells [1] we reasoned that CD3 antibodies are a good alternative to CD4 antibodies to identify antigen-specific tetramer+ CD4+ T cells. Fig 3A shows an image of a whole NALT section stained with 2W:I-Ab tetramers and counterstained with anti-CD3 and anti-IgG antibodies. Fig 3B shows a representative 2W:I-Ab-binding T cell that was co-stained CD3 antibodies. Fig 3C shows representative tetramer-binding cells that were not co-stained with CD3.


In Situ Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4+ T Cells in Tissues.

Dileepan T, Kim HO, Cleary PP, Skinner PJ - PLoS ONE (2015)

Population of 2W:I-Ab tetramer+ cells detected in situ in NALT co-stained with CD3 but not IgG antibodies.(A) Representative image of a whole NALT section from a GAS-2W infected mouse stained with 2W:I-Ab 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 (B) shows a 2W:I-Ab tetramer-binding T cell that is co-stained with CD3 antibodies but not IgG antibodies. The enlargement in (C) shows 2W:I-Ab tetramer-binding cells that are not co-stained with CD3 or IgG antibodies. (B) and (C) are confocal z-scans collected using the same parameters with a 60X objective and zoom of 3. 2W:I-Ab tetramer+CD3+IgG- cells are indicated with arrowheads, and 2W:I-Ab tetramer+CD3-IgG- cells are indicated with arrows.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465905&req=5

pone.0128862.g003: Population of 2W:I-Ab tetramer+ cells detected in situ in NALT co-stained with CD3 but not IgG antibodies.(A) Representative image of a whole NALT section from a GAS-2W infected mouse stained with 2W:I-Ab 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 (B) shows a 2W:I-Ab tetramer-binding T cell that is co-stained with CD3 antibodies but not IgG antibodies. The enlargement in (C) shows 2W:I-Ab tetramer-binding cells that are not co-stained with CD3 or IgG antibodies. (B) and (C) are confocal z-scans collected using the same parameters with a 60X objective and zoom of 3. 2W:I-Ab tetramer+CD3+IgG- cells are indicated with arrowheads, and 2W:I-Ab tetramer+CD3-IgG- cells are indicated with arrows.
Mentions: In order to differentiate tetramer+ antigen-specific CD4+ T cells from other cells that bind tetramers, we co-stained tissue sections with anti-CD4 or anti-CD3 antibodies to label T cells and anti-IgG antibodies to label IgG expressing B cells and plasma cells. 2W:I-Ab tetramer staining combined with anti-CD4 staining revealed populations of 2W:I-Ab tetramer+ cells co-stained with anti-CD4 antibodies distributed amongst other CD4+ cells in NALT tissues from GAS-2W infected mice (Fig 2). In addition, many non-CD4 cells were also 2W:I-Ab tetramer+ but these were easily distinguishable from antigen-specific tetramer+CD4+ T cells through CD4 co-staining (Fig 2 panels B and C). Similar 2W:I-Ab tetramer+ CD4- cells were also detected in negative control animals (S1 Fig). We have historically found that CD3 antibody staining is generally superior in quality and more reliable than CD4 staining of T cells in tissue sections. Given this, we also stained sections with CD3 antibodies. Since class II tetramers do not bind CD8 T cells [1] we reasoned that CD3 antibodies are a good alternative to CD4 antibodies to identify antigen-specific tetramer+ CD4+ T cells. Fig 3A shows an image of a whole NALT section stained with 2W:I-Ab tetramers and counterstained with anti-CD3 and anti-IgG antibodies. Fig 3B shows a representative 2W:I-Ab-binding T cell that was co-stained CD3 antibodies. Fig 3C shows representative tetramer-binding cells that were not co-stained with CD3.

Bottom Line: This method complements other methods developed that similarly use MHC class II reagents to stain antigen-specific CD4+ T cells in situ.The results showed 2W:I-Ab 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Minnesota, Minneapolis, MN, United States of America.

ABSTRACT
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 in situ 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 in situ. 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 in situ staining of 2W:I-Ab specific CD4+ T cells. The results showed 2W:I-Ab 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.

No MeSH data available.


Related in: MedlinePlus