In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Summary

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

Abstract

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled "Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8⁺ Cells In Vivo." The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

Introduction

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of peptide/MHC class I tetramer staining of antigen-specific CD8 T cells¹ and the more recent development of MHC class II tetramer staining of CD4 T cells² by flow cytometry revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry does....

Protocol

1. Day 1: Fresh Tissue Sectioning and Primary Incubation

1. Use a scalpel to cut fresh tissue into small (approximately 0.5 cm wide by 0.5 cm tall) pieces. Separately glue each tissue to a plunger and embed them with 3 - 5 mL of 4% low-melt agarose in PBS. Label the plunger with the tissue information using a sticker. Put it in a chilled holder in an ice bucket to solidify.
2. Turn on the microtome and set the thickness of the sections to 200 µm. Install a razor blade on the microtome and insert the plunger mounted with tissue in the microtome bath.
3. Prepare phosphate-buffered saline with heparin (PBS-H) by adding 100 µg/mL or 18.7 U....

Results

Figure 1 shows how to collect confocal images using a confocal microscope. Figure 2 demonstrates quantitative image analysis using ImageJ. Figures 3 and 4 show representative images of lymph node tissues from an SIV infected rhesus macaque stained with MHC tetramers, CD8 antibodies, and CD20 antibodies, and serve to demonstrate the specificity of the MHC-tetramer staining. Fi.......

Discussion

IST combined with IHC provides an essential tool for detecting, characterizing, and quantifying antigen-specific CD8 T cells in native environments with the context of other cells and tissue structures. Here, we described detailed procedures for IST combined with IHC, followed by quantitative image analysis, to determine the location, abundance, and phenotype of antigen-specific CD8 T cells in lymph nodes from rhesus macaques. Similar staining can be applied to human, mouse, or other species tissues for which MHC-I tetra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MHC-I monomer	NIH tetramer core facility		Materials for MHC-tetramer preparation
ExtrAvidin-FITC	Sigma-Aldrich	E2716	Materials for MHC-tetramer preparation
Normal goat serum	Jackson Immunoresearch	005-000-121
Low melt agarose	Promega	V3121
Heparin	Sigma-Aldrich	SLBL6391V
Triton X-100	Sigma-Aldrich	T-6878
Urea	J.T.Baker	4204-05
Glycerol gelatin	Sigma-Aldrich	SLBH2672V
n-propyl gallate	Sigma-Aldrich	P3130
rat-a-h-CD8 (1:500)	Acris	0714	Antibody unstable, use single use frozen aliquot
m-a-h-CD20 (1:500)	NOVOCASTRA	6026819
m-a-h-Ki67 (1:500)	Vector	6022201
goat-a-m-A488 (1:2,000)	Jackson Immunoresearch	124083
goat-a-rb-Cy3 (1:5,000)	Jackson Immunoresearch	106232
goat-a-rat-Cy5 (1:5,000)	Jackson Immunoresearch	118088
goat-a-h-IgM-Dylight649 (1:5,000)	Jackson Immunoresearch	86579
Compresstome: VF-300 Microtome	Precisionary Instruments, LLC	1079
Quick Set Instant Adhesive	Loctite	46551
24-well flat bottomed tissue culture plates	Falcon	353226
Microscope slide	Globe scienfitic Inc.	#1321
Razor  blade	Ted Pella, Inc	121-6
Feather Disposable Scalpel	FEATHER SAFETY RAZOR CO. LTD.	No. 21
Round paintbrush #2	PRINCETON ART & BRUSH CO.	4350R	Can trim as needed with razor
Confocal Microscope	Olympus	FV1000
FV10-ASW_Viewer4.0	Olympus