Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Summary

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

Abstract

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry. This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor. While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents. Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection. Mononuclear cells are purified from peripheral blood by Ficoll underlay. CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads. Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2. The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25. After labeling, the cells can be directly analyzed by flow cytometry. The tetramer positive cells typically form a distinct population among the expanded CD4+ cells. Tetramer positive cells are usually CD25+ and often CD4 high. Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer. Multiple variations of this basic assay are possible. Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays. Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.

Protocol

1. Peripheral blood mononuclear cell (PBMC) isolation

1. Obtain a blood sample – blood should be collected in syringes or blood tubes and anti-coagulated with heparin (1:50 ratio) to prevent clotting. Expect a yield of about 1×10⁶ PBMC per mL of blood – about 40% of which will be CD4 positive (CD4+) T cells.
2. Aliquot the blood into 50 mL conical tubes, 25 mL per tube. If blood has separated, gently mix before aliquoting to distribute the plasma
3. Add PBS, bringing the total volume to 40 mL and underlay by drawing up 11 mL of Ficoll, inserting into blood tube, and carefully removing the pipet Aid from the pipette. The ficoll will sl....

Discussion

Understanding the role of CD4+ T cells in immunity is fundamentally important. However, antigen specific CD4+ T cells can be difficult to detect and isolate using traditional methods. In contrast, MHC class II tetramers allow the direct visualization of CD4+ T cells with the desired antigen specificity. This video demonstrates the isolation, purification, and in vitro amplification of CD4+ T cells and their subsequent visualization using tetramers. Class II Tetramers are fluorescent protein conjugates consisting of solub.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Class 2 Laminar flow biosafety cabinet	equipment	Thermo Fisher Scientific, Inc.	1200	Or equivalent
50 mL conical Tube	equipment	VWR international	47747-182	Or equivalent
1X PBS (Ca/Mg free)	Reagent	Hyclone	SH30028.02	Or equivalent
Ficoll-paque PLUS	Reagent	GE Healthcare	17-1440-03	foil wrapped to protect from light
Pipet Aid XP	equipment	Drummond Scientific	4-000-101	Or equivalent
10 mL pipet	equipment	Corning	CLS4492	Or equivalent
Aerosolve Canisters	equipment	Beckman Coulter Inc.	359481	with 50 mL inserts
Centrifuge	equipment	Beckman Coulter Inc.	GS-6R	Or equivalent
Transfer pipets	equipment	Samco Scientific, Thermo Fisher Scientific	202-20S	Or equivalent
Pasteur pipets	equipment	VWR international	14672-200	Or equivalent
Hemolytic Buffer	Reagent	N/A	N/A	8.3 g/L NH4Cl, 1.0 g/L NaHCO3, 0.04 g/L disodium EDTA
Trypan blue	Reagent	Sigma-Aldrich	T6146	0.2% in PBS
Hemocytometer	equipment	VWR international	15170-208
15 mL conical tube	equipment	VWR international	05-527-90	Or equivalent
Running Buffer	Reagent	N/A	N/A	PBS + 2mM EDTA + 5g/L BSA
MACS CD4+ T Cell Isolation Kit II	Reagent	Miltenyi Biotec	130-091-155	Or equivalent
EasySep® Magnet	equipment	Stem Cell Technologies	18000
5 mL polypropylene tube	equipment	Falcon BD	352063
RPMI 1640	Reagent	Invitrogen	22400-089	with 25 mM HEPES
Pooled human serum	Reagent	N/A	N/A	drawn from healthy donors, heat inactivated and filtered
Pen Strep	Reagent	Invitrogen	1570-063	Or equivalent
500mL 0.22 micron bottle top filter	equipment	Nalge Nunc international	595-3320	Or equivalent
48-well plate	equipment	Costar	3548	Or equivalent
37°C CO2 incubator	equipment	Sanyo	MCO-18AIC	Or equivalent
20 mg/mL synthetic peptide	Reagent	N/A	N/A	Custom synthesis from any peptide vendor
5 mL FACS tube	equipment	Falcon BD	352008	Polystyrene
Peptide loaded class II tetramer (as described)	Reagent	N/A	N/A	Prepared by the BRI tetramer core
Anti-CD3	Reagent	BD Biosciences	347344	Or equivalent
Anti-CD4	Reagent	eBioscience	17-0049-73	Or equivalent
Anti-CD25	Reagent	eBioscience	11-0259-73	Or equivalent
Facs Calibur Flow Cytometer	equipment	BD Biosciences	342975	Or equivalent