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.
1. Peripheral blood mononuclear cell (PBMC) isolation
2. CD4+ T cell separation*
* Alternatively, MACS columns and beads (Miltenyi Biotec), the AutoMACS cell separator (Miltenyi Biotec), or Robosep cell separator (Stem Cell Technologies) can be used according to manufactures instructions in place of steps 2.2 – 2.11.
3. In vitro Expansion culture
4. Visualizing T cells by tetramer staining
5. Flow Cytometer Acquisition and Analysis
Figure 1. Representative negative control tetramer staining results. The upper left panel shows forward scatter verses side scatter and the lymphocyte size gate (R1). The upper right panel is gated for R1 and shows ant-CD4 versus anti-CD3 and the CD3+ (R2) and CD4+ (R3) gates. The lower left panel is gated for R2 and shows anti-CD4 versus tetramer. The lower right panel is gated for R3 and shows anti-CD25 versus tetramer. The quadrants for both tetramer plots were set to 0.5%.
6. Representative Results
Figure 2. Representative positive tetramer staining results. The panel layout and gating strategy are identical to Figure 1. The tetramer positive cells appear as a distinct CD4 high population on the anti-CD4 versus tetramer panel and a distinct CD25+ population on the anti-CD25 versus tetramer panel.
Figure 3. Representative “false positive” tetramer staining results. The panel layout and gating strategy are identical to Figure 1. The anti-CD4 versus tetramer panel shows an indistinct “mounded” staining. The anti-CD25 versus tetramer also shows a “mounded” staining, with no clear trend toward being CD25+.
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 soluble biotinylated class II α/β dimers conjugated around a fluorescent labeled streptavidin core (Figure 4). The tetramers used in this video were produced by the Teramer Core Laboratory at the Benaroya Research Institute using insect cell cultures (1). These tetramers are prepared as a 0.5 mg/mL solution and can be stored at 4°C for 6-18 months. Tetramers should never be frozen, as freeze-thaw stresses may strip the PE label away from the steptavidin core. Class II staining reagents can be obtained from several sources (Table 1) and are designated as tetramers, “ultimers”, and multimers. It should be noted that the tetramer assay procedure described here is distinct from the procedures used for class I tetramers. Class I tetramer staining is usually optimal at 4°C, while class II tetramer staining generally requires 37°C. Class I tetramers bind more tightly to T cells than Class II tetramers because of the stronger cooperative effect of CD8 as compared to CD4. The tetramer assay described here is effective in visualizing T cell specific for either foreign or self antigens, but the strength of interaction is inherently lower for self antigens. Several variations of the assay are possible. For example, CD4+ T cells can be further fractionated prior to stimulation (e.g. into memory and naïve populations), stimulated using a whole protein or peptide pools (2), or various treatments may be added to different wells to measure their influence on the antigen specific response. Since only a portion of each well is needed for tetramer analysis, the remaining cells can stained with tetramer for sorting or reserved for other purposes. The setup for each individual experiment will be dictated both by the characteristics of the sample and by the scientific question being asked. For design purposes it is critical to know the HLA type of the blood donor because this will influence the epitopes used for expansion and will dictate the corresponding tetramer that must be used. Knowledge of the disease or immunization status may also be crucial for interpreting results. It is important to carefully maintain the cells during the amplification step, since unhealthy cells can give very poor results. Also, proper machine setup, gating and negative controls are vitally important for obtaining high quality flow cytometry results.
Figure 4. Schematic of MHC class II tetramer. The red core represents the streptavidin-PE molecule. The green and black extensions represent the MHC class II α/β dimers. The complex is joined by the high affinity interaction between biotin and streptavidin.
Table 1: Various sources of class II staining reagents
Tetramer Source | Product Type | Web Address |
Beckman Coulter | Tetramers | www.beckman.com |
NIH Tetramer Facility | Tetramers | www.research.yerkes.emory.edu/tetramer_core |
Proimmune | Ultimers | www.proimmune.com |
Benaroya Research Institute | Multimers | www.benaroyaresearch.org |
We thank Dick Foley and Leigh Kimball for their key roles in filming and video production.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Class 2 Laminar flow biosafety cabinet | equipment | Thermo Forma | 1200 | Or equivalent |
50 mL conical Tube | equipment | VWR | 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 | 4-000-101 | Or equivalent |
10 mL pipet | equipment | Corning/Costar | CLS4492 | Or equivalent |
Aerosolve Canisters | equipment | Beckman | 359481 | with 50 mL inserts |
Centrifuge | equipment | Beckman | GS-6R | Or equivalent |
Transfer pipets | equipment | Samco | 202-20S | Or equivalent |
Pasteur pipets | equipment | VWR | 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 | T6146 | 0.2% in PBS |
Hemocytometer | equipment | VWR | 15170-208 | |
15 mL conical tube | equipment | VWR | 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 | 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 | Nalgene | 595-3320 | Or equivalent |
48-well plate | equipment | Costar | 3548 | Or equivalent |
37°C CO2 incubator | equipment | Sanyo Scientific | 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 | 352008 | Polystyrene |
Peptide loaded class II tetramer (as described) | Reagent | N/A | N/A | Prepared by the BRI tetramer core |
Anti-CD3 | Reagent | BD pharmingen | 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 |