Describe what the data show and provide some discussion of how each of the costimulatory ligands (B7.1, B7.2 and CTLA-4) influence T cell activation. Suggest other experiments that might be used to examine T cell activation.

The effects of costimulatory ligands on T cell activation.

Introduction

For full T cell activation, both CD4⁺ and CD8⁺ effector T cells require two main interactions with antigen presenting cells. The first interaction is the “cognate” interaction, often referred to as signal 1. This is the interaction between the T cell receptor and the MHC complex presenting a peptide that is capable of being recognised by and consequently activating the T cell. This is the basis of the adaptive immune system involving T cells – only a small subset of T cell clones will be capable of responding to a particular peptide antigen presented by an MHC molecule.

Although signal 1 is crucial to a productive antigen-specific T cell response, for full activation T cells must receive a costimulatory ligand, which will allow full activation to take place. This second interaction, often called signal 2 is brought about by interaction between the CD28 receptor on T cells and two ligands called B7.2 (CD86) and B7.1 (CD80) on the antigen presenting cell. B7.2 is constitutively presented on APC such as dendritic cells, whereas B7.1, which has higher affinity for CD28 is upregulated when the APC is activated, e.g., by Toll-like receptor ligands binding a bacterial element as a sign of infection.

Prolonged, uncontrolled activation of T cells can be dangerous to the host and so on activated T cells another receptor, CTLA-4 (CD152), which shares homology with CD28 and also binds B7.1 and B7.2, is upregulated. This second receptor on T cells is inhibitory and it regulates the activation status of the T cell to prevent the T cell from causing damage to host tissues.

The aim of this practical is evaluate the differences in T cell activation status following costimulation with B7.1 and B7.2 ligands and to determine whether blocking their engagement with CD28 will suppress T cell activity. As a measure of T cell activity we will examine IL-2 production by ELISA. We will use the Jurkat leukaemia T cell line for the investigation. Jurkat cells are commonly used in laboratories to investigate many aspects of T cell activity. While they express CD3, a T cell marker, they do not express CD4 (T helper) or CD8 (Cytotoxic) T cell markers. Jurkat cells are not specific for a particular antigen but they can receive “signal 1” by stimulating them with anti-CD3 antibody. This non-specifically activates the CD3 T cell receptor signalling complex – in effect, it’s a kind of short circuit that can be used for any T cell population and is widely used by Immunology research laboratories.

Learning outcomes

1. Ability to design and develop an immune cell assay to investigate T cell activation
2. Analyse and evaluate the results
3. Critical appraisal of data to support research paper writing

Diary

Day 1. (A) Set up the assay and (B) prepare the first step of the ELISA

Day 2. Perform the ELISA to analyse Jurkat T cell responses

Day3. Finish the ELISA, collect data and analyse your result

Reagents

What you are provided with:

1. Jurkat cells
2. A 48 well plate (each well holds 1 mL of cell culture) coated with anti-CD3 antibody (1 mg/mL)
3. Reagents
   7. Recombinant B7.1-Ig
   8. Recombinant B7.2-Ig
   9. Recombinant CTLA4-Ig
4. IL-2 ELISA kit reagents
5. Protocol for setting up the assay (Day 1)
6. You are provided with Jurkat cells, which you will need to add to wells of a 48 well plate. After you have added the Jurkat cells, you will need to add the B7.1/B7.2 and CTLA4-Ig reagents (the anti-CD3 antibody has already been added to the appropriate wells of each plate by us).
7. Add 1 ml containing 5 ´ 10⁵ Jurkat cells to each of the 10 wells of a 48 well plate (see cell culture 48 well plate plan).
8. Follow the plate plan to add each of your reagents to the wells
   1. Each B7 reagent is at a concentration of 0.1 mg/mL (10 mL = 1 mg), while CTLA4-Ig is at 0.2 mg/mL (10 mL = 2 mg).
9. Incubate overnight at 37°C, 5% CO₂.
10. Begin analysis of the samples for IL-2 production by ELISA (Biolegend – Human IL-2 ELISA Max deluxe set, cat. 431804).
11. Protocol for the IL-2 ELISA

For the IL-2 ELISA – coat Nunc Maxisorp plates with capture antibody on the day of the assay – incubate overnight in fridge.

1. Day 1: Coat the wells to be used in Columns 1 to 8 of a Nunc Maxisorp ELISA plate (see ELISA 96 well plate plan) with 100 µL/well of capture antibody in Coating Buffer. Cover with plate lid and incubate overnight at 4°C.
2. Day2: Aspirate wells and wash 2 times with PBS Wash Buffer. Blot plate on absorbent paper to remove any residual buffer.
3. Block wells with 100 µL of ELISA blocking Diluent. Incubate at room temperature (RT) for 1 hour.
4. Wash the plate twice with PBS wash buffer containing 0.05% Tween 20 detergent (PBS-T which is added at this stage to prevent non-specific binding).
5. Prepare the standard curve, which will be placed into columns 1 and 2 of the plate with H1 and H2 as blank control wells (see ELISA 96 well plate plan).
   1. Begin by aliquoting 100 mL of ELISA diluent into each well of columns 1 and 2 except wells A1 and A2 (leave empty)
   2. Next aliquot 200 mL of your Standard (provided at 250 pg/ml) into wells A1 and A2
   3. Moving down in each column, remove 100 mL from well A1 and transfer to well B1, gently pipette up and down three times and then remove 100 mL from well B1 and transfer into well C1 and so on until well G1. Repeat for column two starting with well A2.
   4. Discard 100 mL from wells G1 and G2
   5. Leave wells H1 and H2 alone as negative controls (i.e., they simply contain ELISA diluent and no standard.
6. Now add 100 mL of your first test sample to wells A3, A4 and A5. Use the ELISA plate plan below and ensure for each test sample that you add each test sample in triplicate.
7. Incubate the plate overnight at 4˚
8. Day3: Wash the plate three times with PBS-T.
9. Add 100 mL of Detection antibody to the used wells in columns 1 to 8 of the plate and incubate for 1 hour at RT.
10. Wash the plate four times with PBS-T.
11. Add 100 mL of streptavidin-HRP to test wells in columns 1 to 8 of the plate and incubate for 30 minutes at RT.
12. Wash the plate five times with PBS-T.
13. Add 100 mL of TMB substrate solution to test wells in columns 1 to 8 of the plate and incubate at RT in dark. Please note that this colour reaction can be quite fast. Incubate for up to 15 minutes in dark, but check after five minutes to determine whether or not the reaction should be stopped.
14. Stop the reaction by adding 50 mL of 2N H₂SO₄ (STOP).
15. Read the plate using a multiwell spectrophotomer at a set wavelength of 450 nm with a second “subtraction” reading at 595 nm.

Write up plan:

This is an essay-type assignment so we require you to introduce, present and interpret and discuss your data in the format you would see in a published research article. (An example article is Yang et al., 2018, Immunology 155, 263-272 but clearly this has a lot more results than you have.) This needs to be submitted in a word document/PDF via MyAberdeen (max word count 2000).

1. Provide an abstract (summary) and an introduction with some background on T cell activation and the need for costimulation. Also include a section on why IL-2 is a useful cytokine to measure as a marker of T cell activation.
2. Write out the methods as you would see written in a research paper.
3. Results: Construct a standard curve using 4 cycle semi-log graph paper (provided). Use the X axis for IL-2 concentration (pg/mL) and the Y axis for the spectrophotometer values (label the axis OD₄₅₀ nm).
4. Use the standard curve to calculate how much IL-2 has been produced in each of your treatment samples. You can combine replicate samples to provide a mean and SEM for each sample.
5. Plot the data as a bar chart so that each treatment can be compared.
6. Describe what the data show and provide some discussion of how each of the costimulatory ligands (B7.1, B7.2 and CTLA-4) influence T cell activation. Suggest other experiments that might be used to examine T cell activation.
7. Provide a references section