Immunology Module: Analyzing CD4 helper T cell differentiation

Objectives
Activities
Discussion
Questions

Introduction

During the course of normal immune responses, CD4 T cells can differentiate into different types of effector cells, namely Th1 and Th2. Th1 and Th2 cells secrete different cytokines, IFN-γ and TNF for Th1, and IL-4, IL-5, IL-13, and IL-10 for Th2. They have different effects on antigen-presenting cells, on B cells, and on pathogen clearance. They also cause different diseases, when dysregulated. For example, type 1 diabetes mellitus (also known as insulin-dependent diabetes mellitus, IDDM) and multiple sclerosis seem to depend on Th1 cells while asthma and allergy depend on Th2 cells to cause disease. Th1 and Th2 cells can regulate each other (reciprocal regulation).

Objectives: top

1. Understand CD4 T cell differentiation and Th1/Th2 paradigm.
2. Understand the mechanism behind the Enzyme-Linked Immunosorbent Assay (ELISA).
3. Go through ELISA steps.
4. Analyze Th1/Th2 differentiation with ELISA.

Activities: top

Finished steps by instructor
1. Coat 96 well plate with capture antibody over night;
2. Block the plate with bovine serum albumin for 2 hr;
3. Wash the plate;
4. Add culture media from Th1/Th2 cells and incubate for 2 hr;
5. Wash the plate;
6. Add detecting antibody and incubate for 2 hr;
7. Wash the plate;
8. Add avidin-HRP.

Start hands-on steps
1. Wash plate (3X)
2. Add substrate to develop
3. (While waiting, watch lung histology of asthma, a Th2 response induced disease)
4. Read plate on a plate reader and analyzing data

Layout of the sample plate

Discussion: top

1. Based upon the microenvironment and the nature of the antigens, activation of naïve CD4 helper T cells lead to differentiation into Th1 or Th2 effectors.

2. A fine balanced immune system protects us from infectious agents. Tipping the balance leads to various immune response caused diseases.

3. Th1 and Th2 cells can regulate each other.

4. How does ELISA work? Watch the following animation.

---Click to watch animated movie for ELISA---

Questions: top

1. How specific is sandwich ELISA? Why?
2. Why we need to block the plate after coating? What will happen if we do not block the plate?
3. Can we use the same antibody in both capture and detection steps? Why?
4. After infected by pathogens (for example viruses), an individual’s serum will contain antibodies against the pathogen even if the pathogen has been cleared. Can you design an ELISA to test whether an individual has been infected by a selected pathogen?
5. In our experimental setting, we only tested the existence of given cytokines (IL-4 vs IFN-γ) in the samples. Do you think we can use ELISA to quantify the level of cytokines in the samples? If yes, how would you do that?