Reading Assignment
Review the white blood cells and organs of the immune system (Janeway pp 2-12; Parham pp. 10-20)
Read about CD Antigens in the Antigen tutorial on the 419 web site; Janeway Appendix II. Read about Flow Cytometry in the ToolBox; Janeway Figure A.25, Parham Figure 2.13. Read about Antigen Binding in the Toolbox.
Writing Assignment
All lymphocytes look the same under the light microscope, yet cytotoxic T cells, helper T cells, and B cells have very different functions and activated macrophages behave differently than non-activated macrophages. Stem cells have little function, but can be stimulated with growth factors that bind to membrane receptors to become mature cells. Immunologists use the presence of membrane proteins (called "markers") to differentiate distinct functional cell types. These proteins are often receptors for antigens, cytokines, or adhesion molecules.
The purpose of this assignment is to help you learn some surface markers on leukocytes and how they can be detected by flow cytometry. Cells can be phenotyped (their functions identified) by their membrane molecules ("markers"). In addition, flow cytometry is a useful technique for detecting T cell antigen binding and cytokine production.
Draw circles for a B cell, a Th cell, a Tc cell, a macrophage, a neutrophil (PMN), an NK cell, and a dendritic cell (DC) and label each with its markers from this list: Ig μ chain, Ig δ chain, CD79a, CD19, CD3, CD4, CD8, CD16, CD56, CD64, CD209, Class I MHC, and Class II MHC. (This part of AT4 may be hand drawn and need not go to turnitin.com).
Flow cytometry can also be used to count lymphocytes specific for a given antigen. B cells bind antigen directly to their BCR, so you could add fluorescent-tagged antigen to a population of lymphocytes (or purified B cells), wash off the antigen that did not bind, and count the frequency of antigen-binding cells for that antigen. In a naïve (non-immunized) individual, the frequency of specific antigen-binding cells for tetanus toxoid would be about 1/105 B cells. In someone who had recently received a tetanus booster, the frequency of tetanus toxoid-binding B cells would closer to 1/103 or perhaps even 1/102 B cells. The total number of B cells in an immunized person would not be measurably higher than in a naïve person.
T cells do not bind antigen directly; to measure antigen-binding T cells, you need to use tetramer MHC-peptide antigen (see Toolbox Antigen Binding). Frequencies of antigen-binding T cells using this assay are comparable to frequencies of antigen-binding B cells.
Antibodies to cytokines cannot pass through the plasma membrane, so traditional flow detects membrane markers. Activated (effector) T cells secrete cytokines that can be measured in the ELISA and ELISPOT tests (review AT3). These techniques require cell culture for cytokine to be secreted. Flow cytometry can be used to detect cytokine-producing T cells while simultaneously measuring cell markers. Cytokines are synthesized inside cells, so the cells must be permeabilized to allow anti-cytokine antibody to enter the cell. The population of stimulated T cells would be incubated in a secretion inhibitor (brefeldin A) to allow cytokine to accumulate inside the cell. Cells would then be fixed (their proteins cross-linked) with formalin and holes made in the lipid membranes with detergent. Finally, the cells would be incubated with fluorochrome-tagged antibodies to specific cytokines and analyzed by flow cytometry.
Questions to answer and turn in:
1. You want to know what the frequency of H5-binding B cells is in someone you have just immunized with H5 Influenza.
2. You want to know what frequency of H5-specific cytotoxic T cells are present in the immunized person.
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