Paul's problems with respiratory infections began when he was about 2 years old. He was treated repeatedly with antibiotics for middle ear infections and viral pneumonia. He had been a healthy infant and had had no problems with childhood immunizations to polio virus (Salk vaccine = killed polio virus), DPT (diphtheria toxoid, Bordetella pertussis acellular vaccine, and tetanus toxoid), and HiB (Haemophilus influenza B capsule antigen). When he was hospitalized for yet another bout with viral pneumonia, his pediatrician performed some tests to assess the state of his immune system.
Paul's serum IgG and lymphocyte count were slightly elevated. He had circulating IgG antibodies to DPT antigens and to polio virus. His circulating T lymphocytes were 95% CD4+, compared to a normal ratio of 2:1 CD4+:CD8+ circulating T cells. His B cell and neutrophil counts and his complement functions were normal. His cellular immunity, measured in a skin test to poison ivy, to which he had had a skin rash six months previously, was also normal. This test measures memory Th1 cells. However, levels of MHC Class I on his white blood cells were reduced by about 90%.
Cell lines produced from Paul's peripheral blood T cells had normal levels of mRNA for MHC class I α chain molecules and β2M. However, no mRNA was found for TAP-1. Without TAP-1, Paul's cells could not express MHC Class I on their plasma membranes. Without membrane MHC Class I, development of CD8+ Tc could not occur in Paul's thymus. MHC Class II was present and CD4+ T cells developed properly. Th1 inflammatory cells and Th2 helper cells were able to provide normal immunity against bacterial and fungal (exogenous) antigens and contact antigens like poison ivy.
Figure 1: Functions of T lymphocytes.
MIC 419 Case 5 Questions for In-Class Discussion
Wednesday 2/7 TCR and MHC Structure and Antigen Processing
1. What kinds of vaccines had Paul received? Would these vaccines induce humoral (antibodies) or cellular (Tc and/or Th1) functions? (HINT: To induce cellular immunity, the vaccine must contain live pathogen.]
2. Draw and label an ab TCR molecule. Compare and contrast the structures of TCR and BCR. Compare and contrast antigen recognition by TCR and BCR.
3. Discuss the functions of CD3, CD4, and CD8 on T lymphocytes.
Figure 2. T Cell immunological synapse: binding interactions between CD8 T cells and target (virus-infected) cells and between CD4 T cells and professional APC.
4. Draw and label MHC Class I and MHC Class II molecules. To what chains in each molecule does antigen peptide bind?
5. What cells normally
6. Describe antigen processing and presentation on MHC Class I. Where does the antigen protein originate? Where is it cut into peptides? Where are the peptides combined with MHC I? What other molecules are required for this process and what are their functions?
7. Describe antigen processing and presentation on MHC Class II. Where does the antigen protein originate? Where is it cut into peptides? Where are the peptides combined with MHC I? What other molecules are required for this process and what are their functions?
Figure 3. Antigen processing and presentation pathways for exogenous (endocytosed) and endogenous (cytosolic) antigens.
8. What limits the binding of peptides to the Class I MHC peptide-binding site? to the class II MHC peptide-binding site? to TCR? Which molecule(s) have the most specific (most limited) peptide binding?
Figure 4. Comparison of the peptide-binding sites of MHC Class I and Class II.
9. How does the lack of TAP interfere with Class I MHC expression on Paul's cells? Why was Class II MHC expression on Paul's cells normal?
Friday 2/9 T Cell Development
Figure 5. Somatic recombination of TCR beta chain and expression with pre T alpha chain in pre T cells.
10. Compare TCR and BCR generation of diversity.
Figure 6. Positive selection of T cells. Inbred mice homozygous for Class I MHCa or MHCb are irradiated to kill hematopoietic cells and then bone marrow cells from a heterozygous MHC axb mouse are adoptively transferred into the irradiated recipients. The bar graphs show the MHC alleles expressed by the mice following adoptive transfer.
11. What is the MHC on the thymus epithelial cells of each mouse above? What is the MHC on the T cells of each mouse? What MHC do the T cells of each mouse recognize as "self"?
12. Describe how a T cell becomes committed to be a Th or a Tc and how this is related to positive selection.
13. Why did Paul have normal-high numbers of CD4+ T cells but low numbers of CD8+ T cells?
Figure 7. Negative selection of T cells. Bone marrow from an MHCaxb mouse is transferred into an irradiated MHCa mouse (called a chimera because it has cells from two different individuals). Subsequently, MHCb skin is grafted onto that mouse; the MHCa mouse containing the MHCaxb bone marrow cells does not reject the MHC skin.
14. What is the MHC on the thymus epithelial cells of each mouse above? What is the MHC on the T cells of each mouse? What MHC do the T cells of each mouse recognize as "self"?
13.Describe the limitations on negative selection that could result in autoimmunity.
14. How could Paul make IgG antibodies, which require T cell help, but not be able to respond to viruses? How did the polio virus vaccination protect Paul from polio virus if he has no CD8 T cells?
Monday 2/12 MHC Genetics
Figure 8. MHC Genes.
Figure 9. Numbers of MHC alleles that have been identified world-wide.
15. MIC Class I HLA A, B, and C and MHC Class II HLA DP, DQ and DR are cell membrane molecules. How many alleles of Class I and Class II MHC molecules are on one of your liver cells? on one of your B cells?
16. How can our T cells respond to so many different peptides when we have so few MHC alleles to present the peptides? Do you think people with different MHC alleles respond equally well to all antigens? Why or why not?
17. Mice and humans tend to select mates with different MHC than their own; why is this beneficial (and to whom)?
18. Why are tissue matches more likely between closely related people? less likely between people of different ethnic backgrounds?
Flow cytometry is a technique used to quantify circulating leukocytes based on their size, granularity, and cell surface markers (see Toolbox Flow Cytometry). In the diagram at right below, forward and side scatter characteristics have been used to identify lymphocytes, monocytes and granulocytes (each dot represents one cell).
Figure 10. Flow cytometry. Left is a flow cytometer; right is a diagram showing cell separation by size and granularity, used to identify different WBC types.
Cells labeled with fluorochrome-tagged antibodies to cell surface markers can be counted and the amount of marker on their surfaces measured.
Figure 11. The diagram above left shows data for CD8+ cells in normal peripheral blood. The horizontal axis shows fluorescence intensity on a log scale; the vertical axis shows numbers of cells. An electronic "gate" is set indicating the fluorescence intensity of cells considered positive for CD8, in this case 38% of the blood lymphocytes. [Other gates are set to exclude granulocytes and monocytes from being counted.] The diagram at right below shows two-color fluorescence of normal blood lymphocytes, where anti-CD3 is tagged with PE (phycoerythrin, red) and anti-CD8 is tagged with FITC (fluorescein isothiocyanate, green). The gates are shown by the horizontal and vertical lines, and the % of cells in each box is also indicated. [Ignore the fact that the dots are red; this has nothing to do with their fluorescence color.]
19. Redraw the two diagrams in Figure 3 as they would appear for Paul's blood lymphocytes. Draw another 2-color flow diagram for Paul's cells substituting CD4 FITC for CD8 FITC.
20. What other protein deficiencies would result in the same phenotype as the lack of TAP? What genetic deficiencies would prevent Paul from producing Class II MHC but allow him to produce Class I MHC in normal amounts?