You Have Labelled Red Fluorescence Anti-cd4 Antibodies And Labelled Green Fluorescence Anti-cd8 Antibodies. You Use These Antibodies To Stain Thymocytes And Lymph Node Cells From Normal Mice And Rag-1 Knockout Mice. In The Forms Below Indicate The Facs Pl

Ashton-Rickardt, P. G., A. Bandeira, J. R. Delaney, L. Van Kaer, H. P. Pircher, R. M. Zinkernagel, and S. Tonegawa. 1994. Evidence for a differential avidity model of T-cell selection in the thymus. Cell 74:577.

Drappa, M. D., A. K. Vaishnaw, K. E. Sullivan, B. S. Chu, and K. B. Elkon. 1996. Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity. New England Journal of Medicine 335:1643.

Dutton, R. W., L. M. Bradley, and S. L. Swain. 1998. T-cell memory. Annu. Rev. Immunol. 16:201.

Ellmeier, W., S. Sawada, and D. R. Littman. 1999. The regulation of CD4 and CD8 coreceptor gene expression during T-cell development. Annu. Rev. Immunol. 17:523.

Hayday, A. 2000.78 Cells: A right time and right place for a conserved third way of protection. Annu. Rev. Immunol. 18:1975.

Herman, A., J. W. Kappler, P. Marrack, and A. M. Pullen. 1991. Superantigens: mechanism of T-cell stimulation and role in immune responses. Annu. Rev. Immunol. 9:745.

Lanzavecchia, A., G. Lezzi, and A. Viola. 1999. From TCR engagement to T-cell activation: a kinetic view of T-cell behavior. Cell 96:1.

Myung, P. S., N. J. Boerthe, and G. A. Koretzky. 2000. Adapter proteins in lymphocyte antigen-receptor signaling. Curr. Opin. Immunol. 12:256.

Osborne, B. A. 1996. Apoptosis and maintenance of homeostasis in the immune system. Curr. Opin. Immunol. 8:245.

Osborne, B., A. 2000. Transcriptional control of T-cell development. Curr. Opin. Immunol. 12:301.

Owen, J. J. T., and N. C. Moore. 1995. Thymocyte-stromal-cell interactions and T-cell selection. Immunol. Today 16:336.

Salomon, B., and J. A. Bluestone. 2001. Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu. Rev. Immunol. 19:225.

Schreiber, S. L., and G. R. Crabtree. 1992. The mechanism of action of cyclosporin A and FK506. Immunol. Today 13:136.

Thompson, C. B. and J. C. Rathmell. 1999. The central effectors of cell death in the immune system. Annu. Rev. Immunol. 17:781.

Vaishnaw, A. K., J. R. Orlinick, J. L. Chu, P. H. Krammer, M. V. Chao, and K. B. Elkon. 1999. The molecular basis for apoptotic defects in patients with CD95 (Fas/Apo-1) mutations. Journal of Clinical Investigation 103:355.

USEFUL WEB SITES

http://www.ncbi.nlm.nih.gov/Omim/

http://www.ncbi.nlm.nih.gov/htbinpost/Omim/getmim

The Online Mendelian Inheritance in Man Web site contains a subsite that features ten different inherited diseases associated with defects in the TCR complex or associated proteins.

http://www.ultranet.eom/~jkimball/BiologyPages/A/

Apoptosis.html http://www.ultranet.eom/~jkimball/BiologyPages/B/ B_and_Tcells.html

These subsites of John Kimball's Biology Pages Web site provide a clear introduction to T-cell biology and a good basic discussion of apoptosis.

http://www.bioseienee.org/knoekout/knoehome.htm

Within the Frontiers in Bioscience Database of Gene Knockouts, one can find information on the effects of knockouts of many genes involved in the development and function of cells of the T cells.

3. Antigenic activation of TH cells leads to the release or induction of various nuclear factors that activate gene transcription.

a. What transcription factors that support proliferation of activated TH cells are present in the cytoplasm of resting Th cells in inactive forms?

b. Once in the nucleus, what might these transcription factors do?

4. You have fluorescein-labeled anti-CD4 and rhodaminelabeled anti-CD8. You use these antibodies to stain thymocytes and lymph-node cells from normal mice and from RAG-1 knockout mice. In the diagrams below, draw the FACS plots that you would expect.

Study Questions

Clinical Focus Question Over a period of several years, a group of children and adolescents are regularly dosed with compound X, a life-saving drug. However, in addition to its beneficial effects, this drug interferes with Fas-mediated signaling.

a. What clinical manifestations of this side effect of compound X might be seen in these patients?

b. If white blood cells from an affected patient are stained with a fluorescein-labeled anti-CD4 and a phycoerythrin-labeled anti-CD8 antibody, what might be seen in the flow-cytometric analysis of these cells? What pattern would be expected if the same procedure were performed on white blood cells from a healthy control?

1. You have a CD8+ CTL clone (from an H-2kmouse) that has a T-cell receptor specific for the H-Y antigen. You clone the ap TCR genes from this cloned cell line and use them to prepare transgenic mice with the H-2k or a. How can you distinguish the immature thymocytes from the mature CD8+ thymocytes in the transgenic mice?

b. For each transgenic mouse listed in the table below, indicate with (+) or (—) whether the mouse would have immature double-positive and mature CD8+ thymocytes bearing the transgenic T-cell receptor.

c. Explain your answers for the H-2k transgenics.

d. Explain your answers for the H-2d transgenics.

2. Cyclosporin and FK506 are powerful immunosuppressive drugs given to transplant recipients. Both drugs prevent the formation of a complex between calcineurin and Ca2+/calmodulin. Explain why these compounds suppress T-cell-mediated aspects of transplant rejection. Hint: see Figure 10-11.

Thymus

Normal mice RAG-1 knockout mice

Lymph node

Normal mice RAG-1 knockout mice

5. In order to demonstrate positive thymic selection experimentally, researchers analyzed the thymocytes from normal H-2b mice, which have a deletion of the class II IE gene, and from H-2b mice in which the class II IA gene had been knocked out.

a. What MHC molecules would you find on antigen-presenting cells from the normal H-2b mice?

b. What MHC molecules would you find on antigen-presenting cells from the IA knockout H-2b mice?

c. Would you expect to find CD4+ T cells, CD8+ T cells, or both in each type of mouse? Why?

6. In his classic chimeric-mouse experiments, Zinkernagel took bone marrow from mouse 1 and a thymus from mouse

2 and transplanted them into mouse 3, which was thymec-tomized and lethally irradiated. He then challenged the reconstituted mouse with LCM virus and removed its spleen cells. These spleen cells were then incubated with LCM-in-fected target cells with different MHC haplotypes, and the lysis of the target cells was monitored. The results of two

Transgenic mouse

Immature thymocytes

Mature CD8+ thymocytes

H-2k female

H-2k male

H-2d female

H-2d male

Thymectomized, x-irradiated recipient

Lysis of LCM-infected target cells

Experiment

Bone-marrow donor

H-2d H-2k H-2b

A

C57BL/6 X BALB/c

C57BL/6 X BALB/c

+

B

C57BL/6 X BALB/c

C57BL/6 X BALB/c

+

such experiments using H-2b strain C57BL/6 mice and H-2d strain BALB/c mice are shown in the table on the above.

a. What was the haplotype of the thymus-donor strain in experiment A and experiment B?

b. Why were the H-2b target cells not lysed in experiment A but were lysed in experiment B?

c. Why were the H-2ktarget cells not lysed in either experiment?

7. Fill in the blank(s) in each statement below (a-k) with the most appropriate term(s) from the following list. Terms may be used once, more than once, or not at all.

protein phosphatase(s)

CD8

Class I MHC

CD45

protein kinase(s)

CD4

Class II MHC

B7

CD28

IL-2

IL-6

CTLA-4

b ._is a T-cell membrane protein that has cytosolic domains with phosphatase activity.

c. Dendritic cells express_constitutively, whereas B

cells must be activated before they express this membrane molecule.

d. Calcineurin, a_, is involved in generating the ac tive form of the transcription factor NFAT. Activation of TH cells results in secretion of _

and expression of its receptor, leading to proliferation and differentiation.

f. The co-stimulatory signal needed for complete TH-cell activation is triggered by interaction of_on the T

cell and_on the APC.

g. Knockout mice lacking class I MHC molecules fail to produce thymocytes bearing_.

h. Macrophages must be activated before they express _molecules and_molecules.

T cells bearing _

_ are absent from the lymph nodes of knockout mice lacking class II MHC molecules.

PIP2 is split by a_to yield DAG and IP3.

k. In activated TH cells, DAG activates a_, which acts to generate the transcription factor NF-kB. l. _stimulates and_inhibits T-cell activation when engaged by _

, or on antigen-presenting cells.

8. You wish to determine the percentage of various types of thymocytes in a sample of cells from mouse thymus using the indirect immunofluorescence method.

a. You first stain the sample with goat anti-CD3 (primary antibody) and then with rabbit FITC-labeled anti-goat Ig (secondary antibody), which emits a green color. Analysis of the stained sample by flow cytometry indicates that 70% of the cells are stained. Based on this result, how many of the thymus cells in your sample are expressing antigen-binding receptors on their surface? Would all be expressing the same type of receptor? Explain your answer. What are the remaining unstained cells likely to be?

b. You then separate the CD3+ cells with the fluorescence-activated cell sorter (FACS) and restain them. In this case, the primary antibody is hamster anti-CD4 and the secondary antibody is rabbit PE-labeled anti-hamster-Ig, which emits a red color. Analysis of the stained CD3+ cells shows that 80% of them are stained. From this result, can you determine how many TC cells are present in this sample? If yes, then how many TC cells are there? If no, what additional experiment would you perform in order to determine the number of TC cells that are present?

9. Many of the effects of engaging the TCR with MHC-peptide can be duplicated by the administration of ionomycin plus a phorbol ester. Ionomycin is a Ca2+ ionophore, a compound that allows calcium ions in the medium to cross the plasma membrane and enter the cell. Phorbol esters are analogues of diacylglycerol (DAG). Why does the administration of phorbol and calcium ionophores mimic many effects of TCR engagement?

10. What effects on cell death would you expect to observe in mice carrying the following genetic modifications? Justify your answers.

a. Mice that are transgenic for BCL-2 and over-express this protein.

b. Mice in which caspase 8 has been knocked out.

c. Mice in which caspase 3 has been knocked out.

11. Several basic themes of signal transduction were identified and discussed in this chapter. What are these themes? Consider the signal-transduction processes of T-cell activation and provide an example for each of six of the seven themes discussed.

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Responses

  • Johan
    What is the haplotype of the thymusdonor strain in experiment a solution?
    8 years ago
  • ralf lehmann
    What is the haplotype of the thymusdonor strain in experiment a answer?
    8 years ago

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