Conclusion

Mature T lymphocytes have a diverse TCR repertoire that is tolerant to self antigens, yet restricted to self-MHC. They achieve this balance by passing a series of stringent tests in the thymus, reminiscent of natural selection processes in evolution. Developing T cells (thymocytes) arise from multipotent CD4⁻CD8⁻ precursors that migrate from the bone marrow to the thymus, where Notch signals commit them to the T-cell lineage. Immature thymocytes proliferate, up-regulate CD4 and CD8, and undergo random T-cell receptor gene rearrangements, thus generating a large and diverse pool of DP thymocytes, each expressing a distinct TCR.

The fate of a DP thymocyte depends on the affinity of this TCR for self-peptide/MHC complexes it encounters while browsing stromal cells in the two major microenvironments of the thymus: the cortex and medulla. If a DP thymocyte fails to bind peptide/MHC complexes with enough affinity, it dies by neglect—the fate of the large majority (>90%) of DP thymocytes. If a DP thymocyte binds peptide/MHC complexes with intermediate affinity, it undergoes positive selection and is given permission to travel from the cortex to the medulla, to complete maturation to a single-positive (SP) CD4⁺ or CD8⁺ lineage. If a DP thymocyte binds peptide/MHC complexes with very high affinity, it undergoes negative selection.

Positive selection is mediated exclusively by interactions between thymocytes and cortical thymic epithelial cells (cTECs). Negative selection, however, can be mediated by multiple cells in both the cortex and medulla, and can target thymocytes at both the DP and SP stages. Importantly, only medullary thymic epithelial cells (mTECs) have the capacity to present antigens expressed by other tissues and are responsible for removing tissue-specific autoreactive T cells from the repertoire. Mechanisms that remove autoreactive T cells during development—central tolerance—are not infallible, and are reinforced in the periphery by a variety of mechanisms, including the activity of regulatory T cells. Some regulatory T cells arise in the thymus in response to high-affinity TCR interactions—an exception to the “rule” that high-affinity interactions drive negative selection.

The development of positively selected thymocytes to the CD4⁺ or CD8⁺ lineage is also determined by TCR signaling, and is best explained by the kinetic signaling model of lineage commitment. CD4⁺ and CD8⁺ thymocytes that survive positive and negative selection are allowed to migrate from the thymus into the bloodstream and complete their maturation in the periphery.

REFERENCES

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Useful Websites

• https://www.youtube.com/watch?v=08H5CmDaRjU This is one of the wonderful Handwritten Tutorials. It includes very basic information about cell progression and doesn’t go into detail about the selection processes, but is a good video for beginners.
• https://youtu.be/9E_UxnC_L2o A lovely 3-D animation of T-cell development generated as part of a master’s project by Janice Yau (also found at http://janiceyau.com/research.html). This is from the University of Toronto at Mississauga’s Biomedical Communications Graduate Program, where other videos of other biological processes can be found.
• www.bio.davidson.edu/courses/movies.html A full list of animations assembled, and in many cases generated, by individuals associated with Davidson College. See T Cell Development and Selection (http://www.bio.davidson.edu/courses/Immunology/Flash/Main.html). (Requires FLASH.)

STUDY QUESTIONS

1. Each of the following statements is false in one or more ways. Correct them (and explain your correction[s]).
   1. Knockout mice lacking MHC class I molecules fail to produce CD4⁺ mature thymocytes.
   2. β-Selection initiates negative selection.
   3. Negative selection to tissue-specific antigens occurs only in the cortex of the thymus.
   4. Most thymocytes successfully mature to the CD4⁺ or CD8⁺ T-cell lineage.
   5. Precursors of T cells express both CD4 and CD8 and first enter the medulla of a thymus.
   6. Thymocytes that bind to peptide/MHC complexes with high affinity are positively selected.
   7. DN thymocytes progress through several stages distinguished by changes in expression of immunoglobulin and CD25.
   8. All thymocytes with autoreactive T-cell receptors undergo negative selection.
   9. Regulatory T cells help to maintain central tolerance.
   10. Commitment to the CD4⁺ T-cell lineage is regulated by Runx3.
2. Fill in the blanks: Precursors of thymocytes enter the thymus at the junction. Interactions with ligands are required to commit them to the T-cell lineage. If positively selected, DP thymocytes travel from the thymic cortex to the . Up-regulation of allows them to leave the thymus and enter circulation.
3. Whereas the majority of T cells in our bodies express an αβ TCR, up to 5% of T cells express the γδ TCR instead. Explain the difference between these two cell types, in terms of development and antigen recognition.
4. You have fluorescein-labeled anti-CD4 and phycoerythrin-labeled anti-CD8. You use these antibodies to stain thymocytes and lymph node cells from normal mice and from RAG-1 knockout mice. In the forms below, draw the fluorescence-activated cell sorting (FACS) plots that you would expect.

   

   All four F A C S plot C D 4 along the vertical axis against C D 8 along the horizontal axis and are separated into quadrants. The top two plots, for the Thymus, are titled, normal mice, and R A G 1 knockout mice. The bottom two plots, for the Lymph node, are titled, normal mice, and R A G 1 knockout mice.

5. What stages of T-cell development (DN1, DN2, DN3, DN4, DP, CD4 SP, or CD8 SP) would be affected in mice with the following genetic modifications? Justify your answers.
   1. Mice that do not express MHC class II.
   2. Mice that do not express AIRE.
   3. Mice that do not express the TCR α chain.
6. You stain thymocytes with phycoerythrin (PE; red)-conjugated anti-CD3 antibodies and fluorescein isothiocyanate (FITC; green)-conjugated anti-TCR β chain antibodies. Most cells stain with both. However, you find a proportion of cells that stain with neither antibody. You also find a small population that stain with anti-CD3, but not with anti-TCR β antibodies. What thymocyte populations might each of these populations represent?
7. You immunize an H2^k mouse with chicken ovalbumin (a protein against which the mouse will generate an immune response) and isolate a CD4⁺ mature T cell specific for an ovalbumin peptide. You clone the αβ TCR genes from this cell line and use them to prepare transgenic mice with the H2^k or H2^d haplotype.
   1. What approach can you use to distinguish immature thymocytes from mature CD4⁺ thymocytes in the transgenic mice?
   2. Would thymocytes from a TCR transgenic mouse of the H2^k background have a proportion of CD4⁺ thymocytes that is higher, lower, or the same as in a wild-type mouse?
   3. Would thymocytes from a TCR transgenic mouse of the H2^d background have a proportion of CD4⁺ thymocytes that is higher, lower, or the same as in a wild-type mouse? Speculate and explain your answer.
   4. You find a way to “make” the medullary epithelium of an H2^k TCR transgenic mouse express and present the ovalbumin peptide for which your T cell is specific. What would the CD4-versus-CD8 profile of a TCR transgenic thymus look like in these mice?
   5. You also find a way to “make” the cortical epithelium express this ovalbumin peptide in its MHC class II dimer. What might the CD4-versus-CD8 profile of this TCR transgenic thymus look like?
8. In his classic chimeric mouse experiments, Zinkernagel took bone marrow from a mouse of one MHC haplotype (mouse 1) and the thymus from a mouse of another MHC haplotype (mouse 2) and transplanted them into a third mouse, which was thymectomized and lethally irradiated. He then immunized this reconstituted mouse with the lymphocytic choriomeningitis virus (LCMV) and examined the activity of the mature T cells isolated from the spleen and lymph nodes of the mouse.

   He was specifically interested to see if the mature CD8⁺ T cells in these mice could kill target cells infected with LCMV with the MHC haplotype of mouse 1, 2, or 3. The results of two such experiments using H2^b strain C57BL/6 mice and H2^d strain BALB/c mice as bone marrow and thymus donors, respectively, are shown in the following table.

   1. Why were the H2^b target cells not lysed in experiment A but lysed in experiment B?
   2. Why were the H2^k target cells not lysed in either experiment?

   Lysis of LCMV-infected target cells
   Experiment	Bone marrow donor	Thymus donor	Thymectomized x-irradiated recipient	H2d	H2k	H2b
   A	C57BL/6 (H2b)	BALB/c (H2d)	C57BL/6 × BALB/c	+	−	−
   B	BALB/c (H2d)	C57BL/6 (H2b)	C57BL/6 × BALB/c	−	−	+

9. You have a CD8⁺ CTL clone (from an H2^k mouse) that has a T-cell receptor specific for the H-Y antigen. You clone the αβ TCR genes from this cloned cell line and use them to prepare transgenic mice with the H2^k or H2^d haplotype.
   1. How can you distinguish the immature thymocytes from the mature CD8⁺ thymocytes in the transgenic mice?
   2. For each of the following transgenic mice, indicate with (+) or (-) whether the mouse would have immature double-positive and mature CD8⁺ thymocytes bearing the transgenic T-cell receptor: H2^k female, H2^k male, H2^d female, H2^d male.
   3. Explain your answers for the H2^k transgenics.
   4. Explain your answers for the H2^d transgenics.
10. To demonstrate positive thymic selection experimentally, researchers analyzed the thymocytes from normal H2^b mice, which have a deletion of the class II H2-E gene, and from H2^b mice in which the class II H2-A gene had been knocked out.
    1. What MHC molecules would you find on antigen-presenting cells from the normal H2^b mice?
    2. What MHC molecules would you find on antigen-presenting cells from the H2-A knockout H2^b mice?
    3. Would you expect to find CD4⁺ T cells, CD8⁺ T cells, or both in each type of mouse? Why?
11. You wish to determine the percentage of various types of thymocytes in a sample of cells from mouse thymus using the indirect immunofluorescence method.
    1. You first stain the sample with goat anti-CD3 (primary antibody) and then with rabbit FITC-labeled anti-goat immunoglobulin (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?
    2. 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 immunoglobulin, 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 T_C cells are present in this sample? If yes, then how many T_C cells are there? If no, what additional experiment would you perform to determine the number of T_C cells that are present?

CLINICAL FOCUS QUESTIONS

1. The susceptibility to autoimmune diseases often has a genetic basis and has been linked to many different gene loci. Identify two genes that could be involved in an increased susceptibility to autoimmune disease. Explain your reasoning.
2. Susceptibility to many autoimmune diseases has been linked to MHC gene variants. One of the best examples of such a linkage is provided by multiple sclerosis (MS), a human autoimmune disease caused by autoreactive T cells whose activity ultimately damages the myelin sheaths around neurons. Susceptibility to MS has been consistently associated with variants in the HLA-DR2 gene. Although this link was first recognized in 1972, we still don’t fully understand the basis for this susceptibility. One perspective on the reasons for the link between MHC variations and autoimmune disease was offered in a recent review article. The authors state, “The mechanisms underlying MHC association in autoimmune disease are not clearly understood. One long-held view suggests a breakdown in immunological tolerance to self antigens through aberrant class II presentation of self- or foreign peptides to autoreactive T lymphocytes. Thus, it seems likely that specific MHC class II alleles determine the targeting of particular autoantigens resulting in disease-specific associations.” (Fernando, M. M., et al. 2008. Defining the role of the MHC in autoimmunity: a review and pooled analysis. PLoS Genetics 4:e1000024.)
   1. Paraphrase this perspective, using your own words. What, specifically, might the authors mean by “aberrant class II presentation . . . to autoreactive T lymphocytes”?
   2. (Advanced question.) Although this speculation has some merit, it does not resolve all questions. Why? Pose one question that this explanation inspires or does not answer.
   3. (Very advanced question.) Offer one addition to the explanation (or an alternative) that helps resolve the question you posed above.