Some Central Issues in Thymic Selection Remain Unresolved

Although a great deal has been learned about the developmental processes that generate mature CD4+ and CD8+ T cells, some mysteries persist. Prominent among them is a seeming paradox: If positive selection allows only thymocytes reactive with self-MHC molecules to survive, and negative selection eliminates the self-MHC-reactive thymocytes, then no T cells would be allowed to mature. Since this is not the outcome of T-cell development, clearly, other factors operate to prevent these two MHC-dependent processes from eliminating the entire repertoire of MHC-restricted T cells.

Experimental evidence from fetal thymic organ culture (FTOC) has been helpful in resolving this puzzle. In this system, mouse thymic lobes are excised at a gestational age of day 16 and placed in culture. At this time, the lobes consist predominantly of CD4~8~ thymocytes. Because these immature, double-negative thymocytes continue to develop in the organ culture, thymic selection can be studied under conditions that permit a range of informative experiments. Particular use has

Influenza-infected target cell

Influenza-infected target cell

Mhc Lcm Infection

H-2 k transgenic +

H-2d transgenic

Thymocytes in transgenics

FIGURE 10-6

H-2 k transgenic +

H-2d transgenic

Effect of host haplotype on T-cell maturation in mice carrying transgenes encoding an H-2b class I-restricted T-cell receptor specific for influenza virus. The presence of the rearranged TCR transgenes suppressed other gene rearrangements in the transgenics; therefore, most of the thymocytes in the transgenics expressed the aß T-cell receptor encoded by the transgene. Immature doublepositive thymocytes matured into CD8+ T cells only in transgenics with the haplotype (H-2k) corresponding to the MHC restriction of the TCR transgene.

H-Y specific H-2Db restricted

H-Y specific H-2Db restricted

Tcr Transgenic Mice

Clone TCR a and P genes

Male cell (H-2Db)

Male cell (H-2Db)

Clone TCR a and P genes

Female cell (H-2Db)

Use to make a H-Y TCR transgenic mice i

Male H-2Db

Female,' H-2Db

H-Y expression

+

-

Thymocytes

CD4-8-

+ +

+

CD4+8+

+

+ +

CD4+

+

+

CD8+

-

Experimental demonstration that negative selection of thymocytes requires self-anti-

gen plus self-MHC. In this experiment, H-2 male and female transgenics were prepared carrying TCR transgenes specific for H-Y antigen plus the Dfc molecule. This antigen is expressed only in males. FACS analysis of thymocytes from the transgenics showed that mature CD8+ T cells expressing the transgene were absent in the male mice but present in the female mice, suggesting that thymocytes reactive with a self-antigen (in this case, H-Y antigen in the male mice) are deleted during thymic selection. [Adapted from H. von Boehmer and P. Kisielow, 1990, Science 248:1370.]

been made of mice in which the peptide transporter, TAP-1, has been knocked out. In the absence of TAP-1, only low levels of MHC class I are expressed on thymic cells, and the development of CD8+ thymocytes is blocked. However, when exogenous peptides are added to these organ cultures, then peptide-bearing class I MHC molecules appear on the surface of the thymic cells, and development of CD8+ T cells is restored. Significantly, when a diverse peptide mixture is added, the extent of CD8+ T-cell restoration is greater than when a single peptide is added. This indicates that the role of peptide is not simply to support stable MHC expression but also to be recognized itself in the selection process.

Two competing hypotheses attempt to explain the paradox of MHC-dependent positive and negative selection. The avidity hypothesis asserts that differences in the strength of the signals received by thymocytes undergoing positive and negative selection determine the outcome, with signal strength dictated by the avidity of the TCR-MHC-peptide interaction. The differential-signaling hypothesis holds that the outcomes of selection are dictated by different signals, rather than different strengths of the same signal.

The avidity hypothesis was tested with TAP-1 knockout mice transgenic for an ap TCR that recognized an LCM virus peptide-MHC complex. These mice were used to prepare fetal thymic organ cultures (Figure 10-8). The avidity of the TCR-MHC interaction was varied by the use of different concentrations of peptide. At low peptide concentrations, few MHC molecules bound peptide and the avidity of the TCR-MHC interaction was low. As peptide concentrations were raised, the number of peptide-MHC complexes displayed increased and so did the avidity of the interaction. In this experiment, very few CD8+ cells appeared when peptide was not added, but even low concentrations of the relevant peptide resulted in the appearance of significant numbers of CD8+ T cells bearing the transgenic TCR receptor. Increasing the peptide concentrations to an optimum range yielded the highest number of CD8+ T cells. However, at higher concentrations of peptide, the numbers of CD8+ T cells produced declined steeply. The results of these experiments show that positive and negative selection can be achieved with signals generated by the same peptide-MHC combination. No signal (no peptide) fails to support positive selection. A weak signal (low peptide level) induces positive selection. However, too strong a signal (high peptide level) results in negative selection.

The differential-signaling model provides an alternative explanation for determining whether a T cell undergoes positive or negative selection. This model is a qualitative rather than a quantitative one, and it emphasizes the nature of the signal delivered by the TCR rather than its strength. At the core of this model is the observation that some MHC-peptide complexes can deliver only a weak or partly activating signal while others can deliver a complete signal. In this model, positive selection takes place when the TCRs of developing thymocytes encounter MHC-peptide complexes that deliver weak or partial signals to their receptors, and negative selection results when the signal is complete. At this point it is not possible to decide between the avidity model and the differential-signaling model; both have experimental support. It may be that in some cases, one of these mechanisms operates to the complete exclusion of the other. It is also possible that no single mechanism accounts for all the outcomes in the cellular interactions that take place in the thymus and more than one mechanism may play a significant role. Further work is required to complete our understanding of this matter.

The differential expression of the coreceptor CD8 also can affect thymic selection. In an experiment in which CD8 ex pression was artificially raised to twice its normal level, the concentration of mature CD8+ cells in the thymus was one-thirteenth of the concentration in control mice bearing normal levels of CD8 on their surface. Since the interaction of T cells with class I MHC molecules is strengthened by participation of CD8, perhaps the increased expression of CD8 would increase the avidity of thymocytes for class I molecules, possibly making their negative selection more likely.

Another important open question in thymic selection is how double-positive thymocytes are directed to become either CD4+8~ or CD4~8+ T cells. Selection of CD4+8+ thymocytes gives rise to class I MHC-restricted CD8+ T cells and class II-restricted CD4+ T cells. Two models have been proposed to explain the transformation of a double-positive precursor into one of two different single-positive lineages

(a) Experimental procedure—fetal thymic organ culture (FTOC)

Remove thymus

Place in FTOC

Porous membrane

^ Growth medium

(b) Development of CD8+ CD4- MHC I-restricted cells

FIGURE 10-8

Role of peptides in selection. Thymuses harvested before their thymocyte populations have undergone positive and negative selection allow study of the development and selection of single positive (CD4+CD8~ and CD4~CD8+) T cells. (a) Outline of the experimental procedure for in vitro fetal thymic organ culture (FTOC). (b) The development and selection of CD8+CD4~ class I-restricted T cells depends on TCR peptide-MHC I interactions. TAP-1 knockout mice are unable to form peptide-MHC complexes unless peptide is added. The mice used in this study were transgenic for the a and p chains of a TCR that recognizes the added peptide bound to MHC I molecules of the TAP-1 knockout/TCR transgenic mice. Varying the amount of added pep-tide revealed that low concentrations of peptide, producing low avidity of binding, resulted in positive selection and nearly normal levels of CD4~CD8+ cells. High concentrations of peptide, producing high avidity of binding to the TCR, caused negative selection, and few CD4~CD8+ T cells appeared. [Adapted from Ashton Rickardt et al. (1994) Cell 25:651.]

Thymocyte

Thymus donor

Amount of peptide added

Thymic stromal cell

Normal r

Weak signal

None

Peptide r

Weak signal

Peptide

TCR-transgenic TAP-l deficient r

No signal

None r

No signal

Weak signal

Optimal

Weak signal

Strong signal

High

Strong signal

Degree of CD8+ T-cell development

Normal

Minimal

Approaches normal

Minimal

INSTRUCTIVE MODEL

INSTRUCTIVE MODEL

engagement signal

engagement signal

engagement signal

CD4lo8hi CD4- 8+ T cell

STOCHASTIC MODEL

M y Random

CD4+8+ CD4lo8hi

Random 4- CD8

CD4hi8to

Able to bind Ag + class I MHC

Not able to bind Ag + class I MHC

Able to bind Ag + class II MHC

Not able to bind Ag + class II MHC

CD4-8+T cell

CD4-8+T cell

Apoptosis

Apoptosis

Apoptosis

Apoptosis

FIGURE 10-9

Proposed models for the role of the CD4 and CD8 coreceptors in thymic selection of double positive thymocytes leading to single positive T cells. According to the instructive model, interaction of one coreceptor with MHC molecules on stromal cells results in down-regulation of the other coreceptor. According to the stochastic model, down-regulation of CD4 or CD8 is a random process.

(Figure 10-9). The instructional model postulates that the multiple interactions between the TCR, CD8+ or CD4+ coreceptors, and class I or class II MHC molecules instruct the cells to differentiate into either CD8+ or CD4+ singlepositive cells, respectively. This model would predict that a class I MHC-specific TCR together with the CD8 coreceptor would generate a signal that is different from the signal induced by a class II MHC-specific TCR together with the CD4 coreceptor. The stochastic model suggests that CD4 or CD8 expression is switched off randomly with no relation to the specificity of the TCR. Only those thymocytes whose TCR and remaining coreceptor recognize the same class of MHC molecule will mature. At present, it is not possible to choose one model over the other.

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