Experiments Revealed the Essential Elements of Positive and Negative Selection

Direct evidence that binding of thymocytes to class I or class II MHC molecules is required for positive selection in the thymus came from experimental studies with knockout mice incapable of producing functional class I or class II MHC molecules (Table 10-1). Class I-deficient mice were found to have a normal distribution of double-negative, double-positive, and CD4+ thymocytes, but failed to produce CD8+ thymocytes. Class II-deficient mice had double-negative, double-positive, and CD8+ thymocytes but lacked CD4+ thymocytes. Not surprisingly, the lymph nodes of these class II-deficient mice lacked CD4+ T cells. Thus, the absence of class I or II MHC molecules prevents positive selection of CD8+ or CD4+ T cells, respectively.

Further experiments with transgenic mice provided additional evidence that interaction with MHC molecules plays a role in positive selection. In these experiments, rearranged ap-TCR genes derived from a CD8+ T-cell clone specific for influenza antigen plus H-2^ class I MHC molecules were injected into fertilized eggs from two different mouse strains,

TABLE 10-1

Effect of class I or II MHC deficiency on thymocyte populations*

KNOCKOUT MICE

Cell type

Control mice

Class I deficient

Class II deficient

*Plus sign indicates normal distribution of indicated cell types in thymus. Minus sign indicates absence of cell type.

one with the H-2k haplotype and one with the H-2d haplo-type (Figure 10-6). Since the receptor transgenes were already rearranged, other TCR-gene rearrangements were suppressed in the transgenic mice; therefore, a high percentage of the thymocytes in the transgenic mice expressed the T-cell receptor encoded by the transgene. Thymocytes expressing the TCR transgene were found to mature into CD8+ T cells only in the transgenic mice with the H-2k class I MHC haplotype (i.e., the haplotype for which the transgene receptor was restricted). In transgenic mice with a different MHC haplotype (H-2d), immature, double-positive thymocytes expressing the transgene were present, but these thymocytes failed to mature into CD8+ T cells. These findings confirmed that interaction between T-cell receptors on immature thymocytes and self-MHC molecules is required for positive selection. In the absence of self-MHC molecules, as in the H-2d transgenic mice, positive selection and subsequent maturation do not occur.

Evidence for deletion of thymocytes reactive with self-antigen plus MHC molecules comes from a number of experimental systems. In one system, thymocyte maturation was analyzed in transgenic mice bearing an ap TCR transgene specific for the class I Dh MHC molecule plus H-Y antigen, a small protein that is encoded on the Y chromosome and is therefore a self-molecule only in male mice. In this experiment, the MHC haplotype of the transgenic mice was H-2h, the same as the MHC restriction of the transgene-encoded receptor. Therefore any differences in the selection of thymocytes in male and female transgenics would be related to the presence or absence of H-Y antigen.

Analysis of thymocytes in the transgenic mice revealed that female mice contained thymocytes expressing the H-Y-specific TCR transgene, but male mice did not (Figure 10-7). In other words, H-Y-reactive thymocytes were self-reactive in the male mice and were eliminated. However, in the female transgenics, which did not express the H-Y antigen, these cells were not self-reactive and thus were not eliminated. When thymocytes from these male transgenic mice were cul tured in vitro with antigen-presenting cells expressing the H-Y antigen, the thymocytes were observed to undergo apoptosis, providing a striking example of negative selection.

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Essentials of Human Physiology

Essentials of Human Physiology

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