E
c
Ûd
0.8
0.6
0.40.2
6 . 5 D is c u ss io n
The experiments described in this chapter have demonstrated again the utility of MHC I transgenic mice in assessing mechanisms of tolerance in vitro, because self-reactive effector cells can be easily detected with cytotoxic T cell assays. CD4+8- effector mechanisms must be detected by more complex assays.
The experiments described in this section are in agreement with the hypothesis proposed by Simpson et aL, (1993), that self-reactive thymocytes are eliminated, or functionally inactivated, once a certain level of antigen expression on the surface of thymocytes was reached. This level is above that expressed by CD2K^-3 mice, and is probably the level of H-2K^ expressed by most CD2K^-4 mice. The CTL response by one CD2Kb-4 to H-2K^ could not be reproduced and is inconsistent with results from other tests. The mouse which responded to antigen was five months old at the time of the test, by no means too old to give an accurate assay, and was a heterozygote: some siblings tested negative for the transgene by PGR analysis. The mouse which responded may have expressed a little less H-2K^ than other CD2K^-4 mice, perhaps due to a mutation that knocked out some copies of the transgene. Alternatively, due to confusion at the PGR genotyping stage, a GBA/Ga sibling was used for the assay, instead of a GD2K^-4 mouse. This seems unlikely, but would explain the similar responses by GD2K^-4 and GBA/Ga mice to H-2K^ on EL4 target cells (y = 28.6 and 32.5% respectively, both with R^ values >0.7) (In contrast, GD2K^-3, which respond to antigen by lysing H-2K^-bearing targets have a y value of 15.9, compared to the GBA/Ga value of 42.5; both have R^ values greater than 0.7). The response by GD2K^-4.4 and GBA/Ga cells to cross-reacting H-2^ targets (y values of 13.2 and 10.9) are also similar. Despite this, it is worth bearing in mind that as many as 25% of GD2K^-4 mice may be capable of mounting a cytotoxic response to self antigen. This sporadic response to H-2K^^ by GD2K^-4 mice resembles responses to minor histocom patibilty antigens (Dr. A-M. Sponaas, personal communication). However, GD2K^-4 mice that did not respond to H-2K^ in vitro did not respond even after priming with GBK cells in vivo.
Figure 17 shows that low levels of antigen expression on thymocytes from CD2K^-3 heterozygotes permits self-reactive T cells to escape deletion in the thymus. This result is in direct conflict with those of the previous chapter, which show that self-reactive thymocytes are deleted in the thymuses of CD2K^ mice. Both BM3.6 and DES TCRs are deleted upon contact with H-2K^ in CD2K^-3 thymuses. This clearly shows that experiments with TCR-tg mice cannot predict the fate of all self-reactive thymocytes. Such experiments can indicate the extent to which the deletion of a particular clonotype is influenced by antigen density, but not the extent to which different TCRs, with the same antigen specificity, are dependent on antigen expression levels for deletion (Sponaas et aL, 1994b).
The level of H-2K^ expression on thymocytes from CD2K^-3 homozygotes appears to prevent the escape of self-reactive T cells. Auphan et al. (1992) had previously observed that antigen density affects the degree of negative selection. Expression of H- 2K^ on dendritic cells in CD2K^-3 mice appears to be unrelated to the ability of self reactive thymocytes to escape deletion, despite the crucial role they appear to play in negative selection (Zoller, 1991), and their position at the cortico-medullary junction. CD2K^-4 mice, which do not express H-2K^ on bone marrow-derived DCs do not have self-reactive T cells in the periphery.
There may be several mechanisms by which self-reactive thymocytes escape negative selection in the thymus of CD2K^-3 mice. They may down-regulate expression of the CDS co-receptor, decreasing the TCR s avidity for antigen, and escaping signals that trigger apoptosis. Expression of the self-specific TCR itself may decrease, or may be abnormally low in a subset of CD2K^-3 thymocytes. Flow cytometric analysis of CDS and aPTC R expression would have been informative. These mechanisms would also occur in thymocytes from the other CD2K^ lines, and so the level of H-2K^ expression on thymocytes from CD2K^-3 heterozygotes must be crucial. This is confirmed by the experiments with CD2K^>-3 homozygotes, with twice the transgene copies, and almost no
Self-reactive thymocytes may not be deleted in the thymus of the remaining CD2K^ lines. Higher levels of transgene expression may merely lead to anergy - possibly a complete loss of CDS or TCR expression, but the loss from the thymus of self-reactive cells bearing the BM3.6 or DES TCR, suggests that the cells are probably deleted. Thymuses from CD2K^ mice have not been examined for reduced size - the majority of DES or BM3.6 x CD2K^ thymocytes only express one clonotype, and it's deletion from the repertoire is easily observed. This would not be the case in CD2K^ mice; where small thymuses have been seen in CD2K^-7 mice there is an alternative explanation for loss of thymocytes (4.8).