The complete repertoire of T cells selected on the 99ATg and specific for IEk
+ MCC was not assessed in this paper since the T cells we isolated came from mice that contained fetal liver cells bearing IEk
engaged by normal mouse peptides, albeit at low levels. The T cells in the chimeras were thus positively selected by IEk
bound to a single peptide, and were tolerant to IEk
bound to many normal mouse peptides at low levels, but not necessarily at high levels. This was manifested by the fact that none of the T cell hybridomas obtained from the chimeras reacted with IEk
on IEwtTg Ii+
cells, although some of the hybridomas did react with IEk
on B10.BR cells. Nevertheless, it is likely that expression of even the low levels of IEk
found in the chimeras did cause deletion of some IEk
+ mouse peptide– reactive T cells, and thus reduce the total diversity of TCRs found in the chimeras. Moreover, all the mice contained an additional class II protein, IAf
, and it is likely that at least some selected T cells were deleted by recognition of this protein, bound to its array of mouse peptides (11
Assessment of the ability of IEk
bound to 99A to select IEk
+ MCC–reactive T cells was also limited by the fact that some TCRs specific for this latter ligand also react with high affinity with the 99A selecting ligand (32
). This was demonstrated in this paper by the deletion in 99ATg mice of T cells bearing the well known IEk
+ MCC reactive TCRs, AND or AD10 (20
Overall, therefore, the repertoire for IEk + MCC in the transgenic chimeras was limited to some extent by negative selection on two types of ligands, low levels of wild-type IEk or IAf bound to mouse peptides, and IEk-99A.
In spite of these two limitations, it was clear that T cells reactive with IEk
+ MCC could be positively selected by IEk
-99A, a closely related ligand. A previous publication showed that MCC with substitutions of E for K at position 99, or E for T at position 102, and indeed even MCC itself could positively select T cells reactive with IEk
+ MCC (15
). Assuming that the affinity/avidity hypothesis of positive selection is correct, these results show that TCRs with high affinity for IEk
+ MCC can have an appropriate selecting affinity for IEk
+ any of these MCC variants or even for IEk
+ MCC itself at the concentrations that these ligands were expressed in the thymus.
Selection is peptide specific. T cells specific for IEk
+ MCC cannot be selected by IEk
bound to any peptide. Such T cells were not positively selected in animals expressing IEk
but not Ii (reference 14
and this paper). Apparently none of the peptides that were bound to IEk
in IiKO animals could participate in selecting T cells that can react with IEk
+ MCC. Also, as shown in this paper, IEk
+ MCC–reactive T cells were not obtained from animals expressing IEk
bound to 72A. Likewise, T cells with this specificity were not selected by IEk
bound to a lambda repressor peptide (15
Our result with the 72A peptide is quite surprising since it has previously been shown that unaltered Hb 67–76 could select MCC-reactive T cells (15
). The 72A peptide differed from that previously used by the conservative change of A for N at the central, T cell contact residue of the peptide, and by a longer NH2
-terminal tail protruding from the IEk
-binding groove. MCC itself has a basic amino acid, K, at the equivalent position. Perhaps TCRs that will react with MCC are selected better by Hb with the hydrophilic amino acid N at this position than Hb with the shorter, more hydrophobic A. 99A, however, also has an A at the equivalent position and, as shown in this paper, 99A selected MCC-reactive TCRs well.
Alternatively or as well, perhaps the difference was due to the level of expression of the IEk–peptide conjugates. The IEk-99A transgenic protein was expressed at much higher levels per cell than the IEk-72A conjugate. This may have caused positive selection of thymocytes bearing TCRs with very low affinity for the 99A peptide, whereas low affinity positive selection may not have been possible in the 72ATg mice. Recent measurement of the affinity of a TCR on one of the IEk + MCC–specific T cells described in this paper showed that it did indeed have extremely low affinity for IEk-99A.
Finally, the difference may be due to the fact that the T cells used in this paper were tolerant to IEwt and, less likely, to IAf. Tolerance to IEk plus all the peptides to which it is bound in normal mice may have deleted all the T cells selected on IEk-72A that could react with IEk + MCC. We do not think this is the explanation, however, because preliminary experiments with 72ATg T cells that are not tolerant to IEwt suggest that these T cells are also unable to react with IEk + MCC.
Some of the TCRs specific for IEk
+ MCC that were positively selected in the 99ATg mice were quite similar to those found in normal animals. Again, this was quite surprising since many T cells with this specificity that bore TCRs like those in wild-type animals must have been deleted in the 99ATg animals, as exemplified by the disappearance of T cells bearing the transgenic TCRs, AD10 and AND, in these mice. It is worth noting, however, that the TCR repertoire of the 99ATg animals was not completely included within that of normal animals since two different TCRs which used Vβ8.3 were obtained from the 99ATgs, and IEk
+ MCC–reactive T cells bearing this Vβ have not previously been reported from normal mice, although one T cell with this specificity and this Vβ was reported from mice heterozygous for Ii expression (15
Finally, although IEk + MCC–reactive T cells bearing identical TCRs have yet to be independently isolated, these data support the notion that the complete TCR repertoire for this MHC + peptide combination is nearing saturation. Several of the chains of the TCRs isolated from our transgenic mice were identical in sequence to chains that have been described previously. Presumably the numbers of TCR α and β chains that can contribute to TCRs that can react with IEk + MCC are limited, and this fact is reflected in the fact that particular sequences for α or β are being reported with increased frequency.