We provide evidence for the surprising conclusion that in CD4−/−
mice, the CD8 population contains a large fraction of MHC class II–restricted cells in addition to the conventional class I–restricted cells. In both of the immunization systems we studied, these CD8+
class II–restricted T cells were readily apparent. It has also been reported that the response to murine hepatitis virus in CD4−/−
mice shows a similar, readily detectable CD8 T cell response to class II–restricted antigen, though in this case the epitopes were not defined (22
). The class II–restricted response of CD8 lineage cells, which is readily seen in CD4−/−
mice, is not obvious in control animals, where it is difficult to detect any CD8 response to the same MHC class II–restricted peptides. We have shown that this difficulty in detecting class II–restricted CD8 cells in a wild-type mouse is not because conventional CD4 T cells suppress the CD8 response to the class II/peptide epitopes; even when the CD4 T cell population is depleted just before immunization, no class II–restricted CD8 T cell response is revealed (). On the contrary, we observed that the few events in the IFN-γ staining profiles suggesting that CD8+
T cells in normal mice can respond to class II–restricted peptides were decreased in acutely CD4-depleted mice. This suggests that these events were caused by bystander activation of CD8+
T cells driven by peptide activation of CD4 cells in the cultures. However, we do not rule out the possibility that there is a very weak response of CD8+
T cells to class II–restricted peptides in normal B6 mice.
Indeed, it is likely that the class II–restricted CD8 T cells in the CD4−/−
animals arise as a result of misdirection during positive selection in the thymus (4
). We assume that the cells are selected by recognition of class II in the thymus, where some immature thymocytes express a randomly generated TCR with sufficient affinity for class II molecules on the thymic cortical epithelial cells that they can be selected into the CD8 lineage when the CD4 coreceptor is absent.
We propose that the main reason why the CD8 response to class I–restricted peptides in CD4−/− mice appears to be weaker than the response in control mice is that their TCR repertoire is heavily contaminated by MHC class II–restricted cells. It is not explained by the deficiency in T cell help, as demonstrated best by their weaker response even when immunized alongside equal numbers of CD8 T cells from wild-type mice (). It is difficult to estimate precisely the extent of this contamination. When we compare the fraction of total CD8 cells making a peptide-specific response in intact wild-type or mutant mice, as we did in and , we find that CD4−/− give roughly half the wild-type level. However, this does not take into account the relative starting numbers of CD8 cells or their degree of expansion after immunization. When equal starting numbers of CD8 T cells were mixed and immunized together, we found that for the Ova257–264 and GP33–41 epitopes, the wild-type CD8 T cells gave 2.5-fold and 4.8-fold more progeny than the mutant CD8 T cells, respectively. This comparison suggests that the CD8 repertoire in CD4−/− mice may actually contain more class II–restricted receptors than class I–restricted receptors!
One intriguing question that remains is which immature thymocytes bearing class II–restricted receptors in CD4−/−
mice are selected into the CD8 lineage, and which ones are selected into the double-negative CD4 lineage. According to the strength of signal model for CD4/CD8 lineage choice, double-positive thymocytes with higher avidity for the selecting ligand would commit to the CD4 lineage, and lower avidity cells would preferentially commit to the CD8 lineage (5
). Although one can readily observe the response of the CD8 lineage cells to class II–restricted peptides in CD4−/−
mice, a response by the double-negative CD4 lineage population is not apparent in our studies. One caveat to this is that we have mainly used IFN-γ staining to detect these cells, and it is possible that the response of CD4 lineage cells would be detectable using tetramers for these specificities, or by staining for other cytokines. However, we have evidence that a similar conclusion is reached even when intracellular IL-2 staining is used for detection (unpublished data). In addition, previous work on the response of CD4 lineage cells in CD4−/−
mice has suggested that, whereas they can efficiently mount a Th1 response typified by IFN-γ synthesis, they are much less able to mount a Th2 response (23
). Finally, it is clear that the response of CD4 T cells in wild-type animals is readily detected by intracellular staining for IFN-γ. Another factor that may determine our inability to detect the response to class II–restricted peptides in the double negative CD4 lineage cells in CD4−/−
mice is simple numbers. They make up only 10–20% of the peripheral T cell pool, whereas we estimate ≥50% of CD8 lineage cells are class II–restricted. At present, it is not precisely known what factors determine the outcome of MHC class II recognition in the thymus of CD4−/−
mice, whether it be deletion, selection into the double-negative “CD4 wannabe” lineage, selection into the CD8 lineage, or no selection. Similarly, what controls the response to antigen presented on MHC class II molecules of peripheral T cells that lack the CD4 coreceptor and are either double negative or CD8+