Specificity and flexibility in thymic selection

Positive selection of T cells occurs at the CD4⁺8⁺ stage of development and results in phenotypic changes (including downregulation of CD4 or CD8) and functional maturation^2,3,11,12. We previously described a TCR-transgenic mouse (OVA-tcr-1) with a receptor specific for ovalbumin residues 257–264 (OVAp) plus H–2K^b. Thymocytes bearing this receptor are positively selected in fetal thymic organ cultures (FTOCs) expressing K^b (Fig. 1a, d) (ref. 9) but maturation is impaired in FTOCs from β₂M^−/− mice (Fig. 1b, e), where class I expression is drastically reduced^{7,13 15}. Selection is restored by addition of exogenous β₂-microglobulin (β₂M) and certain variants of OVAp, exemplified by peptides E1 (Fig. 1c, f) and R4 (Fig. 1g) (ref. 9). This process shows exquisite peptide fine specificity; neither E1R4 (which combines both the E1 and R4 substitutions) nor K4 (which is chemically very similar to R4) are capable of positively selecting the OVA-tcr-1 receptor (Fig. 1h, i). Two theories have been pro-posed to explain peptide specificity in positive selection^8,9,16. The TCR may interact directly with both MHC and peptide residues (‘peptide interaction’ model), or it may only recognize MHC residues, in which case the peptide's role is merely to avoid impeding the TCR–MHC interaction (‘peptide avoidance’ model). Our results are hard to reconcile with the latter model; thus if both E1 and R4 simply keep out of the TCR's way, it is likely that E1R4 would do so too, and hence should positively select. Therefore, our data support a peptide interaction model.

We previously showed that peptides that induce positive selection, including E1 and R4, also act as TCR antagonists^9,10 (Fig. 2). K4 and E1R4 did not behave as antagonists (Fig. 2), demonstrating similar fine specificity for positive selection and TCR antagonism. This suggests that the two phenomena may have a similar mechanistic basis, possibly based on TCR affinity for the peptide–MHC ligand^9,10,17,18. However, some peptides that are weak agonists induce positive selection at low concentrations and negative selection at higher concentrations (refs 9, 19, 20 and K.A.H. et al., manuscript in preparation). Thus, positive selection is conditioned by both qualitative (perhaps affinity) and quantitative (ligand density) aspects of the TCR interaction.

E1 not only antagonizes OVA-tcr-1 T-cell responses (Fig. 2), but is also a weak agonist, stimulating mature transgenic cytotoxic T lymphocytes (CTLs)⁹ and deleting immature thymocytes in suspension culture (data not shown). We investigated how the presence of E1 during development alters the reactivity of mature OVA-tcr-1 thymocytes (Fig. 3a, b). Thymocytes from TCR-transgenic β₂M^−/− FTOCs made a very weak response to both OVAp and E1. Addition of E1 during FTOC increased the absolute numbers of CD8⁺ T cells per lobe and thus the response to OVAp. But these cells were virtually non-responsive to E1 (Fig. 3a). This lack of E1 reactivity is all the more remarkable as the antigen-presenting cells used are β₂M^+/+ and so present peptides 50–100-fold better than β₂M^−/− cells^7,9. Similarly, inclusion of E1 in OVA-tcr-1 β₂M^+/− FTOC ablated the E1 response but only partially reduced proliferation to OVAp (Fig. 3b).

What is the basis of this selective non-reactivity to E1? We investigated whether thymocyte TCR and/or CD8 expression levels were affected. TCR-α-chain expression was essentially equivalent in all the populations (Fig. 3d), but CD8 expression was significantly lowered on thymocytes from FTOCs in which E1 was included (Fig. 3c). This is seen most dramatically in the β₂M^+/− FTOC, but is also apparent in cells from β₂M^−/− lobes in which E1 is the positively selecting ligand. These data suggest that thymocytes can regulate co-receptor expression levels to avoid overt reactivity to the positively selecting ligand. Mature cells bearing low levels of CD8 (CD8^lo) could arise from selective survival of CD8^lo precursors or by CD8 downregulation on CD8^hi precursors. Relative to β₂M^+/− FTOC we observed a decrease in the number of TCR^hi CD8⁺ cells in β₂M^−/− plus E1 (∼20%) or β₂M^+/− plus E1 (∼50%) cultures (see Fig. 3 legend), which supports the former model but does not exclude the latter.

To test whether CD8 expression levels alone could explain the differential response to E1 and OVAp, we analysed mature T-cell clones from OVA-tcr-1 animals (Fig. 4a, b). TG-O.C2 is typical of most clones in that it responded to E1 as an antagonist. However, TG-I.C4 responded to E1 as a weak agonist. This response is completely blocked by an antibody to the transgenic α-chain (data not shown), suggesting that E1 reactivity is not due to expression of an endogenous TCR α-chain. Fluorescence-activated cell sorting (FACS) analysis indicated that CD8 (Fig. 4c) and TCR (data not shown) expression was higher on TG-I.C4 than TG-O.C2. To assess CD8 contribution in E1 reactivity, we tested the effects of anti-CD8 antibody. Partial CD8 blocking dramatically inhibited the TG-1.C4 response to E1 but, at this dose, did not inhibit OVAp reactivity (Fig. 4d). Interestingly, this treatment resulted in E1 now acting as a potent antagonist for TG-1.C4. Thus, the CD8 interaction can apparently determine whether a ligand is perceived as an agonist or an antagonist.

During positive selection, a T cell must interact with self MHC while avoiding negative selection on one hand and autoreactivity on the other. This could be achieved by regulation of receptor expression levels and/or signal transduction machinery. Our data suggest that flexibility in CD8 expression levels is one way for thymocytes to fine-tune the efficacy of their interaction with MHC–peptide ligands present in the thymus and achieve positive selection while maintaining self tolerance. In what may be an analogous situation, lowering CD8 expression on mature T cells converts TCR agonists into antagonists. We note that selection by agonist ligands in a different TCR transgenic model yields CD8^lo single positive thymocytes also, although the functional potential of these cells was not addressed^19,20. In keeping with our observations, CD8 overexpression apparently converts a positively selecting environment into a negatively selecting one^21,22. Also, CD8 and/or TCR downregulation in peripheral T cells allows survival in the face of potentially tolerizing antigens^23,24.