The Stimulation of Low-Affinity, Nontolerized Clones by Heteroclitic Antigen Analogues Causes the Breaking of Tolerance Established to an Immunodominant T Cell Epitope

H-2K mice injected, intravenously in saline or intraperitoneally in incomplete Freund's adjuvant, with large quantities of the immunodominant I-E^k–restricted epitope from moth cytochrome c (MCC) 88–103 fail to respond to subsequent immunization with this epitope when administered in complete Freund's adjuvant. This state of tolerance can be broken by immunization with certain MCC 88–103 analogues that are heteroclitic antigens as assessed on representative MCC 88–103 specific T cell clones. In this paper, the mechanism of breaking tolerance by heteroclitic antigens was investigated. The following observations were made: (a) T cell hybridomas derived from tolerance-broken animals required higher concentrations of MCC 88–103 to be stimulated than hybridomas derived from normal immune animals, suggesting that they have T cell receptors (TCRs) of lower affinity; (b) in contrast to normal immune animals whose MCC-specific TCRs are typically Vβ3⁺/Vα11⁺, none of the hybridomas derived from tolerance-broken animals expressed Vβ3, although they were all Vα11⁺. Also, the Vβ complementarity determining region 3 (CDR3) regions from the tolerance-broken animals did not contain the canonical structure and length characteristics of the normal MCC 88–103 immune repertoire; and (c) adoptive transfer and tolerization of MCC-specific Vβ3⁺/Vα11⁺ transgenic T cells followed by immunization with heteroclitic antigen failed to terminate the state of tolerance. Collectively, these data strongly suggest that the mechanism involved in breaking tolerance in this system is the stimulation of nontolerized, low-affinity clones, rather than reversal of anergy. Further support for this mechanism was the finding that after activation, T cells apparently have a lowered threshold with respect to the affinity of interaction with antigen required for stimulation.