Monoclonal, antigen-specific, T cell contrasuppressor factor expresses determinants of TCR alpha-chain (not necessarily TCR beta-chain), having a molecular mass of about 40 kDa.

Abstract

Contrasuppression is a regulatory T cell activity that acts on helper and contact sensitivity effector T cells to protect them from the action of suppressor T cells. In this study, we examined a monoclonal Ag-specific T cell-secreted contrasuppressor factor (TcsF) with Ag specificity for the hapten TNP (trinitrophenyl). This factor positively influences the adoptive cell transfer of contact sensitivity in the presence of active suppression. Results presented in this report demonstrate a relationship between TcsF and the alpha,beta-T cell receptor (TCR). We found that TcsF bound to mAb anti-TCR-alpha (H28) and to mAb anti-TCR-beta (H57) immunoaffinity columns, but not to a mAb anti-TCR-gamma,delta (UC7) column. The bound contrasuppressor activity could be recovered from these affinity columns by base elution. Reduction of TcsF with DTT followed by alkylation with methylmethanethiosulfonate (MMTS), demonstrated that the active subunit of TcsF bound to and eluted from anti-TCR-alpha, but not to anti-TCR-beta columns. The active TcsF that bound to anti-TCR-alpha but not to the anti-TCR-beta column was shown subsequently to have the ability to bind to specific Ag columns TNP-BSA and TNP-BGG Sepharose 4B. The TcsF could be successfully recovered later from Ag columns; thus suggesting that the Ag-binding domain for mAb, and the biologic activity, resides on the TCR-alpha chain of TcsF. Separation of TcsF on SDS-PAGE under reducing conditions, followed by elution and renaturation of proteins from the gel slices, showed that a 35 to 40 kDa protein had the T cell contrasuppressive activity. Western blot analysis of nonreduced TNP-binding TcsF revealed TCR-alpha,beta determinants on an 80-kDa native molecule similar to TCR-alpha,beta from a helper T cell clone. In summary, Ag-specific, Ag-binding T cell-derived contrasuppressor factor has serologic determinants of TCR-alpha and TCR-beta chains, but the TCR-alpha chain and not the TCR-beta chain is required for biologic function. This TCR-alpha determinant-containing factor is absorbed by specific Ag (TNP) columns. SDS-PAGE analysis under reducing conditions suggested a molecular weight of about 35 to 40 kDa for the TNP-specific TcsF. The mechanism of action of the TCR-alpha-containing contrasuppressor factor in the regulation contact sensitivity is discussed.