Modulation of Ia and photoreactive antigen on antigen-presenting cells: Fun with a photoprobe

Abstract

To identify the antigen-specific recognition complex containing elements from T cells and antigen-presenting cells (APC), a photoactivatable antigen system was developed which could potentially crosslink the complex during the specific cellular responses. In this paper we describe the development of this system using murine T-cell hybridomas responding to stimulator cells chemically conjugated with N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and genetically restricted by I-A d. In initial experiments it was found that several I-A d-positive B-cell lines were nonstimulatory when coupled with HSAB, but that I-A d-positive P388D 1 macrophage-like cells were efficient stimulators of HSAB-specific T-cell responses. These results suggested that the relevant HSAB coupled surface structure was not likely I-A d. To substantiate this point, Ia-positive or Ia-negative P388D 1 cells were initially coupled with HSAB and the expression of Ia was modulated by the addition and withdrawal of Con A-stimulated spleen cell supernatant fluid through several days of culture. Under these conditions, efficient stimulation was only observed when Ia was expressed, although the HSAB antigen was continuously present. In other experiments it was found that exposure of HSAB-coupled APC to light selectively eliminated their stimulatory capacity for HSAB-specific T hybridomas, suggesting that the light-induced crosslinking by HSAB directly eliminates the antigenic determinant. This antigen system allows a unique opportunity to manipulate the antigen during specific cellular interactions, and to introduce covalent crosslinking of the specific antigen recognition complex that may allow its isolation and characterization.