Inhibition of responder cell activity in mixed leukocyte culture reactions

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Abstract
In a previous report we described a human allo-antiserum which showed MHC restricted dual specific inhibition of mixed leukocyte culture (MLC) reactions (de Rooij et al. 1980). Responder cells were preincubated with the antiserum and washed. Inhibition of MLC reactions occurred if HLA-B8 positive responder cells and HLA-B7 or B40 positive stimulator cells were used. The mechanism of inhibition by this antiserum was investigated and described in this report. The results strongly suggested that the observed inhibition was due to HLA-B7 specific antibody molecules cross-reacting with HLA-B8 antigens. We decided on the following mechanism: HLA-B7 specific antibody molecules bind to HLA-B8 antigens on responder cells at 4 degrees C. Bound antibody molecules area readily released at 37 degrees C and the bind preferentially to HLA-B7 or -B40 positive stimulator cells. Stimulator cells are then lysed by antibody dependent cellular cytotoxicity (ADCC), resulting in impaired MLC reaction. This conclusion was derived from the following observations: 1. HLA-B7 positive cells could be lysed due to antibody molecules bound to B8 positive cells: a. Cells coated with antibody molecules lysed B7 or B40 positive target cells. b. Antibody molecules released from B8 positive cells lysed B7 or B40 positive target cells in ADCC. 2. The active antibody molecules were HLA-B7 specific: a. The inhibiting antibody molecules in the antiserum could be absorbed on and eluted from HLA-B7 positive platelets (de Rooij et al. 1980). b. Antibody molecules bound to and released from HLA-B8 positive cells carried the same B7 specific inhibiting and cytotoxic activity as the original antiserum. 3. The site of recognition on HLA-B8 positive cells is the HLA-B8 antigen: a. F(ab')2 preparations from HLA-B8 and -Bw6 specific antisera inhibited antibody binding to B8 positive cells. b. Antibody binding was not restricted to B, T, FcR+, FcR- or non adherent cell subpopulations. 4. The antibody molecules bind to B8 positive cells with the antigen binding site and not with the Fc part of the molecule: a. Antibody binding could be blocked partially with a F(ab')2 preparation from the original antiserum but not with a Fc preparation. b. The antigen binding site of antibody molecules bound to HLA-B8 positive cells was not freely available, since the MLC inhibiting activity of sensitised B8 positive cells could not be blocked by soluble HLA-B7 antigens. c. The antiserum was cytotoxic for HLA-B8 positive target cells in ADCC. The observed inhibition of MLC by antibody molecules with apparent dual specificity is thus the consequence of a shared or a similar antigenic determinant of HLA-B7 and HLA-B8 antigen molecules.