Idiotype-specific immune responses following Id-KLH immunisation. (A) Patient 5 humoral response measured by ELISA. Pre- or post-vaccination serum was serially diluted and tested for binding (indicated by optical density, O.D.) to the patient's own tumor Id vs. another patient's (irrelevant) Id protein as a control. Only post-vaccine serum shows a high degree of tumor Id-specific binding. (B) Specificity of an anti-Id antibody response after immunisation. Post-immunisation sera (patient 1) was serially diluted and tested by ELISA for binding (indicated by optical density, O.D.) to the patient's own tumor (relevant) Id versus three other patient's tumor Id proteins as controls. The solid black line indicates a highly tumor-specific antibody response, with minimal binding to other patient's tumor Ids (irrelevant; dashed lines). (C) Flow cytometric demonstration that anti-Id antibodies induced by recombinant Id-KLH vaccine specifically recognise autologous tumor cells. Tumor cells from patients 2 and 5 (upper and lower sets of panels, respectively) were incubated with either autologous post-vaccine serum or that of the other patient as a control. Bound anti-Id IgG antibodies were detected by anti-IgG-PE (Y-axis), and tumor B cells (IgM+) were counterstained by anti-IgM-FITC (X-axis). Each patient's serum specifically recognises only the autologous tumor cells, but not the control, irrelevant tumor cells. (D) Tumor Id-specific T cell proliferative response post-vaccination. Proliferation of PBMC cultured with autologous tumor Id or irrelevant control Id proteins (100 μg/mL) from other patient's tumors were measured by 3H-thymidine incorporation (counts per minute, c.p.m.). The kinetics of the T cell proliferative response to tumor versus control Id proteins during the course of the five immunisations is depicted. Maximal T cell response is achieved after the 5th injection.