Restricted V(H)3 gene usage in phage-displayed Fab that are selected by intravenous immunoglobulin

Objective. To perform a comparative analysis of 1) intravenous Ig (IVIG)-bo und Fab fragments from a patient with autoimmune thrombocytopenia that had progressed to systemic lupus erythematosus (SLE) and 2) MG-selected Fabs fr om an SLE patient without thrombocytopenia. Methods. MG preparations have been successfully used to treat certain cases of autoimmune thrombocytopenia and SLE, Specific interactions of IVIG with the components of the immune system are not well characterized. To investi gate these, me had previously cloned a large number of phage-displayed IgG Fab fragments, derived from 3 patients with autoimmune thrombocytopenia, th at were specifically bound by MG molecules during panning. Many of these Fa bs reacted with platelets. Sequencing revealed that the most frequently use d V-H germline gene segments of all IVIG-bound Fabs were 3-23 and 3-30/3-30 .5, One patient's autoimmune thrombocytopenia had progressed to SLE, Using the same cloning and panning procedures, we performed a comparative analysi s of this patient's MG-bound Fab fragments and the MG-selected Fabs from an SLE patient without thrombocytopenia, Results. We observed an exclusive selection of antibodies derived from 3-23 and 3-30/3-30.5 germline segments. In contrast to the Fab fragments from t he autoimmune thrombocytopenia patient who developed SLE, none of the MG-se lected Fabs from the SLE patient without thrombocytopenia bound to thromboc ytes. Conclusion. Our results suggest a preferential interaction of a subfraction of IVIG-representative of normal Ig repertoires-with antibodies and B cell receptors derived from these 2 gene segments. Importantly, these are the m ost frequently rearranged V-H germline genes among human B cells, This kind of interaction is characteristic of a B cell superantigen, since light cha ins, antigen specificity, and the high variation in the third complementari ty-determining region 3 showed little influence on the selection of 3-23- o r 3-30/3-30.5-derived Fabs by MG. However, at least some of the contact res idues on Fabs for IVIG appear to be different from those for staphylococcal protein A and human immunodeficiency virus gp 120, The IVIG-selected Fabs may now be used to clone antibodies representative of this IVIG subfraction to study their possible regulatory influence on the B cell repertoire duri ng normal development and disease.