Splenic Immunoglobulin Variable Region Genes Encoding Red Blood Cell Binding Fab Fragments in Autoimmune Hemolytic Anemia

Abstract

An absolute requirement for the V_H4–34 immunoglobulin (Ig) variable (V) region heavy chain (V_H) gene has been demonstrated in pathogenic cold agglutinin autoantibodies. Investigation of IgG binding anti-Rhesus (Rh) alloantibodies provides further evidence of V gene restriction in red blood cell (RBC) binding antibodies and demonstrates that the V_H4–34 gene used to form cold agglutinins may also encode RBC antibodies of varied specificities. We reasoned that a similar V gene restriction may be evident in the gene segments encoding IgG anti-RBC autoantibodies mediating autoimmune hemolytic anemia (AIHA). To further examine this question IgG Fab fragment phage display libraries were constructed from the spleen of a patient with AIHA. The index autoantibody appeared to have incomplete anti-C specificity and bound all panel RBCs except Rh null. The Fab fragment phage display libraries were therefore panned twice on CDE/CDe RBCs and binding phage were eluted. Binding of the phage displayed Fab fragments to RBCs was confirmed by immunoflourescence and flow cytometry. Specificity was confirmed by the absence of binding to Rh null cells, murine RBCs and to human peripheral blood lymphocytes. Molecular analysis of Ig V genes encoding the pan RBC binding Fab fragments revealed a relative V_H gene restriction and evidence of somatic mutation. The V_H3 family member V_H26 was prominent in RBC binding Fabs. The V_H3 family member hV3005 and the V_H4 family DP-65 gene segments also encoded RBC binding Fabs. The J_H4 gene segment was present in all binding clones. Varied kappa and lambda light chain (V_L) genes were identified by sequencing and no single light chain was prevalent. Three of the ten V_L and two of the three V_H identified by sequencing appeared to derive from germline genes previously noted to have RBC binding specificity. We conclude that splenic Ig V genes can encode pan RBC binding antibodies with specificities similar to autoantibodies found in AIHA and that V_H gene segment utilization by these antibodies is derived from a limited pool of somatically mutated V_H gene segments.

Keywords:

• Autoimmunity
• hemolysis
• immunoglobulin
• heavy chain
• gene