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

An absolute requirement for the V(H(4-34) ) 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(H(4-34) ) 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(H(3) ) family member V(H(26) ) was prominent in RBC binding Fabs. The V(H(3) ) family member hV3005 and the V(H(4) ) family DP-65 gene segments also encoded RBC binding Fabs. The J(H(4) ) 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.