Anti-CD34+ Fabs generated against hematopoietic stem cells in HIV-derived combinatorial immunoglobulin library...

Bone marrow suppression associated with HIV infection does not appear to be solely due to direct viral cytopathic effects. Autoantibodies may play a role in myelosuppression, however it is unclear whether autoantibodies produced in HIV infection represent a primary pathogenic process or merely reflect polyclonal B cell activation. To address these questions, we generated combinatorial immunoglobulin libraries using the pComb3 phagemid from an HIV+ individual with evidence of circulating autoantibodies. From one library, three anti-CD34 Fabs were identified using fresh CD34+ cells as antigenic targets by a method of phage subtraction. The anti-CD34 Fabs are specific by immunoblotting and Elisa and are of high affinity, with calculated Kds in the range of 10(-7) -10(-8) M. Nucleic acid sequencing revealed all three to be of the VH3 family and to have lambda light chains with some gene segments expressing little somatic mutation, while other segments were somatically mutated in patterns suggestive of antigen selection. These findings indicate that (1) A subset of HIV-associated anti-CD34 autoantibodies are monospecific and antigen-selected and are not merely a consequence of polyclonal B cell activation and elevated Ig levels in HIV. Autoreactivity in HIV therefore includes both polyspecific, low affinity antibodies as well as monospecific antigen-selected high affinity antibodies. (2) Although bone marrow suppression in HIV is likely to be multifactorial, autoantibodies to hematopoietic stem cells may contribute to its pathogenesis. (3) Library sampling of VH gene family rearrangements shows no evidence for under-representation of the VH3 family in the immune dysregulation of HIV infection. Phage subtraction is corroborated to be an effective means of identifying, cloning, and characterizing antibodies to hematopoietic differentiation antigens.