All ETDs from UAB

Advisory Committee Chair

Harry W Schroeder

Advisory Committee Members

Peter Burrows

Thomas P Atikinson

John F Kearney

David E Briles

Document Type

Dissertation

Date of Award

2016

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Part I. Mouse Studies Complementarity determining region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used ELISA or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity, but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germline sequence.This may help explain why antibodies in HIV infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization. Part II. Human studies In addition to the mouse studies, we have tested Ig repertoire usage in our patients with Common Variable Immune Deficiency, or CVID. CVID is a diagnostic category containing a mixture of patients of differing genotypes who present as a common phenotype of recurrent sino-pulmonary infections (RESPI) and panhypogammaglobulinemia, but who have several different underlying pathogenic etiologies. With regards to our studies, we have identified HLA-B*44 positive identical female twins who suffer sino-pulmonary infections and are discordant for CVID and RESPI. Additionally, discordance in terms of Ig repertoire gene usage was observed between the twins. Furthermore, to gain a broader insight on the CVID molecular and genetic mechanisms underneath, we have enlarged the patient cohorts and tested NK killer immunoglobulin-like receptors (KIR) and KIR/HLA compound genes distribution frequency discordance between patients group and healthy controls. Significant differences in terms of gene frequency between patients and controls were seen for KIR2DL5, 2DS1, 3DS1 (p<0.05). To our knowledge, this is the first time an association of KIR with CVID has been identified. Several KIR/HLA compound genotypes, including KIR3DS1+HLA-Bw4 (p<0.05), 3DS1+HLA-Bw*480T (p<0.005), 2DL1+Group C2 (p<0.005), homozygous 2DL3+Group C1 showed significant associations. Interestingly the activating KIR3DS1/Bw4 genotype was associated with increased risk of disease, whereas the inhibitory KIR2DL1/C2 genotype was protective, suggesting a discordant role for NK cell activation in disease pathogenesis. These findings warrant functional studies to define the role of NK cells and/or T cells in the pathogenesis of CVID, and suggest the possibility of applying both KIR and HLA ligand genotyping to predict disease risk.

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