Clonal Progression during the T Cell-Dependent B Cell Antibody Response Depends on the Immunoglobulin DH Gene Segment Repertoire.

Ahmad Trad,Hans Lange,Michael Zemlin,Radu Iulian Tanasa,Harry W Schroeder,Hilmar Lemke

doi:10.3389/fimmu.2014.00385

Abstract

The diversity of the third complementarity determining region of the IgH chain is constrained by natural selection of immunoglobulin diversity (DH) sequence. To test the functional significance of this constraint in the context of thymus-dependent (TD) immune responses, we immunized BALB/c mice with WT or altered DH sequence with 2-phenyloxazolone-coupled chicken serum albumin (phOx-CSA). We chose this antigen because studies of the humoral immune response to the hapten phOx were instrumental in the development of the current theoretical framework on which our understanding of the forces driving TD responses is based. To allow direct comparison, we used the classic approach of generating monoclonal Ab (mAb) from various stages of the immune response to phOx to assess the effect of changing the sequence of the DH on clonal expansion, class switching, and affinity maturation, which are hallmarks of TD responses. Compared to WT, TD-induced humoral IgM as well as IgG antibody production in the D-altered ΔD-DμFS and ΔD-iD strains were significantly reduced. An increased prevalence of IgM-producing hybridomas from late primary, secondary, and tertiary memory responses suggested either impaired class switch recombination (CSR) or impaired clonal expansion of class switched B cells with phOx reactivity. Neither of the D-altered strains demonstrated the restriction in the VH/VL repertoire, the elimination of VH1 family-encoded antibodies, the focusing of the distribution of CDR-H3 lengths, or the selection for the normally dominant Ox1 clonotype, which all are hallmarks of the anti-phOx response in WT mice. These changes in clonal selection and expansion, as well as CSR indicate that the genetic constitution of the DH locus, which has been selected by evolution, can strongly influence the functional outcome of a TD humoral response.

Highlights

In immunoglobulins, juxtaposition of the three complementary determining regions (CDRs) of the L chain and the three of the H chain creates the site at which antigen binds [1, 2]
We found that changing conserved elements of the sequence of the diversity gene segment locus led to the failure to select for the use of VHOx1/VκOx1 gene combination, the failure to yield the normal focusing of CDRH3 sequence, and the loss of Ox1 idiotype (IdOx1) dominance
In WT BALB/c, the preference for IdOx1 anti-phOx Ab reflects both the failure of B cells producing phOx-binding IgM antibodies that use VH1-family genes to contribute to the IgG Ab repertoire and a focusing of CDR-H3 content [10]

Summary

Introduction

Juxtaposition of the three complementary determining regions (CDRs) of the L chain and the three of the H chain creates the site at which antigen binds [1, 2]. While CDRs 1 and 2 are entirely of germline origin and CDR-L3 is largely so, CDR-H3 is the direct product of VDJ rearrangement and N nucleotide addition [3]. This makes CDR-H3 the focus for preimmune Ig diversity. In combination, this diversity and its physical location at the center of the antigen binding site tends to endow CDR-H3 with the ability to define the antigen binding specificity and affinity of the antibody. Analyses of anti-hapten immune responses have been crucial for the dissection of the roles played by T cells in initiating and regulating humoral immune maturation. Immune maturation in the classic humoral immune response of BALB/c mice to the hapten

Methods

Results

Conclusion