Forced usage of positively charged amino acids in immunoglobulin CDR-H3 impairs B cell development and antibody production

Abstract

Tyrosine and glycine constitute 40% of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3), the center of the classic antigen-binding site. To assess the role of DH RF1-encoded tyrosine and glycine in regulating CDR-H3 content and potentially influencing B cell function, we created mice limited to a single DH encoding asparagine, histidine, and arginines in RF1. Tyrosine and glycine content in CDR-H3 was halved. Bone marrow and spleen mature B cell and peritoneal cavity B-1 cell numbers were also halved, whereas marginal zone B cell numbers increased. Serum immunoglobulin G subclass levels and antibody titers to T-dependent and T-independent antigens all declined. Thus, violation of the conserved preference for tyrosine and glycine in DH RF1 alters CDR-H3 content and impairs B cell development and antibody production.

Categorical selection of the antibody repertoire in splenic B cells

“…B cells bearing short or charged CDR-H3 appear to be acceptable or even to accumulate in the marginal zone, whereas the FO population is depleted of both and bears extensively similarity to recirculating bone marrow fraction F. This provides further support for the FO fraction serving as the major contributor to the pool of recirculating conventional B cells. In our studies of mice forced to express highly charged CDR-H3, we have observed depletion of splenic FO and bone marrow recirculating B cells coupled with enhanced numbers of MZ [7]. These studies would support the view that expression of Ig with highly polar or charged antigen binding sites either facilitates entry into the MZ compartment or is actively selected against in the FO and recirculating B cell pools.…”

“…We and others have speculated that highly hydrophobic CDR-H3 are thus less likely to create optimal paratopes [22,23] If so, the decline in hydrophobic CDR-H3 may reflect death from neglect rather than active negative selection per se. Resolution of this issue will require manipulation of the hydrophobicity characteristics of the repertoire as a whole, such as that which we have previously achieved by altering the sequence of the D H [7], combined with kinetic analysis. Unlike the T1 repertoire, which appears to reflect the composition of newly formed bone marrow immigrants, the CDR-H3 repertoires of MZ and FO differ not only from bone marrow fraction E and splenic T1 cells, they also differ from each other.…”

“…1). We compared these sets of sequences to a compendium of 255 unique, translatable sequences from bone marrow CD19 + sIgMhisIgD-fraction E and 254 sequences from CD19 + sIgM losIgD hi fraction F, including 57 fraction E and 60 fraction F sequences newly obtained for this work [6,7,9,10].…”

“…Our initial analysis of bone marrow CDR-H3 repertoire development had been performed on four separate mice derived from the same breeding pool [6]. Additional samples were independently obtained from five individual 8-10-wk-old BALB/cJ mice bred in our own mouse colony [7,10]. To increase the power of our comparisons for this work, we sorted bone marrow cells from two additional, individual 8-wk-old internally bred BALB/cJ mice.…”

“…Transition through bone marrow checkpoints acted to focus the repertoire by reducing or eliminating outliers to what appeared to be a preferred distribution of VDJ gene segment usage, D H reading frame preference, amino acid composition, length, predicted base and loop structure, and hydrophobicity. Comparisons of the repertoire expressed in IgM + IgD -immature B cells and that expressed by IgM lo IgD hi mature, recirculating B cells suggested that the process of CDR-H3 focusing might continue in the periphery [7].…”