Abstract
Sequential developmental checkpoints are used to “optimize” the B cell antigen receptor repertoire by minimizing production of autoreactive or useless immunoglobulins and enriching for potentially protective antibodies. The first and apparently most impactful checkpoint requires μHC to form a functional pre-B cell receptor (preBCR) by associating with surrogate light chain, which is composed of VpreB and λ5. Absence of any of the preBCR components causes a block in B cell development that is characterized by severe immature B cell lymphopenia. Previously, we showed that preBCR controls the amino acid content of the third complementary determining region of the H chain (CDR-H3) by using a VpreB amino acid motif (RDR) to select for tyrosine at CDR-H3 position 101 (Y101). In antibodies bound to antigen, Y101 is commonly in direct contact with the antigen, thus preBCR selection impacts the antigen binding characteristics of the repertoire. In this work, we sought to determine the forces that shape the peripheral B cell repertoire when it is denied preBCR selection. Using bromodeoxyuridine incorporation and evaluation of apoptosis, we found that in the absence of preBCR there is increased turnover of B cells due to increased apoptosis. CDR-H3 sequencing revealed that this is accompanied by adjustments to DH identity, DH reading frame, JH, and CDR-H3 amino acid content. These adjustments in the periphery led to wild-type levels of CDR-H3 Y101 content among transitional (T1), mature recirculating, and marginal zone B cells. However, peripheral selection proved incomplete, with failure to restore Y101 levels in follicular B cells and increased production of dsDNA-binding IgM antibodies.
Highlights
In bone marrow and fetal liver, immunoglobulin (Ig) heavy (H) chain genes are generated de novo through variable (V), diversity (D), and joining (J) gene segment rearrangement and non-germline encoded N addition [1]
We report an analysis of the developing repertoire in BALB/c B lineage cells deficient in λ5 (λ5KO) and denied central pre-B cell receptor (preBCR) selection
The sequences we obtained from transitional (T1), mature recirculating bone marrow, and marginal zone (MZ) B cells demonstrate no statistically significant differences between the prevalence of Y101 in λ5KO versus wild-type (WT) BALB/c mice. These findings suggest that in addition to the preBCR checkpoint, one or more peripheral quality control (QC) checkpoints between the immature cell stage and mature, IgM/IgD bearing splenic B cell stages favor the survival of B cells bearing Igs with CDR-H3 Y101
Summary
In bone marrow and fetal liver, immunoglobulin (Ig) heavy (H) chain genes are generated de novo through variable (V), diversity (D), and joining (J) gene segment rearrangement and non-germline encoded N addition [1]. The site of VDJ juxtaposition, which is the focus for repertoire diversification, is termed H chain complementarity determining region 3 (CDR-H3). Peripheral Selection Upon preBCR Deficiency at the center of the antigen binding site allows it to often play a determinative role in antigen recognition and binding [2]. Highly variable in sequence, in both human and mouse, CDR-H3 is remarkably enriched for tyrosine [3]. To test the role of DH RFs in tyrosine enrichment, we created mice with altered DH to promote use of non-tyrosineenriched RF sequence. Tyrosine usage declined [7], supporting the view that natural selection of DH sequence partly predetermines the amino acids at the center of the antigen binding site
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