Germline-enforced enrichment for charged amino acids in TCR beta chain (TCRß) complementarity determining region 3 (CDR-B3) alters T cell development, repertoire content, and antigen recognition.

T cell receptor beta chain (TCRß) diversity (Dß) gene segments are highly conserved across evolution, with trout Dß1 sequence identical to human and mouse Dß1. A key conserved feature is enrichment for glycine in all three Dß reading frames (RFs). Previously, we found that replacement of mouse Dß1 with a typical immunoglobulin DH sequence, which unlike Dß is enriched for tyrosine, leads to an increase in the use of tyrosine in TCRß complementarity determining region 3 (CDR-B3) after thymic selection, altering T cell numbers, CDR-B3 diversity, and T cell function. To test whether the incorporation of charged amino acids into the Dß sequence in place of glycine would also influence T cell biology, we targeted the TCRß locus with a novel glycine-deficient DßDKRQ allele that replaces Dß1 coding sequence with charged amino acids in all three reading frames. Developing T cells using DßDKRQ expressed TCR CDR-B3s depleted of tyrosine and glycine and enriched for germline-encoded lysine, arginine, and glutamine. Total thymocytes declined in number during the process of ß selection that occurs during the transition from the DN3bc to DN4 stage. Conventional thymocyte and T cell numbers remained reduced at all subsequent thymic stages and in the spleen. By contrast, regulatory T cell numbers were increased in Peyer's patches and the large intestine. In terms of functional consequences, T cell reactivity to an ovalbumin immunodominant epitope was reduced. These findings buttress the view that natural selection of Dß sequence is used to shape the pre-immune TCRß repertoire, affecting both conventional and regulatory T cell development and influencing epitope recognition.

Loss of early B cell protein λ5 decreases bone mass and accelerates skeletal aging.

The early B cell protein λ5 is an essential component of the surrogate light chain and the preB cell receptor (preBCR), which is critical for optimal B cell development. To investigate the effect of λ5 and/or B cells on bone acquisition over time, we developed a panel of JH -/- , λ5-/-, JH -/- λ5-/-, and wild-type (WT) BALB/c mice and then studied postnatal bone development and aging in these mice at one, six, twelve, and twenty-two months of age. The trabecular bone volume over total volume (BV/TV) in JH -/- mice was similar to WT mice at all ages. In contrast, at six months of age and thereafter, λ5-/- and JH -/- λ5-/- mice demonstrated a severe decrease in trabecular bone mass. Surprisingly, bone mass in six-month-old λ5-/- and JH -/- λ5-/- mice was similar to or even lower than in aged (twenty-two-months) WT mice, suggesting accelerated skeletal aging. The postnatal development and the acquisition of cortical bone mass in JH -/- λ5-/- mice were generally comparable to WT. However, JH -/- λ5-/- mice showed a significant decrease in cortical BV/TV at six- and twelve months of age. To examine the contribution of λ5 and B cells to postnatal bone synthesis, we separately transplanted whole bone marrow cells from JH -/- λ5-/- and WT mice into irradiated JH -/- λ5-/- and WT recipients. WT recipients of JH -/- λ5-/- marrow cells failed to show acquisition of trabecular bone mass, whereas transplanting WT marrow cells into JH -/- λ5-/- recipients led to the recovery of trabecular bone mass. Transfer of WT marrow cells into JH -/- λ5-/- mice promoted synthesis of new cortical and trabecular bone. Our findings indicate that λ5 plays a major role in preserving bone mass during postnatal development and skeletal aging which is distinct from its role in B cell development. The absence of both λ5 and B cells in JH -/- λ5-/- mice leads to delayed acquisition of cortical bone during postnatal development. Dissecting the mechanism(s) by which λ5 regulates bone homeostasis may provide new avenues for the treatment of age-related loss of bone mass and osteoporosis.

Corrigendum: Loss of early B cell protein λ5 decreases bone mass and accelerates skeletal aging.

[This corrects the article DOI: 10.3389/fimmu.2022.906649.].

Bim establishes the B-cell repertoire from early to late in the immune response.

In T cell-dependent antibody responses, some of the activated B cells differentiate along extrafollicular pathways into low-affinity memory and plasma cells, whereas others are involved in subsequent germinal center (GC) formation in follicular pathways, in which somatic hypermutation and affinity maturation occur. The present study demonstrated that Bim, a proapoptotic BH3-only member of the Bcl-2 family, contributes to the establishment of the B-cell repertoire from early to late stages of immune responses to T cell-dependent antigens. Extrafollicular plasma cells grew in the spleen during the early immune response, but their numbers rapidly declined with the appearance of GC-derived progeny in wild-type mice. By contrast, conditional Bim deficiency in B cells resulted in expansion of extrafollicular IgG1+ antibody-forming cells (AFCs) and this expansion was sustained during the late response, which hampered the formation of GC-derived high-affinity plasma cells in the spleen. Approximately 10% of AFCs in mutant mice contained mutated VH genes; thus, Bim deficiency appears not to impede the selection of high-affinity AFC precursor cells. These results suggest that Bim contributes to the replacement of low-affinity antibody by high-affinity antibody as the immune response progresses.

Replacement of TCR Dß With Immunoglobulin DH DSP2.3 Imposes a Tyrosine-Enriched TCR Repertoire and Adversely Affects T Cell Development.

Enrichment for tyrosine in immunoglobulin CDR-H3 is due in large part to natural selection of germline immunoglobulin DH sequence. We have previously shown that when DH sequence is modified to reduce the contribution of tyrosine codons, epitope recognition is altered and B cell development, antibody production, autoantibody production, and morbidity and mortality following pathogen challenge are adversely affected. TCRß diversity (Dß) gene segment sequences are even more highly conserved than DH, with trout Dß1 identical to human and mouse Dß1. We hypothesized that natural selection of Dß sequence also shapes CDR-B3 diversity and influences T cell development and T cell function. To test this, we used a mouse strain that lacked Dß2 and contained a novel Dß1 allele (DßYTL) that replaces Dß1 with an immunoglobulin DH, DSP2.3. Unlike Dß1, wherein glycine predominates in all three reading frames (RFs), in DSP2.3 there is enrichment for tyrosine in RF1, threonine in RF2, and leucine in RF3. Mature T cells using DßYTL expressed TCRs enriched at particular CDR-B3 positions for tyrosine but depleted of leucine. Changing Dß sequence altered thymocyte and peripheral T cell numbers and the T cell response to an ovalbumin immunodominant epitope. The differences in tyrosine content might explain, at least in part, why TCRs are more polyspecific and of lower affinity for their cognate antigens than their immunoglobulin counterparts.

Preimmune Control of the Variance of TCR CDR-B3: Insights Gained From Germline Replacement of a TCR Dß Gene Segment With an Ig D H Gene Segment.

We have previously shown that the sequence of the immunoglobulin diversity gene segment (D H ) helps dictate the structure and composition of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3). In order to test the role of germline D sequence on the diversity of the preimmune TCRß repertoire of T cells, we generated a mouse with a mutant TCRß DJC locus wherein the Dß2-Jß2 gene segment cluster was deleted and the remaining diversity gene segment, Dß1 (IMGT:TRDB1), was replaced with DSP2.3 (IMGT:IGHD2-02), a commonly used B cell immunoglobulin D H gene segment. Crystallographic studies have shown that the length and thus structure of TCR CDR-B3 places amino acids at the tip of CDR-B3 in a position to directly interact with peptide bound to an MHC molecule. The length distribution of complementarity determining region 3 of the T cell receptor beta chain (CDR-B3) has been proposed to be restricted largely by MHC-specific selection, disfavoring CDR-B3 that are too long or too short. Here we show that the mechanism of control of CDR-B3 length depends on the Dß sequence, which in turn dictates exonucleolytic nibbling. By contrast, the extent of N addition and the variance of created CDR3 lengths are regulated by the cell of origin, the thymocyte. We found that the sequence of the D and control of N addition collaborate to bias the distribution of CDR-B3 lengths in the pre-immune TCR repertoire and to focus the diversity provided by N addition and the sequence of the D on that portion of CDR-B3 that is most likely to interact with the peptide that is bound to the presenting MHC.

IL-23 Promotes a Coordinated B Cell Germinal Center Program for Class-Switch Recombination to IgG2b in BXD2 Mice.

IL-23 promotes autoimmune disease, including Th17 CD4 T cell development and autoantibody production. In this study, we show that a deficiency of the p19 component of IL-23 in the autoimmune BXD2 (BXD2-p19-/- ) mouse leads to a shift of the follicular T helper cell program from follicular T helper (Tfh)-IL-17 to Tfh-IFN-γ. Although the germinal center (GC) size and the number of GC B cells remained the same, BXD2-p19-/- mice exhibited a lower class-switch recombination (CSR) in the GC B cells, leading to lower serum levels of IgG2b. Single-cell transcriptomics analysis of GC B cells revealed that whereas Ifngr1, Il21r, and Il4r genes exhibited a synchronized expression pattern with Cxcr5 and plasma cell program genes, Il17ra exhibited a synchronized expression pattern with Cxcr4 and GC program genes. Downregulation of Ighg2b in BXD2-p19-/- GC B cells was associated with decreased expression of CSR-related novel base excision repair genes that were otherwise predominantly expressed by Il17ra + GC B cells in BXD2 mice. Together, these results suggest that although IL-23 is dispensable for GC formation, it is essential to promote a population of Tfh-IL-17 cells. IL-23 acts indirectly on Il17ra + GC B cells to facilitate CSR-related base excision repair genes during the dark zone phase of GC B cell development.

B Cell Development and Maturation.

Since the identification of B cells in 1965 (Cooper  et al. 1965), three has been tremendous progress in our understanding of B cell development, maturation and function. A number of B cell subpopulations, including B-1, B-2 and regulatory B cells, have been identified. B-1 cells mainly originate from the fetal liver and contain B-1a and B-1b subsets. B-2 cells are derived from the bone marrow (BM) and can be further classified into follicular B (FOB) and marginal zone B (MZB) cells. Regulatory B cells (Bregs) function to suppress immune responses, primarily by production of the anti-inflammatory cytokine IL-10. B cell tolerance is established at several checkpoints, during B cell development in the BM (central tolerance) as well as during B cell maturation and activation in the periphery (peripheral tolerance). This chapter will focus on the regulation of important processes during the development and maturation of B-1 and B-2 cells.

Disruption of the preB Cell Receptor Complex Leads to Decreased Bone Mass.

In the bone marrow, preB cells are found adjacent to the bone endosteum where bone synthesizing osteoblast and bone resorbing osteoclasts reside. Although there is evidence of interactions between preB and bone cells, the factors that contribute to such interactions are poorly understood. A critical checkpoint for preB cell development assesses the integrity of the nascent immunoglobulin µ heavy chain (HC) by testing whether it can participate in the formation of a preB cell receptor (preBCR), composed of the µ HC and surrogate light chain (LC). In this work, we tested whether loss of preBCR components can affect bone synthesis. A panel of gene targeted mice with sequential blocks in preBCR formation or function [surrogate light chain component lambda 5 deleted (λ5-/-), transmembrane domain of µHC deleted (IgM-mem-/-), and CD19 preBCR co-receptor deleted (CD19-/-)] were evaluated for effects on postnatal bone synthesis. Postnatal bone mass was analyzed in 6 month old mice using µ-CT, histomorphometry and double calcein labeling. Both cortical and trabecular bone mass were significantly decreased in the femurs of the λ5 and IgM-mem deficient mice. Histomorphometric analysis showed a decrease in the numbers of osteoblasts and osteoclasts in all three mutant strains. Double calcein labeling revealed a significant decrease in dynamic synthesis and mineralization of bone in λ5-/- mice. Our data strongly suggest that interference with preBCR formation or function affects bone homeostasis independent of the presence or absence of mature B cells, and that components of the preBCR play important, and potentially distinct, roles in regulating adult bone mass.

The sequences encoded by immunoglobulin diversity (DH ) gene segments play key roles in controlling B-cell development, antigen-binding site diversity, and antibody production.

Although at first glance the diversity of the immunoglobulin repertoire appears random, there are a number of mechanisms that act to constrain diversity. For example, key mechanisms controlling the diversity of the third complementarity determining region of the immunoglobulin heavy chain (CDR-H3) include natural selection of germline diversity (DH ) gene segment sequence and somatic selection upon passage through successive B-cell developmental checkpoints. To test the role of DH gene segment sequence, we generated a panel of mice limited to the use of a single germline or frameshifted DH gene segment. Specific individual amino acids within core DH gene segment sequence heavily influenced the absolute numbers of developing and mature B-cell subsets, antibody production, epitope recognition, protection against pathogen challenge, and susceptibility to the production of autoreactive antibodies. At the tip of the antigen-binding loop (PDB position 101) in CDR-H3, both natural (germline) and somatic selection favored tyrosine while disfavoring the presence of hydrophobic amino acids. Enrichment for arginine in CDR-H3 appeared to broaden recognition of epitopes of varying hydrophobicity, but led to diminished binding intensity and an increased likelihood of generating potentially pathogenic dsDNA-binding autoreactive antibodies. The phenotype of altering the sequence of the DH was recessive for T-independent antibody production, but dominant for T-cell-dependent responses. Our work suggests that the antibody repertoire is structured, with the sequence of individual DH selected by evolution to preferentially generate an apparently preferred category of antigen-binding sites. The result of this structured approach appears to be a repertoire that has been adapted, or optimized, to produce protective antibodies for a wide range of pathogen epitopes while reducing the likelihood of generating autoreactive specificities.

In the Absence of Central pre-B Cell Receptor Selection, Peripheral Selection Attempts to Optimize the Antibody Repertoire by Enriching for CDR-H3 Y101.

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.

FCRLA-A Resident Endoplasmic Reticulum Protein that Associates with Multiple Immunoglobulin Isotypes in B Lineage Cells.

FCRLA is homologous to receptors for the Fc portion of IgG (FcγR) and is located in the same region of human chromosome one, but has several unusual and unique features. It is a soluble resident ER protein retained in this organelle by unknown mechanisms involving the N-terminal domain, a disordered domain with three Cys residues in close proximity in the human protein. Unlike the FcγRs, FCRLA is not glycosylated and has no transmembrane region. FCRLA is included in this CTMI volume on IgM-binding proteins because it binds IgM in the ER, but quite surprisingly, given the isotype-restricted ligand specificity of the other FcRs, it also binds all other Ig isotypes so far tested, IgG and IgA. In the case of IgM, there is even preferential binding of the secretory and not the transmembrane form. Among B cells, FCRLA is most highly expressed in the germinal center and shows little expression in plasma cells. Based on these observations, we propose that one human FCRLA function is to stop GC B cells from secreting IgM, which would act as a decoy receptor, thus preventing the B cells from capturing antigen, processing it, and presenting the antigen-derived peptides to T follicular helper cells. Without help from these T cells, there would be limited B cell isotype switching, proliferation, and differentiation. On the other hand, FCRLA is downregulated in plasma cells, where IgM secretion is an essential function. FCRLA may also act as a chaperone involved by unknown mechanisms in the proper assembly of Ig molecules of all isotypes.

Alterations in B cell development, CDR-H3 repertoire and dsDNA-binding antibody production among C57BL/6 ΔD-iD mice congenic for the lupus susceptibility loci sle1, sle2 or sle3.

Systemic lupus erythematosus (SLE) is an autoimmune disease that reflects a failure to block the production of self-reactive antibodies, especially those that bind double-stranded DNA (dsDNA). Backcrossing the lupus-prone NZM2410 genome onto C57BL/6 led to the identification of three genomic intervals, termed sle1, sle2 and sle3, which are associated with lupus susceptibility. We previously generated a C57BL/6 strain congenic for an immunoglobulin DH locus (ΔD-iD) that enriches for arginine at dsDNA-binding positions. We individually introduced the ΔD-iD allele into the three sle strains to test whether one or more of these susceptibility loci could affect the developmental fate of B cells bearing arginine-enriched CDR-H3s, the CDR-H3 repertoire created by the DH and the prevalence of dsDNA-binding antibodies. We found that the combination of the ΔD-iD allele and the sle1 locus led to a decrease in mature, recirculating B cell numbers and an increase in marginal zone cell numbers while maintaining a highly charged CDR-H3 repertoire. ΔD-iD and sle2 had no effect on peripheral B cell numbers, but the CDR-H3 repertoire was partially normalized. ΔD-iD and sle3 led to an increase in marginal zone B cell numbers, with some normalization of hydrophobicity. Mice with ΔD-iD combined with either sle1 or sle3 had increased production of dsDNA-binding IgM and IgG by 12 months of age. These findings indicate that the peripheral CDR-H3 repertoire can be categorically manipulated by the effects of nonimmunoglobulin genes.

VpreB serves as an invariant surrogate antigen for selecting immunoglobulin antigen-binding sites.

Developmental checkpoints eliminate B cells synthesizing defective immunoglobulin heavy (HC) and light (LC) chains. The first checkpoint tests for formation of a VpreB/λ5/µHC-containing preB-cell receptor (preBCR) and predicts whether µHCs will bind conventional LCs to form membrane IgM. VpreB and λ5 also create a sensing site that interacts with µHC antigen-binding region CDR-H3, but whether it plays a role in immunoglobulin repertoire selection and function is unknown. On a position-by-position basis, we analyzed the amino acid content of CDR-H3s from H chains cloned from living and apoptotic preB cells and from IgG:Antigen structures. Using a panel of DH gene-targeted mice, we show that progressively reducing CDR-H3 tyrosine content increasingly impairs preBCR checkpoint passage. Counting from cysteine at Framework 3 position 96, we found that VpreB particularly selects for tyrosine at CDR-H3 position 101, and that Y101 also binds antigen in IgG:Antigen structures. VpreB thus acts as an early invariant antigen. It selects for particular CDR-H3 amino acids and shapes the specificity of the IgG humoral response. This helps explain why some neutralizing antibodies against pathogens are readily produced while others are rare.

High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

During a T cell-dependent immune response, B cells undergo clonal expansion and selection and the induction of isotype switching and somatic hypermutation (SHM). Although somatically mutated IgM(+) memory B cells have been reported, it has not been established whether they are really high affinity B cells. We tracked (4-hydroxy-3-nitrophenyl) acetyl hapten-specific GC B cells from normal immunized mice based on affinity of their B cell receptor (BCR) and performed BCR sequence analysis. SHM was evident by day 7 postimmunization and increased with time, such that high affinity IgM(+) as well as IgG(+) memory B cells continued to be generated up to day 42. In contrast, class-switch recombination (CSR) was almost completed by day 7 and then the ratio of IgG1(+)/IgM(+) GC B cells remained unchanged. Together these findings suggest that IgM(+) B cells undergo SHM in the GC to generate high affinity IgM(+) memory cells and that this process continues even after CSR is accomplished.

Violation of an evolutionarily conserved immunoglobulin diversity gene sequence preference promotes production of dsDNA-specific IgG antibodies.

Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.

Regulation of VH replacement by B cell receptor-mediated signaling in human immature B cells.

VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 µHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.