Influence of pre-B cell receptor deficiency on the immunoglobulin repertoires in peripheral blood B cells before and after immunization.

During B cell development, pre-B cell receptor (pre-BCR), comprising the immunoglobulin heavy chain (HC) and surrogate light chain (SLC), plays a crucial role. The expression of pre-BCR serves as a certification of HC quality, confirming its ability to associate with the SLC and light chain (LC). In mice lacking SLC, the absence of this quality control mechanism leads to a distorted repertoire of HCs in the spleen and bone marrow. In this study, we conducted a comparative analysis of the immunoglobulin gene repertoire in peripheral blood cells of both wild-type mice and pre-BCR-deficient mice. Our findings reveal differences not only in the µ HC repertoire but also in the α HC and κ LC repertoires of the pre-BCR-deficient mice. These results suggest that the pre-BCR-mediated quality check of HC influences the selection of class-switched HC and LC repertoires. To further explore the impact of pre-BCR deficiency, we immunized these mice with thymus-dependent antigens and compared the antigen-responding repertoires. Our observations indicate that the affinity maturation pathways remain consistent between wild-type mice and pre-BCR-deficient mice, albeit with variations in the degree of maturation.

Elucidation of binding mechanism, affinity, and complex structure between mWT1 tumor-associated antigen peptide and HLA-A*24:02.

We have applied our advanced computational and experimental methodologies to investigate the complex structure and binding mechanism of a modified Wilms' Tumor 1 (mWT1) protein epitope to the understudied Asian-dominant allele HLA-A*24:02 (HLA-A24) in aqueous solution. We have applied our developed multicanonical molecular dynamics (McMD)-based dynamic docking method to analyze the binding pathway and mechanism, which we verified by comparing the highest probability structures from simulation with our experimentally solved x-ray crystal structure. Subsequent path sampling MD simulations elucidated the atomic details of the binding process and indicated that first an encounter complex is formed between the N-terminal's positive charge of the 9-residue mWT1 fragment peptide and a cluster of negative residues on the surface of HLA-A24, with the major histocompatibility complex (MHC) molecule preferring a predominantly closed conformation. The peptide first binds to this closed MHC conformation, forming an encounter complex, after which the binding site opens due to increased entropy of the binding site, allowing the peptide to bind to form the native complex structure. Further sequence and structure analyses also suggest that although the peptide loading complex would help with stabilizing the MHC molecule, the binding depends in a large part on the intrinsic affinity between the MHC molecule and the antigen peptide. Finally, our computational tools and analyses can be of great benefit to study the binding mechanism of different MHC types to their antigens, where it could also be useful in the development of higher affinity variant peptides and for personalized medicine.

Carboxylated polyamidoamine dendron-bearing lipid-based assemblies for precise control of intracellular fate of cargo and induction of antigen-specific immune responses.

For the establishment of advanced medicines such as cancer immunotherapy, high performance carriers that precisely deliver biologically active molecules must be developed to target organelles of the cells and to release their contents there. From the viewpoint of antigen delivery, endosomes are important target organelles because they contain immune-response-related receptors and proteins of various types. To obtain carriers for precision endosome delivery, a novel type of polyamidoamine dendron-based lipid having pH-sensitive terminal groups was synthesized for this study. Liposomes were prepared using these pH-sensitive dendron-based lipids and egg yolk phosphatidylcholine. Their pH-responsive properties and performance as an endosome delivery carrier were investigated. pH-Sensitive dendron lipid-based liposomes retained water-soluble molecules at neutral pH but released them under weakly acidic conditions. Particularly, liposomes containing CHexDL-G1U exhibited highly sensitive properties responding to very weakly acidic pH. These dendron lipid-based liposomes released the contents specifically in the endosome. The timing of content release can be controlled by selecting pH-sensitive dendron lipids for liposome preparation. Significant tumor regression was induced in tumor-bearing mice by the administration of CHexDL-G1U-modified liposomes containing the model antigenic protein. Furthermore, CHexDL-G1U-modified liposomes induced WT1 tumor antigenic peptide-specific helper T cell proliferation. The results demonstrate that dendron lipid-based liposomes are useful as a potent vaccine for cancer immunotherapy.

Development of a novel monoclonal antibody that binds to most HLA-A allomorphs in a conformation-dependent yet peptide-promiscuous fashion.

Specificity analyses of peptide binding to human leukocyte antigen (HLA)-A molecules have been hampered due to a lack of proper monoclonal antibodies (mAbs) for certain allomorphs, such as the prevalent HLA-A1 for Caucasians and HLA-A11 for Asians. We developed a mAb that recognizes a conformational epitope common to most HLA-A allomorphs. The mAb, named A-1, does not discriminate peptides by amino acid sequences, making it suitable for measuring peptide binding. A stabilization assay using TAP-deficient cell lines and A-1 was developed to investigate the specificity of peptide binding to HLA-A molecules. Regarding the evolution of HLA-A genes, the A-1 epitope has been conserved among most HLA-A allomorphs but was lost when the HLA-A gene diversified into the HLA-A*32, HLA-A*31, and HLA-A*33 lineages together with HLA-A*29 after bifurcating from the HLA-A*25 and HLA-A*26 branchs. The establishment of A-1 is expected to help researchers investigate the peptide repertoire and develop computational tools to identify cognate peptides. Since no HLA-A locus-specific mAb has been available, A-1 will also be useful for analyzing the locus-specific regulation of the HLA gene expression.

High-affinity IgM+ memory B cells are defective in differentiation into IgM antibody-secreting cells by re-stimulation with a T cell-dependent antigen.

IgM antibodies (Abs) are thought to play a major role in humoral immunity but only at the early stage of the primary immune response. However, two subsets of IgM+ memory B cells (MBCs), one with high affinity gained by means of multiple somatic hypermutation (SHM) and the other with low affinity and no SHMs, are generated through the germinal center (GC)-dependent and GC-independent (non-GC) pathway, respectively, after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP)-chicken γ-globulin. Surprisingly, an analysis of antibody-secreting cells reveals that a large amount of anti-NP IgM Ab with few SHMs is secreted during the recall response, indicating that only non-GC MBCs have terminal differentiation potential. Since secondary IgM Abs are capable of binding to dinitrophenyl ligands, they likely provide broad cross-reactivity in defense against microbial infection.

Distorted antibody repertoire developed in the absence of pre-B cell receptor formation.

The pre-B cell receptor (pre-BCR), consisting of the µ heavy chain (µHC) and the surrogate light chain (SLC, Vpre-B and λ5), plays important roles during B cell development. The formation of the pre-BCR, which enables the nascent immunoglobulin HC to associate with the SLC, is considered a prerequisite for B cell development. However, a significant number of peripheral mature (leaky) B cells exist in SLC-deficient mice. These leaky B cells develop in the absence of pre-BCR and do not undergo the pre-BCR checkpoint. The antibody repertoires of leaky B cells thus reflect the absence of pre-BCR function. To investigate how the absence of the pre-BCR is circumvented by these leaky-B cells and examine the effect of the pre-BCR checkpoint on the antibody system, we analyzed the antibody repertoires of λ5-deficient (λ5-/-) mice using next-generation sequencing. In λ5-/- mice, spleen B cells displayed different patterns of VDJ-usage, relative to those in wild-type (WT) mice. Moreover, leaky B cells were neither derived from unusual B2 cells, characterized by particular LC gene rearrangements in the absence of pre-BCR signaling, nor from B1 cells, originating from different B cell progenitors. Analysis of the CDR-H3 amino acid sequences of µ-chain repertoires revealed that certain bone marrow B cells with particular CDR-H3 profiles undergo clonal expansion in λ5-/- mice. Part of these CDR-H3s contain arginine(s) in the middle of the CDR-H3 loop in λ5-/- mice, whereas few arginine(s) exist in this middle loop in WT CDR-H3s in the absence of clonal expansion. This CDR-H3 feature in λ5-/- mice presumably reflects the role of the pre-BCR in autoantibody regulation, since arginine(s) are often found in the antigen-binding site of autoantibodies. Here, we present a unique viewpoint on the role of pre-BCR, by assessing the whole antibody repertoire formed in SLC-deficient mice.

Deciphering antigen-responding antibody repertoires by using next-generation sequencing and confirming them through antibody-gene synthesis.

Vast diversity and high specificity of antigen recognition by antibodies are hallmarks of the acquired immune system. Although the molecular mechanisms that yield the extremely large antibody repertoires are precisely understood, comprehensive description of the global antibody repertoire generated in individual bodies has been hindered by the lack of powerful measures. To obtain holistic understanding of the antibody-repertoire space, we used next-generation sequencing (NGS) to analyze the deep profiles of naive and antigen-responding repertoires of the IgM, IgG1, and IgG2c classes formed in individual mice. The overall landscapes of naive IgM repertoires were almost the same for each mouse, whereas those of IgG1 and IgG2c differed considerably among naive individuals. Next, we immunized mice with a model antigen, nitrophenol (NP)-hapten linked to chicken γ-globulin (CGG) carrier, and compared the antigen-responding repertoires in individual mice. To extract the complete antigen response, we developed an intelligible method for detecting common components of antigen-responding repertoires. The major responding antibodies were IGHV1-72/IGHD1-1/IGHJ2 for NP-hapten and IGHV9-3/IGHD3-1/IGHJ2 for CGG-carrier protein. The antigen-binding specificities of the identified antibodies were confirmed through ELISA after antibody-gene synthesis and expression of the corresponding NGS reads. Thus, we deciphered antigen-responding antibody repertoires by inclusively analyzing the antibody-repertoire space generated in individual bodies by using NGS, which avoided inadvertent omission of key antibody repertoires.

Somatic hypermutation of immunoglobulin genes in Rad18 knockout mice.

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is triggered by the activity of activation-induced cytidine deaminase (AID). AID induces DNA lesions in variable regions of Ig genes, and error-prone DNA repair mechanisms initiated in response to these lesions introduce the mutations that characterize SHM. Error-prone DNA repair in SHM is proposed to be mediated by low-fidelity DNA polymerases such as those that mediate trans-lesion synthesis (TLS); however, the mechanism by which these enzymes are recruited to AID-induced lesions remains unclear. Proliferating cell nuclear antigen (PCNA), the sliding clamp for multiple DNA polymerases, undergoes Rad6/Rad18-dependent ubiquitination in response to DNA damage. Ubiquitinated PCNA promotes the replacement of the replicative DNA polymerase stalled at the site of a DNA lesion with a TLS polymerase. To examine the potential role of Rad18-dependent PCNA ubiquitination in SHM, we analyzed Ig gene mutations in Rad18 knockout (KO) mice immunized with T cell-dependent antigens. We found that SHM in Rad18 KO mice was similar to wild-type mice, suggesting that Rad18 is dispensable for SHM. However, residual levels of ubiquitinated PCNA were observed in Rad18 KO cells, indicating that Rad18-independent PCNA ubiquitination might play a role in SHM.

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.

An asymmetric antibody repertoire is shaped between plasmablasts and plasma cells after secondary immunization with (4-hydroxy-3-nitrophenyl)acetyl chicken γ-globulin.

Studies on the structural basis of antibody affinity maturation have been carried out by measuring the affinity of secreted antibodies, and information on structures has often been obtained from nucleotide sequences of BCRs of memory B cells. We considered it important to establish whether the repertoire of secreted antibodies from plasma cells is really in accord with that of BCRs on memory B cells at the same time points post-immunization. We isolated plasma cells secreting antibodies specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten by affinity matrix technology using biotin-anti-CD138 and streptavidin-NP-allophycocyanin, to which anti-NP antibodies secreted by autologous plasma cells bound preferentially. We found that plasmablasts occupied >90% of the antibody-secreting cell compartment in the primary response and that they secreted antibodies whose VH regions were encoded by V186.2(+)Tyr95(+) sequences, which provided an increase in the medium level of affinity by somatic hypermutation (SHM) of heavy chains at position 33. After secondary immunization, a further increase in antibody affinity was observed, which was explained by the appearance of a number of plasma cells secreting V186.2(+)Gly95(+) antibodies that acquired high affinity by multiple SHMs as well as plasmablasts secreting V186.2(+)Tyr95(+) antibodies. However, we did not detect any plasmablasts secreting V186.2(+)Gly95(+) antibodies, showing that plasmablasts and plasma cells have a different antibody repertoire, i.e. their respective repertoires are asymmetric. On the basis of these findings, we discussed the relationship between the BCR affinity of memory B cells and plasmablasts as well as plasma cells as pertaining to their ontogeny.