Synthesis of secretory leukocyte protease inhibitor using cell-free protein synthesis system.

Secretory leukocyte protease inhibitor (SLPI) functions as a protease inhibitor that modulates excessive proteolysis in the body, exhibits broad-spectrum antimicrobial activity, regulates inflammatory responses, and plays an important role in the innate immunity. The purpose of the study was to artificially synthesize a SLPI, an antimicrobial peptide, and investigate its effect on antimicrobial activity against Porphyromonas gingivalis and interleukin-6 (IL-6) production. SLPI protein with a molecular weight of approximately 13 kDa was artificially synthesized using a cell-free protein synthesis (CFPS) system and investigated by western blotting and enzyme-linked immunosorbent assay (ELISA). Disulfide bond isomerase in the protein synthesis mixture increased the amount of SLPI synthesized. The synthesized SLPI (sSLPI) protein was purified and its antimicrobial activity was investigated based on the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells. The effect of sSLPI on IL-6 production in human periodontal ligament fibroblasts (HPLFs) was examined by ELISA. Our results showed that sSLPI significantly inhibited the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells and further inhibited IL-6 production by HPLFs. These results suggested that SLPI artificially synthesized using the CFPS system may play a role in the prevention of periodontal diseases through its antimicrobial and anti-inflammatory effects.

Effects of Candidalysin Derived from Candida albicans on the Expression of Pro-Inflammatory Mediators in Human Gingival Fibroblasts.

Candida albicans (Ca) is frequently detected in the peri-implant sulcus with peri-implantitis, a major postoperative complication after oral implant therapy. However, the involvement of Ca in the pathogenesis of peri-implantitis remains unclear. In this study, we aimed to clarify Ca prevalence in the peri-implant sulcus and investigated the effects of candidalysin (Clys), a toxin produced by Ca, on human gingival fibroblasts (HGFs). Peri-implant crevicular fluid (PICF) was cultured using CHROMagar and Ca colonization rate and colony numbers were calculated. The levels of interleukin (IL)-1ß and soluble IL-6 receptor (sIL-6R) in PICF were quantified by enzyme-linked immunosorbent assay (ELISA). Pro-inflammatory mediator production and intracellular signaling pathway (MAPK) activation in HGFs were measured by ELISA and Western blotting, respectively. The Ca colonization rate and the average number of colonies in the peri-implantitis group tended to be higher than those in the healthy group. IL-1ß and sIL-6R levels in the PICF were significantly higher in the peri-implantitis group than in the healthy group. Clys significantly induced IL-6 and pro-matrix metalloproteinase (MMP)-1 productions in HGFs, and co-stimulation with Clys and sIL-6R increased IL-6, pro-MMP-1, and IL-8 production levels in HGFs compared with Clys stimulation alone. These findings suggest that Clys from Ca plays a role in the pathogenesis of peri-implantitis by inducing pro-inflammatory mediators.

A novel agonist with homobivalent single-domain antibodies that bind the FGF receptor 1 domain III functions as an FGF2 ligand.

Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands. FGF2, one of the FGF ligands, is an essential factor for cell culture in stem cells for regenerative medicine; however, recombinant FGF2 is extremely unstable. Here, we successfully generated homobivalent agonistic single-domain antibodies (variable domain of heavy chain of heavy chain antibodies referred to as VHHs) that bind to domain III and induce activation of the FGF receptor 1 and thus transduce intracellular signaling. This agonistic VHH has similar biological activity (EC50) as the natural FGF2 ligand. Furthermore, we determined that the agonistic VHH could support the proliferation of human-induced pluripotent stem cells (PSCs) and human mesenchymal stem cells, which are PSCs for regenerative medicine. In addition, the agonistic VHH could maintain the ability of mesenchymal stem cells to differentiate into adipocytes or osteocytes, indicating that it could maintain the properties of PSCs. These results suggest that the VHH agonist may function as an FGF2 mimetic in cell preparation of stem cells for regenerative medicine with better cost effectiveness.

Porphyromonas gingivalis Outer Membrane Vesicles Stimulate Gingival Epithelial Cells to Induce Pro-Inflammatory Cytokines via the MAPK and STING Pathways.

Porphyromonas gingivalis (Pg) is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. Pg-OMVs are involved in the pathogenesis of periodontitis. Pg-OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of Pg-OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. Pg-OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. Pg-OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that Pg-OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that Pg-OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.

First reported case of Lachnoanaerobaculum gingivalis bacteremia in an acute myeloid leukemia patient with oral mucositis during high dose chemotherapy.

CASE DESCRIPTION: Lachnoanaerobaculum gingivalis is an obligate anaerobe identified in a human dental plaque in 2019. Here, we report the first case of L. gingivalis bacteremia in a patient with oral mucositis during chemotherapy. L. gingivalis was confirmed by 16S rRNA gene analysis but not by MALDI-TOF-MS. CONCLUSION: During chemotherapy in patients with oral mucositis, we should consider the possibility of L. gingivalis bacteremia.

An alpaca single-domain antibody (VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens.

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.

pruR and PA0065 Genes Are Responsible for Decreasing Antibiotic Tolerance by Autoinducer Analog-1 (AIA-1) in Pseudomonas aeruginosa.

Pseudomonas aeruginosa infection is considered a high-risk nosocomial infection and is very difficult to eradicate because of its tolerance to antibiotic treatment. A new compound, autoinducer analog-1 (AIA-1), has been demonstrated to reduce antibiotic tolerance, but its mechanisms remain unknown. This study aimed to investigate the mechanisms of AIA-1 in the antibiotic tolerance of P. aeruginosa. A transposon mutant library was constructed using miniTn5pro, and screening was performed to isolate high tolerant mutants upon exposure to biapenem and AIA-1. We constructed a deletion mutant and complementation strain of the genes detected in transposon insertion site determination, pruR and PA0066-65-64, and performed killing assays with antibiotics and AIA-1. Gene expression upon exposure to biapenem and AIA-1 and their relationship to stress response genes were analyzed. High antibiotic tolerance was observed in Tn5-pruR and Tn5-PA0065 transposon mutants and their deletion mutants, ΔpruR and ΔPA0066-65-64. Complemented strains of pruR and PA0066-65-64 with their respective deletion mutants exhibited suppressed antibiotic tolerance. It was determined that deletion of PA0066-65-64 increased rpoS expression, and PA0066-65-64 affects antibiotic tolerance via the rpoS pathway. Additionally, antibiotics and AIA-1 were found to inhibit pruR and PA0066-65-64. This study proposed that pruR and PA0066-65-64 are members of the antibiotic tolerance suppressors.

A Trade-off Between Thermostability and Binding Affinity of Anti-(4-hydroxy-3-nitrophenyl)Acetyl Antibodies During the Course of Affinity Maturation.

Somatic hypermutation (SHM) is one of the driving forces that increases antibody (Ab) affinity. We studied the effects of SHM on thermostability and affinity using three single-chain Fv fragments (scFvs) of anti-(4-hydroxy-3-nitrophenyl)acetyl Abs, namely 9TG, 9T7, and E11. 9TG has a germline structure that lacks SHM and is an ancestor of 9T7 with 11 mutations. E11, which has 21 mutations, is a mature Ab and has its own ancestor. The thermostabilities and antigen-Ab interactions were analyzed by circular dichroism (CD), differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Far-UV CD spectra showed that all scFvs were folded into a structure referred to as immunoglobulin-fold and were unfolded by heating at different melting temperatures. Comparison of thermodynamic parameters obtained from DSC and ITC revealed that the magnitude of stabilization free energy at 37 °C was in the order, 9TG > 9T7 > E11, while that of the free energy of interaction with antigen was 9TG < 9T7 < E11, suggesting that Abs make a trade-off between stability and affinity during affinity maturation.

Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface.

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based "mini Q-bodies" by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches.

Delicate balance among thermal stability, binding affinity, and conformational space explored by single-domain VHH antibodies.

The high binding affinities and specificities of antibodies have led to their use as drugs and biosensors. Single-domain VHH antibodies exhibit high specificity and affinity but have higher stability and solubility than conventional antibodies as they are single-domain proteins. In this work, based on physicochemical measurements and molecular dynamics (MD) simulations, we have gained insight that will facilitate rational design of single-chain VHH antibodies. We first assessed two homologous VHH antibodies by differential scanning calorimetry (DSC); one had a high (64.8 °C) and the other a low (58.6 °C) melting temperature. We then generated a series of the variants of the low stability antibody and analyzed their thermal stabilities by DSC and characterized their structures through MD simulations. We found that a single mutation that resulted in 8.2 °C improvement in melting temperature resulted in binding affinity an order of magnitude lower than the parent antibody, likely due to a shift of conformational space explored by the single-chain VHH antibody. These results suggest that the delicate balance among conformational stability, binding capability, and conformational space explored by antibodies must be considered in design of fully functional single-chain VHH antibodies.

Development of Antibodies against HPV-6 and HPV-11 for the Study of Laryngeal Papilloma.

Laryngeal papilloma (LP), which is associated with infection by human papillomavirus (HPV)-6 or -11, displays aggressive growth. The precise molecular mechanism underlying the tumorigenesis of LP has yet to be uncovered. Building on our earlier research into HPV-6, in this study, the viral gene expression of HPV-11 was investigated by quantitative PCR and DNA/RNA in situ hybridization. Additionally, newly developed antibodies against the E4 protein of HPV-6 and HPV-11 were evaluated by immunohistochemistry. The average viral load of HPV-11 in LP was 1.95 ± 0.66 × 105 copies/ng DNA, and 88% of HPV mRNA expression was found to be E4, E5a, and E5b mRNAs. According to RNA in situ hybridization, E4 and E5b mRNAs were expressed from the middle to upper part of the epithelium. E4 immunohistochemistry revealed a wide positive reaction in the upper cell layer in line with E4 mRNA expression. Other head and neck lesions with HPV-11 infection also showed a positive reaction in E4 immunohistochemistry. The distribution pattern of HPV DNA, viral mRNA, and E4 protein in LP with HPV-11 infection was quite similar to that of HPV-6. Therefore, it might be possible to apply these E4-specific antibodies in other functional studies as well as clinical applications, including targeted molecular therapies in patients with HPV-6 and HPV-11 infection.

Coordinated Expression of HPV-6 Genes with Predominant E4 and E5 Expression in Laryngeal Papilloma.

Laryngeal papilloma (LP) associated with human papillomavirus (HPV)-6 or -11 infection shows aggressive growth. However, the detailed molecular mechanism of virus-driven tumorigenesis has not been uncovered fully. HPV-6 viral gene expression and dynamic alterations were investigated with in situ localization of viral DNA and RNA in 13 patients with HPV-6-infected laryngeal papilloma. The average viral load was 4.80 × 105 ± 1.86 × 105 copies/ng DNA. E4, E5a, and E5b mRNAs accounted for 96% of the expression of 9 mRNAs. The alteration of viral DNA load during recurrence paralleled the mRNA expression levels, and the expression of all mRNAs showed a similar curve. E4, E5a, and E5b were expressed in the middle to upper part of the epithelium and were co-expressed in the same cells. E4 immunohistochemistry demonstrated an extensively positive reaction in the upper cell layer in accordance with E4 mRNA expression. These results suggest that individual viral genes are coordinately expressed for viral replication, virus release, and immunosurveillance avoidance. The newly developed E4-specific monoclonal antibody can be applied to further functional studies and clinical applications such as targeted molecular therapies.

In vitro selection of anti-gliadin single-domain antibodies from a naïve library for cDNA-display mediated immuno-PCR.

Gluten intolerance, or adverse intestinal reactions to gluten, is a fairly common problem among certain groups of people. Celiac disease is the most severe form of gluten intolerance, which can lead to permanent damage in the digestive system. Since lifelong avoidance of gluten is the only available treatment, development of reliable techniques to identify gluten contamination in food is important. Gliadin, a component of gluten, is known to play a major role in gluten toxicity. In this study, cDNA display method was used to select specific single-domain antibodies against toxic gliadin from an alpaca-derived naïve VHH library. The cDNA display method is a promising in vitro display technique, which uniquely converts an unstable mRNA-protein fusion molecule to a stable mRNA/cDNA-protein fusion molecule using a well-designed puromycin linker. Three candidate VHHs were selected and the affinities of the VHHs were observed by pulldown assay and indirect ELISA method. In addition, a novel cDNA display mediated immuno-PCR method (cD-IPCR) was successfully applied to detect gliadin in food. We believe this work demonstrates the potential application of the cDNA display method in selecting binders against toxic and heterogeneous targets such as gliadin with an immunization-free preparation manner.

Adipose tissue-derived mesenchymal stem cells ameliorate bone marrow aplasia related with graft-versus-host disease in experimental murine models.

Graft-versus-host disease (GVHD) constitutes the most frequent complications after the allogeneic hematopoietic stem cell transplantation for a variety of hematological malignancies. In the present study, we explored the prophylactic potential of adipose tissue-derived mesenchymal stem cells (AD-MSCs) in controlling GVHD in murine models with a special focus on bone marrow aplasia related with acute GVHD. The CB6F1 mice were induced GVHD by the injection intravenously of C57BL/6 (B6-Ly-5.1) splenocytes without conditioning irradiation or chemotherapy. AD-MSCs from C3H mice were injected intravenously via tail veins. GVHD was assessed using flowcytometry analysis of peripheral blood cells and histopathologic analysis of target organs. Histopathological analyses revealed that AD-MSCs markedly suppressed the infiltration of lymphocytes into liver as well as the aplasia in bone marrow. This study is the first to clarify the effectiveness of AD-MSCs against bone marrow aplasia in GVHD, supporting a rationale of AD-MSCs for ameliorating bone marrow suppression and infectivity after allo-HSCT in human clinics.

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.

Characterization and Selection of 3-(1-Naphthoyl)-Indole Derivative-Specific Alpaca VHH Antibodies Using a Phage Display Library.

A new alpaca VHH antibody library against 3-(1-naphthoyl)-indole derivatives was developed from alpaca immunized with 7-(3-(1-naphthoyl)-1H-indol-1-yl)-heptanoic acid-keyhole limpet hemocyanin (Hep-KLH) protein conjugates as the immunogen. From this library, two 3-(1-naphthoyl)-indole derivative-specific clones, named NN01 and NN02, were isolated using biopanning technology. The binding specificity of these clones was confirmed using a competitive enzyme-linked immunosorbent assay (c-ELISA). Based on the results of c-ELISA, a median inhibitory concentration (IC50) of these two VHH antibodies, NN01 and NN02, in the case of 7-(3-(1-naphthoyl)-1H-indol-1-yl)-heptanoic acid (Hep; one of 3-(1-naphthoyl)-indole derivatives) as an inhibitor exhibited an approximate 3 × 10(-7) M and 6 × 10(-7) M, respectively. Thus, VHH antibodies produced in this study could be considered a useful tool for the detection of 3-(1-naphthoyl)-indole derivatives.

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.

Generation of a Tlx1(CreER-Venus) knock-in mouse strain for the study of spleen development.

The spleen is a lymphoid organ that serves as a unique niche for immune reactions, extramedullary hematopoiesis, and the removal of aged erythrocytes from the circulation. While much is known about the immunological functions of the spleen, the mechanisms governing the development and organization of its stromal microenvironment remain poorly understood. Here we report the generation and analysis of a Tlx1(Cre) (ER) (-Venus) knock-in mouse strain engineered to simultaneously express tamoxifen-inducible CreER(T2) and Venus fluorescent protein under the control of regulatory elements of the Tlx1 gene, which encodes a transcription factor essential for spleen development. We demonstrated that Venus as well as CreER expression recapitulates endogenous Tlx1 transcription within the spleen microenvironment. When Tlx1(Cre) (ER) (-Venus) mice were crossed with the Cre-inducible reporter strain, Tlx1-expressing cells as well as their descendants were specifically labeled following tamoxifen administration. We also showed by cell lineage tracing that asplenia caused by Tlx1 deficiency is attributable to altered contribution of mesenchymal cells in the spleen anlage to the pancreatic mesenchyme. Thus, Tlx1(Cre) (ER) (-Venus) mice represent a new tool for lineage tracing and conditional gene manipulation of spleen mesenchymal cells, essential approaches for understanding the molecular mechanisms of spleen development.

Low-affinity IgM antibodies lacking somatic hypermutations are produced in the secondary response of C57BL/6 mice to (4-hydroxy-3-nitrophenyl)acetyl hapten.

Class-switched memory B cells, which are generated through the processes of somatic hypermutation (SHM) and affinity-based selection in germinal centers, contribute to the production of affinity-matured IgG antibodies in the secondary immune response. However, changes in the affinity of IgM antibodies during the immune response have not yet been studied, although IgM(+) memory B cells have been shown to be generated. In order to understand the relationship between IgM affinity and the recall immune response, we prepared hybridomas producing anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) IgM antibodies from C57BL/6 mice and from activation-induced cytidine deaminase (AID)-deficient mice. Binding analysis by ELISA showed that mAbs obtained from the secondary immune response contained IgM mAbs with affinity lower than the affinity of mAbs obtained from the primary response. By analyzing sequences of the IgM genes of hybridomas and plasma cells, we found many unmutated VH genes. VH genes that had neither tyrosine nor glycine at position 95 were frequent. The repertoire change may correlate with the lower affinity of IgM antibodies in the secondary response. The sequence and affinity changes in IgM antibodies were shown to be independent of SHM by analyzing hybridomas from AID-deficient mice. A functional assay revealed a reciprocal relationship between affinity and complement-dependent hemolytic activity toward NP-conjugated sheep RBCs; IgM antibodies with lower affinities had higher hemolytic activity. These findings indicate that lower affinity IgM antibodies with enhanced complement activation function are produced in the secondary immune response.