Akikazu Murakami

The amino acid residue at position 95 and the third CDR region in the H chain determine the ceiling affinity and the maturation pathway of an anti-(4-hydroxy-3-nitrophenyl)acetyl antibody

Two groups of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) Abs each possessing a different amino acid, Tyr or Gly, at position 95, appeared respectively at early and late stages of immunization. The early Abs predominantly harbored Tyr95 and were referred to as the Tyr95 type. These had ∼100-fold lower ceiling affinity than the late Abs harboring Gly95, which were referred to as the Gly95 type. We found that in order to raise affinity, the Tyr95 type utilized a mutation at position 33 in V(H), while the Gly95 type used multiple mutations in both V(H) and V(L), and that the effect of the mutations was reciprocal; the former mutation had a positive effect on Tyr95 type Abs but a negative effect on Gly95 type Abs, and vice versa. The reciprocal effect of these mutations on affinity enabled us to assess the type of Abs prepared by introducing 20 different amino acids at position 95. We found that Abs harboring Lys95, Arg95, Pro95, and Tyr95 belonged to the Tyr95 type and those with Ala95 and Gly95, to the Gly95 type. Since this dependency on the amino acid at position 95 was observed in H chains whose third CDR (CDR 3H) consisted of 9 amino acids and not 11, the CDR 3H region was also considered to play an important role in determining the maturation pathway and the magnitude of the ceiling affinity.

Identification of CMTM7 as a Transmembrane Linker of BLNK and the B-Cell Receptor

BLNK is a pivotal adaptor protein in the signal transduction pathway from the IgM class B-cell receptor. BLNK is phosphorylated by Syk and binds various signaling intermediates, leading to cellular events including MAP-kinase activation, culminating in cellular activation. It remains unclear how BLNK is initially recruited to the surface IgM (sIgM) complex to which Syk is also recruited. Here we show that CMTM7, a tetra-spanning membrane protein of unknown function, co-localized with clathrin and sIgM at the plasma membrane. RNA-interference-mediated knockdown of CMTM7 expression in B cells resulted in an impairment of sIgM-ligation-induced tyrosine phosphorylation of BLNK, which was due to an impaired interaction of BLNK and Syk, and in a failure to activate JNK and ERK, but not upstream kinases such as Src-family kinases and Syk. CMTM7 was bound to BLNK in a membrane fraction, and their association was augmented after sIgM ligation. Exogenous CMTM7 or a mutant with an N-terminal deletion (ΔN), but not one with a C-terminal deletion (ΔC) that is defective in membrane localization, were able to restore BLNK-Syk binding, BLNK phosphorylation and ERK activation in the CMTM7-knockdown B cells. In addition, CMTM7 and the ΔN, but not the ΔC, were constitutively associated with sIgM, and this binding was required for BLNK recruitment to sIgM. From these data, we conclude that CMTM7 functions to link sIgM and BLNK in the plasma membrane, to recruit BLNK to the vicinity of Syk, and to initiate the BLNK-mediated signal transduction.

Evaluation of the conformational equilibrium of reduced hen egg lysozyme by antibodies to the native form

To evaluate the conformation of reduced HEL, the monoclonal antibodies HyC1 and HyC2, which recognize different conformational epitopes on native hen egg lysozyme (HEL), were used, and the kinetics of their interactions with native HEL, S-1,2-dicarboxyethylated HEL (DCE-HEL), and carboxymethylated Cys6 and Cys127 HEL (CM(6,127)-HEL) were assessed using surface plasmon resonance. Although their association rate constants differed 10(5)-fold, their dissociation rate constants were essentially the same, suggesting that DCE-HEL and CM(6,127)-HEL possess conformations similar to that of native HEL when they bind antibodies. We considered that the ratio of the association rate constant of reduced HEL to native HEL represents the proportion of the native format determinant in equilibrium. Reduction of the Cys6-Cys127 disulfide bond would transform the epitope recognized by HyC1 into a non-native conformation similar to that of DCE-HEL. We show that monoclonal antibodies provide a sensitive tool for evaluation of the structural and hydrodynamic changes of proteins.

Characterization of memory B cells responsible for affinity maturation of anti- (4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies

We searched for memory B cells responsible for high-affinity anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody production by C57BL/6 mice immunized with NP-chicken γ-globulin (CGG), using flow cytometry. We first prepared transfectants expressing B-cell antigen receptor (BCR) of known affinity as a memory B-cell model as well as NP-allophycocyanin (APC) of different NP valences, NP(lo), NP(med) and NP(hi). We then used the latter as probes capable of distinguishing BCR affinities: NP(lo)-APC bound to BCRs with an affinity higher than 3.4 × 10(6) M(-1), while NP(med)-APC bound to those with a higher than germline affinity. B cells capable of binding to NP(lo)-APC appeared in spleens on day 14 post-immunization, and harbored Tyr95 (Tyr95 type) as well as a mutation from Trp33 to Leu. B cells with BCRs harboring Gly95 (Gly95 type) appeared only in the NP(med)-APC-binding fraction on day 56 and in the NP(lo)-APC-binding fraction on day 77, indicating that this long duration was necessary for Gly95 type B cells to acquire high affinity and to become a member of the group of memory B cells with high affinity. Administration of NP-CGG on day 77 caused little change in the proportion of the Gly95 type in NP(lo)-APC-binding B cells in the following 2 weeks but brought about an increase in the number of high-affinity antibody-secreting cells (ASC), suggesting that the memory B-cell compartment established was maintained at a later stage and supplied high-affinity ASCs. The relationship between these Gly95 type memory B cells and ASCs is discussed.

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.

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.

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 Tlx1CreER‐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 Tlx1CreER‐Venus knock‐in mouse strain engineered to simultaneously express tamoxifen‐inducible CreERT2 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 Tlx1CreER‐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, Tlx1CreER‐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. genesis 52:916–923, 2014.

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.