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With the use of a mouse FDC line, FL-Y, we have been analyzing roles for FDCs in controlling B cell fate in GCs. Beside these regulatory functions, we fortuitously found that FL-Y cells induced a new type of CD11b⁺ monocytic cells (F4/80⁺, Gr-1⁻, Ly6C⁻, I-A/E(-/lo), CD11c⁻, CD115⁺, CXCR4⁺, CCR2⁺, CX₃CR1⁻) when cultured with a Lin⁻c-kit⁺ population from mouse spleen cells. The developed CD11b⁺ cells shared a similar gene-expression profile to mononuclear phagocytes and were designated as FDMCs. Here, we describe characteristic immunological functions and the induction mechanism of FDMCs. Proliferation of anti-CD40 antibody-stimulated B cells was markedly accelerated in the presence of FDMCs. In addition, the FDMC-activated B cells efficiently acquired GC B cell-associated markers (Fas and GL-7). We observed an increase of FDMC-like cells in mice after immunization. On the other hand, FL-Y cells were found to produce CSF-1 as well as IL-34, both of which are known to induce development of macrophages and monocytes by binding to the common receptor, CSF-1R, expressed on the progenitors. However, we show that FL-Y-derived IL-34, but not CSF-1, was selectively responsible for FDMC generation using neutralizing antibodies and RNAi. We also confirmed that FDMC generation was strictly dependent on CSF-1R. To our knowledge, a CSF-1R-mediated differentiation process that is intrinsically specific for IL-34 has not been reported. Our results provide new insights into understanding the diversity of IL-34 and CSF-1 signaling pathways through CSF-1R.

In germinal centers (GCs), B cells are selected through interaction with follicular dendritic cells bearing immune complexes and follicular helper T (Tfh) cells secreting Tfh cytokines, including IL-21. To analyze these cellular interactions, we have explored culture conditions that can simulate GC B cell selection in vitro using a mouse follicular dendritic cell line, FL-YB. FL-YB cells efficiently enhanced viability of cocultured mouse B cells in a BAFF-dependent fashion. Interestingly, we found that addition of IL-21, a major Tfh cytokine, readily induced death of B cells that were cocultured with FL-YB cells, whereas IL-21 alone sustained viability of B cells in the absence of FL-YB cells. The IL-21-induced death was dependent on a low m.w. soluble factor that was released from FL-YB cells, which was finally identified as PGE(2). Treatment of B cells with IL-21 plus PGE(2), but not either alone, resulted in enhanced expression of a proapoptotic protein Bim and the upstream transcription factor Foxo1. A PGE(2) receptor isoform, EP4, was responsible for IL-21/PGE(2)-induced B cell death. Thus, PGE(2) is an endogenous chemical mediator that can switch pleiotropic actions of IL-21 on B cells. IL-21/PGE(2)-induced B cell death was rescued if B cells were costimulated via CD40. In immunized mice, deficiency of IL-21R in B cells led to a significant decrease in the frequency of activated caspase-3-positive GC B cells concomitant with impaired affinity maturation of Abs. Taken together, results implicate a physiological role of IL-21/PGE(2)-induced B cell death in GC B cell selection.

Chicken B cell line DT40 continuously accumulates mutations in the immunoglobulin variable region (IgV) gene by gene conversion and point mutation, both of which are mediated by activation-induced cytidine deaminase (AID), thereby producing an antibody (Ab) library that is useful for screening monoclonal Abs (mAbs) in vitro. We previously generated an engineered DT40 line named DT40-SW, whose AID expression can be reversibly switched on or off, and developed an in vitro Ab generation system using DT40-SW cells. To efficiently create an Ab library with sufficient diversity, higher hypermutation frequency is advantageous. To this end, we generated a novel cell line DT40-SWDeltaC, which conditionally expresses a C-terminus-truncated AID mutant lacking the nuclear export signal. The transcription level of the mutant AID gene in DT40-SWDeltaC cells was similar to that of the wild-type gene in DT40-SW cells. However, the protein level of the truncated AID mutant was less than that of the wild type. The mutant protein was enriched in the nuclei of DT40-SWDeltaC cells, although the protein might be highly susceptible to degradation. In DT40-SWDeltaC cells, both gene conversion and point mutation occurred in the IgV gene with over threefold higher frequency than in DT40-SW cells, suggesting that a lower level of the mutant AID protein was sufficient to increase mutation frequency. Thus, DT40-SWDeltaC cells may be useful for constructing Ab libraries for efficient screening of mAbs in vitro., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Monoclonal antibodies (mAb) have recently proven to be excellent biopharmaceutical agents. The generation of hybridomas from antigen-stimulated B cells has been a key technology for obtaining mAbs; however, it is a laborious and time-consuming process, and sometimes mAbs for molecules conserved between species are difficult to obtain because of immunological tolerance. Thus, it is of great importance to develop in vitro technologies for generating useful Abs as drug candidates. We have been attempting to develop a novel in vitro antibody generation system using a chicken B cell line DT40, which displays Abs and mutates Ig genes during culture, thereby generating a useful Ab library for screening mAbs. First, we generated an engineered cell line DT40-SW whose mutation machinery can be reversibly switched on and off. The Ab generation system using DT40-SW is useful in the following ways: (1) mAbs for various model antigens including antoantigens can be obtained from the DT40-SW Ab library that is free from immunological tolerance; (2) the switching device of the mutation machinery enables fixing desirable Ig mutants by stopping mutation; (3) by repeated culture and sorting of clones bearing higher affinity for target antigens, affinity maturation can be mimicked in vitro. We have also genetically improved DT40-SW cells for mutation efficiency and Ab production. The Ab generation system will be applicable for obtaining valuable Abs such as antitumor Abs.

The quasimonoclonal mouse is useful to examine B cell selection during T-dependent antibody (Ab) responses because of its limited B cell populations mainly expressing the knockin 17.2.25 V(H)-encoded H chain (V(H)T) paired with the lambda1 or lambda2 L chain. It has been reported that both two V(H)T/lambda1 and V(H)T/lambda2 B cell populations responded to a T-dependent antigen conjugated with a hapten p-nitrophenylacetyl (pNP), but only V(H)T/lambda2 B cells differentiated to secrete high affinity anti-pNP IgG Abs by acquiring a critical mutation (T313A) in the V(H)T. The V(H)T/lambda2 B cells may be more potent in migrating to the germinal centers (GCs) due to about 50-fold higher affinity for pNP than V(H)T/lambda1 B cells. Here, to uncover how V(H)T/lambda2 B cells were preferentially recruited for affinity maturation during the anti-pNP Ab response, we examined the L chain usage and mutation frequency of V(H)T(+) GC B cells at a single cell level. V(H)T/lambda2 B cells bearing the unmutated V(H)T gene were found in the GCs more frequently than V(H)T/lambda1 and mutated V(H)T/lambda2 counterparts in an early phase of the Ab response. In the course of the GC reaction, the number of V(H)T/lambda2 B cells that mutated their V(H)T genes preferentially expanded, and finally V(H)T/lambda2 B cells bearing the T313A mutation occupied V(H)T(+) GC B cell population. Thus, it is suggested that B cells with a higher affinity were selected not only for entry to the GCs but also in the affinity maturation process during a T-dependent Ab response.

The quasimonoclonal (QM) mouse provides a model to analyze B cell selection because major B cell antigen receptors (BCR) are composed of the knockin V(H)DJ(H) 17.2.25 (V(H)T) encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for (4-hydoxy-3-nitrophenyl)acetyl (NP). We have reported that during a T-dependent antibody (Ab) response for a low-affinity NP analog p-nitrophenylacetyl (pNP), although V(H)T/lambda1 and V(H)T/lambda2 IgM were equally produced, V(H)T/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. The initial affinity of V(H)T/lambda2 B cells for pNP was approximately 50-100-fold higher than that of V(H)T/lambda1 B cells, suggesting a role of BCR affinity in recruiting B cells to affinity maturation processes. Here, we investigated whether the intensity of BCR signals could contribute to the selection of V(H)T/lambda2 B cells for affinity maturation. V(H)T/lambda2 B cells were more responsive to pNP than V(H)T/lambda1 B cells in vitro. When CFSE-labeled QM B cells were transferred into the wild type mice where T cells had been primed with chicken gamma-globulin (CGG), QM B cells challenged by pNP-conjugated CGG could be observed to get activated and migrate to GCs in the early phase of the T-dependent response to pNP-CGG. Adoptive transfer of sorted populations revealed that the V(H)T/lambda2 B cell population was more potent in migration into GCs than the V(H)T/lambda1 counterpart. Thus, it is suggested that the higher BCR affinity of V(H)T/lambda2 B cells may be an initial cue for their recruitment to GCs during a T-dependent Ab response.

Here, we report an in vitro screening system for monoclonal antibodies using a hypermutating chicken B cell line, DT40-SW. When switching on hypermutation, cultured DT40-SW cells constituted an antibody library, from which clones secreting antibodies to a test antigen were successfully isolated, and genetically stabilized by switching off the mutation machinery.

During culture, a chicken B cell line DT40 spontaneously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytidine deaminase (AID)-dependent homologous recombination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. A few percent of the initially BFP-expressing cells converted their fluorescence from blue to green after culture for 2-3 weeks when the substrate construct was integrated in the Ig light chain locus, but not in the ovalbumin locus. This was the result of AID-dependent and the GFP gene-templated gene conversion of the BFP gene, thereby leading to the introduction of various sizes of GFP-derived gene segment into the BFP gene. Thus, G/B construct may be used to visualize gene conversion events. After switching off AID expression in DT40 cells, the mutant clones were isolated stably and maintained with their mutations being fixed. Thus, the gene conversion machinery in DT40 cells will be a useful means to engineer non-Ig proteins by a type of DNA shuffling.

A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies (Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on.

The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin V(H)17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had approximately 2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, V(H)T/lambda2 B cells significantly predominated over V(H)T/lambda1 B cells in MZ-(V(H)T/lambda1:V(H)T/lambda2 approximately 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of lambda2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the V(H)T/lambda2 B cells were further augmented by doubling the V(H)T gene, but dampened by the dysfunction of Bruton's tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between V(H)T/lambda1 and V(H)T/lambda2 IgM mAbs revealed a polyreactive nature of the V(H)T/lambda2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.

A population of peripheral B cells have been shown to express recombination activating gene products, RAG-1 and RAG-2, which are considered to be involved in revising the B cell antigen receptor (BCR) in the periphery. BCR engagement has been reported to turn off RAG expression in peripheral B cells, whereas the same treatment has an opposite effect on immature B cells in the bone marrow. In contrast to receptor editing that is involved in the removal of autoreactivity in immature B cells, it has been shown that secondary V(D)J rearrangement in peripheral B cells, termed receptor revision, contributes to affinity maturation of antibodies. Here, we show that RAG-2 expression in murine splenic B cells was abrogated by the coligation of BCR with complement receptors (CD21/CD35) much more efficiently than by the engagement of BCR alone. On the other hand, the same coligation augmented proliferation of anti-CD40-stimulated B cells. These findings suggest a crucial role for CD21/CD35 in directing the conservation or the revision of BCRs in peripheral B cells.