Yoshihiro Aiba

IL-10 augments antibody production in in Vitro immunized lymphocytes by inducing a Th2-type response and B cell maturation

An in vitro immunization (IVI) protocol enables antigen specific antibody production from L-Leucyl-L-Leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBL) upon antigen stimulation in the presence of IL-2, IL-4, and muramyl dipeptide. In the course of our studies, we have found that IL-10 added at the antigen sensitization significantly augmented antibody production level from the LLME-treated PBL. In the present study, we tried to demonstrate the role of IL-10 in the augmentation of antibody production in an IVI protocol by clarifying the cytokine expression profiles in CD4+ and CD8+ T cells. The results showed that IL-10 skewed the Th1/Th2 balance to Th2-type responses by suppressing Th1-type cytokine production and augmenting Th2-type cytokine production in CD4+ and CD8+ T cells, as well as in CD19+ B cells. Furthermore, IL-10 augmented the expression of CD38, an antigen marker of plasma cells, on B cells, which clearly indicates that IL-10 promoted differentiation and maturation of B cells in an IVI protocol. These results indicate that IL-10 plays an important role in setting the cellular milieu to produce antibodies in an IVI protocol.

Different individual immune responses elicited by in vitro immunization

We have previously demonstrated that the addition of muramyl dipeptide (MDP), interleukin-2 (IL-2) and IL-4 effectively raises antibody production from L-Leucyl-L-leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBLs) against specific soluble antigen when immunized in vitro. However, PBLs from individual donors were separate optimal conditions regarding concentrations for IL-2 and IL-4, which in turn required us to optimize each individual PBLs to effectively produce antigen specific human antibody by in vitro immunization. These individual differences in the requirement for IL-2 and IL-4 reflects the differences in individual immune responses against a specific soluble antigen, which can be elicited by in vitro immunization. In the present study, we investigated these individual differences in the requirement for IL-2 and IL-4 to induce antibody productionin vitro in the PBLs of 12 volunteers (9 healthy donors and 3 allergenic patients). IL-2 requirements for antibody production varied dependent upon each donor, while higher amounts of IL-4 inhibited IgM and IgG production in all of the healthy donors. However, some of the characteristic features for PBLs donated from allergenic included lowered IgM production compared to PBLs derived from healthy donors, and very high IgE production in the absence of cytokines and allergen. These results demonstrate that the sensitivity of PBLs against antigen sensitization differs between healthy donors and atopic patients, which suggests that the frequency of antigen sensitization might be reflected in differing activation states and/or differing subpopulations of lymphocytes in vivo.

A rapid and efficient strategy to generate antigen-specific human monoclonal antibody by in vitro immunization and the phage display method.

An in vitro immunization (IVI) protocol was developed for inducing antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs). After antigen sensitization of PBMCs by IVI, B cells producing antigen-specific antibody can be propagated within a week. Here, we attempted to establish a rapid, efficient strategy to obtain antigen-specific antibody by the phage display method using in vitro immunized PBMCs. Heavy and light chain variable region genes were easily amplified from these PBMCs immunized with mite extract (ME). After generating a combinatorial phage library (1.6 x 10(5) members), 4 antigen-specific clones were selected by 5 panning rounds using biotinylated antigen and streptavidin magnetic beads. Next, we combined variable region genes of these selected clones with human IgG constant region genes and produced human IgG-type antibody. Direct and competitive enzyme-linked immunosorbent assays demonstrated that the mAb 1C11 clone bound specifically to ME. We thus established a rapid, efficient method to obtain antigen-specific human antibody genes and produce human monoclonal IgG antibody using the phage antibody library generated from in vitro immunized PBMCs.

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells.

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.

Anti-Peptide Antibody Production Elicited by in Vitro Immunization of Human Peripheral Blood Mononuclear Cells

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-α as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.

Suppression of immunoglobulin production in human peripheral blood mononuclear cells by monocytes via secretion of heavy-chain ferritin.

In vitro antigen stimulation of peripheral blood mononuclear cells (PBMCs) does not induce immunoglobulin (Ig) production. However, pretreatment of PBMCs with l-leucyl-l-leucine methyl ester (LLME) prior to in vitro stimulation removes the suppression of Ig production. In the present study, we attempted to identify the target cells of LLME and determine the mechanisms by which Ig production in PBMCs is suppressed. We found that CD14(+) monocytes are involved in the suppression of Ig production in PBMCs. Furthermore, we confirmed that heavy-chain ferritin derived from CD14(+) monocytes suppresses Ig production in PBMCs, possibly through iron sequestration.

Identification of Genes Involved in the Suppression of Antibody Production from Human Peripheral Blood Lymphocytes

Pretreatment with L-leucyl-L-leucine methyl ester (LLME) is a prerequisite for peripheral blood mononuclear cells (PBMCs) to produce antigen-specific antibodies when sensitized with an antigen. Little information, however, is available regarding the mechanisms involved in LLME-induced augmentation of antibody production from PBMCs that are antigen sensitized. In the present study, we attempted to identify the genes involved in the suppression of antibody production from PBMCs that was not treated with LLME, but sensitized with an antigen. Using subtractive screening, we obtained 63 independent genes, including 17 EST genes, that are specific for LLME-nontreated PBMC. Among these genes, the expression of heavy chain ferritin (H-ferritin), CC chemokine ligand 18 (CCL18), and matrix metalloproteinase 12 (MMP12) were augmented in LLME-nontreated PBMCs, suggesting that inflammatory factors might be involved in the suppression of antibody production in LLME-nontreated PBMCs.

Involvement of IL-10 in the suppression of antibody production by in vitro immunized peripheral blood mononuclear cells

Previously, we have established an in vitro immunization method to induce antigen-specific antibody-producing B cells. In the present study, we have attempted to clarify the mechanisms that regulate antibody production by in vitro immunized peripheral blood mononuclear cells (PBMC). Freshly isolated PBMC did not induce antibody production following in vitro immunization, but expressed the interleukin (IL)-10 gene. On the other hand, PBMC pretreated with l-leucyl-l-leucine methyl ester (LLME) induced antibody production, but did not express the IL-10 gene. IL-10 induced functional impairment of CD4+ Th cells and CD11c+ DC, resulting in the suppression of antibody production by in vitro immunized PBMC.

Establishment of Efficient Cloning Method for Variable Region Genes of Antigen Specific Human Monoclonal Antibody

We have developed an in vitro immunization protocol of human peripheral blood mononuclear cells (PBMC) for generating human antigen-specific antibodies. By using this protocol, B cells producing antigen-specific antibody can be propagated within a week. In the present study, we tried to establish an efficient strategy to clone variable region genes of antigen-specific human monoclonal antibody by applying in vitro immunized PBMC to the phage display method. To evaluate the efficiency of our strategy, variable region genes prepared by PCR from PBMC immunized in vitro with mite-extract (ME) and non-immunized PBMC were applied to the phage display method. After concentrating the ME-specific phage antibody by several rounds of panning, the number of phage antibodies specific for ME and antigen specificity both increased by using in vitro immunized PBMC. These results demonstrate that efficiency in acquisition of variable region genes of antigen specific human monoclonal antibody can be improved by in vitro immunizing PBMC used as a source of antibody genes.

Production of Human Anti-Peptide Antibody for Clinical Use by In Vitro Immunization

Human monoclonal antibodies have a large potential to be used for diagnosis and therapy of various diseases, such as cancer, allergy, and so on. However, there exist some problems on sensitizing antigens to the human in order to obtain the antigenspecific human antibodies because of ethical and moral reasons. In vitro immunization method (IVI), in which primary B cells are activated to produce antigen-specific B lymphocytes, was established to solve this problem. In our previous study, we have established an IVI of human peripheral blood lymphocytes (PBL) against soluble proteins such as Cholera toxin B subunit (CTB), KLH or Rice allergic protein (RA) (Ichikawa et al., 1999). Furthermore, using a fragment peptide of CTB or RA, we have demonstrated that peptide antigen can be useful as sensitising antigen on IVI. However, some of proteins were difficult to produce antibodies because of serious effect on PBL on IVI, such as cytotoxicity, autoimmune response, change in the cytokine balance, and so on. TNF-α is a pleiotropic cytokine primarily produced by activated macrophages to response to a variety of inflammatory agents, so it is difficult to use as sensitizing antigen on IVI. Furthermore, Anti-TNF-α chimeric monoclonal antibody Infliximab have been reported to improve symptoms of rheumatoid arthritis and Crohns disease, and approved for treatment of these disease (Targan et al., 1997, Maini et al., 1999). In this study, we tried to establish an in vitro immunization protocol against peptide fragments derived from tumor necrosis factor-α (TNF-α) for production of antibody for clinical use.