A method for inducing antigen-specific IgG production by in | T Helper Cell | Interleukin 10

Journal of Immunological Methods 386 (2012) 60–69

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Journal of Immunological Methods
journal homepage: www.elsevier.com/locate/jim

Research paper

A method for inducing antigen-specific IgG production by in vitro immunization
Mieko Kato a, b, Huimin Yan a, Noriko M. Tsuji a, Tomoki Chiba b, Yoshiro Hanyu a,⁎
a Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, 305‐8566, Japan b Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, 305‐8572, Japan

a r t i c l e

i n f o

a b s t r a c t
In vitro immunization (IVI) possesses a number of advantages over conventional immunization. However, the number of positive clones derived from IVI is limited, and the affinity of the antibodies from derived clones is relatively low. Moreover, the majority of immunoglobulins produced in culture are IgMs instead of IgGs, which limits the application. Here, we report an improved protocol for IVI using mouse spleen cells. This protocol consists of multiple cycles of repeated antigen stimulation followed by cell expansion, which increases the frequency of plasma cells that produce antigen-specific IgG antibodies. The culture conditions, including the cell density, the type of stimulants, and the initial cell preparation, were found to be important for inducing the IgG response. In addition, an analysis of the genes and cytokines expressed during the IVI showed that the antigen-specific B cells were specifically activated via CD4-positive helper T cells. As evidence for this concept, our IVI protocol enabled us to establish an IgG antibody against keyhole limpet hemocyanin with a dissociation constant in the order of 10−7 M. © 2012 Elsevier B.V. All rights reserved.

Article history: Received 27 April 2012 Received in revised form 22 August 2012 Accepted 29 August 2012 Available online 10 September 2012 Keywords: B cell CpG oligonucleotide Helper T cell Hybridoma IgG In vitro immunization

1. Introduction The fields of research, diagnostics, and therapeutics derive significant benefits from antibodies. Thus, obtaining a good antibody with both high affinity and selectivity to an antigen is essential for meeting a variety of requirements in these different fields. However, the ability to establish an antibody with the desired affinity and selectivity against different types of antigens remains challenging. In particular, difficulties remain in making useful antibodies against certain antigens,

Abbreviations: IVI, in vitro immunization; CpG ODN, CpG oligodeoxynucleotide; PEG, Polyethylene glycol; PBS, Phosphate buffered saline; FBS, Fetal bovine serum; IL-2, Interleukin-2; IL-4, Interleukin-4; IL-5, Interleukin5; IL-10, Interleukin-10; IL-12, Interleukin-12; GM-CSF, Granulocyte macrophage colony-stimulating Factor; IFN-γ, Interferon-γ; TNF-α, Tumor necrosis factor-α; TLR 9, Toll-like receptor 9; NK cell, Natural killer cell; MDP, Muramyl dipeptide; ELISPOT, enzyme-linked immunospot; PBMCs, peripheral blood mononuclear cells; Tr1, Type 1 Regulatory T cells. ⁎ Corresponding author. Tel.: +81 29 861 2716; fax: +81 29 861 2706. E-mail address: y.hanyu@aist.go.jp (Y. Hanyu). 0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jim.2012.08.019

such as those that occur in small amounts, that are toxic, or that show high homology to antigens of the host animals. With in vitro immunization (IVI), immune cells isolated from naïve animals are stimulated with an antigen in vitro, thereby resulting in the induction of B cells that produce antigen-specific antibodies (Zafiropoulos et al., 1997). Subsequently, such cells are fused with myeloma cells to form hybridomas that produce the antigen-specific monoclonal antibodies desired. IVI shows potential merit in the generation of many types of antigens, including those that are problematic for in vivo immunization. That IVI is free from the injection of animals with the antigen is an additional advantage of this method in avoiding antigen toxicity rendered failture. With further development, IVI should especially be useful for obtaining human monoclonal antibodies (Matsumoto et al., 2008). If one can establish human monoclonal antibodies with IVI using human peripheral blood mononuclear cells (PBMCs), complicated processes, such as the humanization or chimerization of antibodies (Kettleborough et al., 1991), could be avoided.

it has been argued that an improved IVI protocol with conditions for inducing class-switching (Geha et al. Flow cytometry was performed using FACSAria (BD Biosciences). 2.) according to the manufacturer's instructions. and IL-21 at 10 ng/mL (PeproTech Inc. . IL-10. alkaline-phosphatase-labeled anti-mouse IgG or IgM antibody (Chemicon. NJ) for 2 days.. and the following stimulants: 0. Thermo Fisher Scientific. After staining with PE-Cy7conjugated anti-CD4 mAb.4. Sigma-Aldrich) in the presence of GolgiStop.5 ng/mL. IL-5. processes that are essential for obtaining antibodyproducing cells in vivo. This protocol enables the induction of antigen-specific IgG antibody production. After the culture period. An analysis of the genes and cytokines expressed during the IVI showed that the antigen-specific B cells were specifically activated via CD4-positive helper T cells. GM-CSF. 0.25 μM MDP. including MDP. cells were added to the plates at a density of 5 × 105 cells/well.25 μM KLH. All experiments were conducted twice.5.M. St. After the plates were washed with PBS. IVI methods have been established by treating the PBMCs with L-leucyl-L-leucine methyl ester (Matsumoto et al. and singlecell suspensions were prepared. For the secondary antigen stimulation. Materials and methods 2. / Journal of Immunological Methods 386 (2012) 60–69 61 To date. In addition. MA) as the antigen. Roche) was applied. The plates were then blocked for 2 h at 37 °C with RPMI 1640 containing 10% FBS. 2. 2003) and affinitymaturation. 2008).3. Minneapolis. 2. according to the manufacturer's instructions. and the plates were incubated at room temperature for 10 min. The mice were sacrificed. MN. When the development was complete. The stimulated cells were collected by centrifugation and then expanded in the culture media with IL-2 at 10 ng/mL. we succeeded in establishing an efficient method for IVI. and the plate was incubated for 2 h. Hokkaido System Science. Nevertheless. TX). 2. 2006) and applying potent stimulators. Afterwards. with 10 μg of keyhole limpet hemocyanin (KLH. the number of spots was scored. Birmingham. A blocking solution (Blocking Reagent for ELISA. Therefore. Flow cytometric analysis Splenocytes were restimulated with PMA (50 ng/ml. MA) was added.. After the plates were washed with PBS-T. Total IgG and IgM levels in the culture supernatants of the stimulated splenocytes were determined using ELISA with a mouse IgG ELISA quantitation kit and a mouse IgM ELISA quantitation kit (BETHYL. and the average signal intensity was used in the analysis. IL-12. the majority of clones from IVI produce IgMs. MO).05% Tween 20 (PBS-T) and incubated with diluted goat anti-mouse IgG antibody conjugated with alkaline phosphatase (Southern Biotech. as well as selecting the most suitable immune stimulants. most protocols still fail to deliver sufficient stimuli to antigen-specific B cells for their expansion and/or differentiation into antibody-producing cells. cells were fixed and permeabilized with the Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences). Sigma Fast BCIP/NBT solution (Sigma) was added. and 0. Canada). Ashland. the cells were then washed twice and incubated individually at 37 °C for 2 days in RPMI-1640 containing 20% FBS. 2. and the plates were read using a microplate reader (Model 680. Murine IL-2. Japan) and 0. Intracellular cytokines were stained with PE-conjugated anti-IL-4. and FITCconjugated anti-IFN-γ mAbs. In this study. ELISA A 96-well enzyme-linked immunosorbent assay (ELISA) plate was coated with 50 μL of 5 μg/mL KLH per well. IVI has not been used widely until now. we developed an IVI protocol that effectively activates the immune cells. For the IVI. OR). The spleens were squeezed.08 μM CpG ODN. CA). these expanded cells were collected by centrifugation and stimulated with 0.. Kato et al. CD8-positive T cells and natural killer cells (NK) cells were removed from the splenocytes with negative selection methods by using anti-CD8 antibody-coated magnet beads and antiCD49b antibody-coated magnet beads (Miltenyi Biotech. and data were analyzed using FlowJo software (Tree Star. The plate was subsequently washed with PBS containing 0. would be a powerful tool for creating useful monoclonal antibodies (Peled et al. APC-conjugated anti-IL-10.25 μM MDP (Sigma Aldrich). Montgomery. In particular. By improving the cell preparation methods and the culture conditions. and re-suspended in 10 mL of RPMI-1640 containing 10% fetal bovine serum (FBS). The cells were cultured for 24 h at 37 °C and 5% CO2. according to the manufacturer's instructions. Cytokine measurements Cytokine levels in the culture supernatants of stimulated splenocytes were measured.05% Tween-20 (PBS-T). and TNF-α levels were measured using a Bio-Plex assay with a mouse cytokine group 1 Th1/Th2 assay kit (Bio-Rad Laboratories. the plates were washed with PBS containing 0. The cells were washed once in RPMI-1640 (Sigma Aldrich. IL-4. IL-2.. The amount of the antigen-specific antibody was measured using the alkalinephosphatase substrate kit (Sigma Aldrich). Sigma-Aldrich) and Ionomycin (200 ng/ml. Louis. After the wells were washed. and IL-4.2.25 μM CpG ODN (5′-tccatgacgttcctgacgtt-3′. Waltham. useful antibodies with high affinity are rarely obtained with the conventional IVI protocols. Bio-Rad Laboratories) at a wavelength of 405 nm. ELISPOT assay The frequency of B cell-producing antigen-specific IgG was determined using an enzyme-linked immunospot (ELISPOT) assay. Billerica. AL) for 2 h at 37 °C.1. IFN-γ. Consequently. 50 μL of PBS containing the supernatant of stimulated splenocytes was added to each well. and their spleens were removed aseptically. because its procedures are complicated and have yielded unsatisfactory results. IL-4 at 2. which show suboptimal affinities to be broadly useful. The red blood cells and the granule cells were removed by using Lympholyte-M (Cedarlane Laboratories. Multiscreen HA filtration plates (Millipore. Rocky Hill. MA) were coated with KLH at a concentration of 10 μg/mL (50 μL/well) and incubated overnight at 4 °C. Mice and IVI Six-week-old female BALB/c mice were obtained from SLC (Japan).

First. GE Healthcare). Next.. After 24 h. Neither IgG nor IgM was produced without the stimulation. expansion with either the cocktail of interleukins or the 2 successive antigen stimulations increased the production of IgG more than that of a single antigen stimulation. Next.005% surfactant P20) and injected as the analyte solution. The sequences of the primers for Blimp-1. Takara Bio) contained 12. CA). the employment of an expansion step between the antigen stimulations appeared to be critical for the production of antigen-specific IgG. The repeated antigen stimulations did not increase antigen-specific IgGproducing cells. The reaction mixture (RT-PCR kit. In contrast. Japan) using SYBR Green for the detection of the PCR products. The hybridomas generated were incubated in a 96-well plate for 7 days. the first antigen stimulation. The cells were fused by PEG methods. Establishment of monoclonal antibodies by IVI For establishing mouse monoclonal antibodies against KLH by IVI.15 M NaCl. Purified monoclonal antibodies were dissolved in HBS-EP buffer (0.5).1. All measurements were performed at a flow rate of 30 μL/min at 25 °C. Antigen stimulation was subsequently applied again for 2 days to induce the production of the antigen-specific IgG. This 3-step activation procedure enabled the efficient induction of antigen-specific IgG-producing cells. 2. The size and color of the spots on the cells after the expansion were smaller and lighter. 3. As shown by ELISPOT. We measured the production of total IgG and IgM production from the stimulated splenocytes during the IVI (Fig.6. The fused cells were suspended in a 96-well plate and grown at 37 °C under a 5% CO2-enriched atmosphere. Cells were routinely cultured in RPMI-1640 supplemented with 10% FBS at concentrations between 1 × 105 and 1 × 106 cells/mL. The 2 cell types were counted using a hemocytometer and mixed together in a ratio of 1:1 for fusion. Omitting any one of these 3 steps either substantially reduced or occasionally diminished the production of antigen-specific IgG. Gene expression After RNA from stimulated cells was purified. The supernatants from the individual wells were screened by ELISA. 0. real-time PCR was carried out with a Terminal Cycle Dice TP800 (Takara Bio Inc. Further. which used CpG ODN and MDP as stimulants. the cell suspensions were pipetted into 10 mL of RPMI-1640 medium (free of phenol red) containing 20% FBS and 5% Briclone (QED Bioscience Inc. we examined the effects by the cell density during the IVI by increase seeding density from 5 × 10 5 cells/mL to 1 × 10 7 cells/mL. a single antigen stimulation evoked the production of both immunoglobulins. / Journal of Immunological Methods 386 (2012) 60–69 2. Sigma) medium was added to each well.5 μL of SYBR Premix Ex Taq (2 ×) (Takara Bio). The KLH was amine coupled to the CM5 sensor chip as instructed by the manufacturer (NHS/EDC coupling kit. Antigen-specific IgGproducing cells could not be induced by a single stimulation with the antigen.5 μL). was applied to these cells for 2 days. and induced the highest production of IgM.01 M HEPES [pH 7. Thus. the stimulated cells with the larger diameters were collected by low speed centrifugation and then expanded with a cocktail of cytokines for 2 days to activate IgG-producing cells. than those of the spots on the cells after the 2nd stimulation.4]. the only splenocytes after the 3-step stimulation produced higher affinity antigen-specific IgG antibodies. 2. splenocytes isolated from 6-week-old BALB/c mice were treated to eliminate cytotoxic lymphocytes. hypoxanthine (HAT. Kinetic analysis by surface plasmon resonance Kinetic analysis was performed using a Biacore J system (GE Healthcare).htm). The spots of the cells after the 3-step stimulation were large and dark in color. the second expansion is not effective for the induction of antigen-specific IgG-producing cells. such as CD8positive T cells and NK cells. Murine myeloma cells P3X63Ag8U. Thus. Kato et al. 10 μM PCR forward primer (0. AID. Data were evaluated using the Biaevaluation software (version 4. GAPDH expression was used for the normalization.2. Next. 3A). 1 shows the experimental procedure used for inducing the production of antigen-specific IgG antibodies in vitro.62 M. the number of antigen-specific IgG-producing cells among the activated splenocytes was determined with ELISPOT (Fig. Moreover. Afterwards.5 μL). were obtained using the Perfect Real Time support system (http://www.8. indicating that antibodies were being produced in large amounts and that the affinities against the antigen were higher than those of the previous steps. and the secreted antibody was purified with a protein G column (GE Healthcare.jp/prt/imtro. 0. through negative selection with antibody-coated magnetic beads. The number of positive cells increased more than 3-fold by the 2nd stimulation. the 2nd stimulation after the expansion of the antigenstimulated splenocytes induced more antigen-specific IgGproducing cells. USA).takara-bio. The hybridomas that produced the monoclonal antibodies were cloned by a limiting dilution method. Procedure for IVI and total IgG and IgM production Fig. Antigen-stimulated splenocytes that had undergone expansion and the second antigen stimulation showed the highest production of IgG but showed decreased IgM production. the corresponding cDNA was synthesized with TAKARA reverse transcriptase. 3 mM EDTA. Using this cDNA as a template. the number of cells producing antigen-specific IgG antibody increased from . 3. and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expansion after the 2nd stimulation led to substantial cell death during culture (data not shown). and the interaction surface was regenerated with glycine-HCl (pH 1. San Diego. Results 3. respectively.1. and cDNA (2 μL) to give a final reaction volume of 25 μL. The 3-step stimulation induced the highest production of IgG but reduced the production of IgM compared to the single and 2-step stimulations. The induction of antigen-specific IgG antibody by in vitro immunization After the stimulation. MA. 2). 10 μM PCR reverse primer (0. Further. splenocytes from BALB/c mice immunized with KLH in vitro were isolated and mixed with an equal number of myeloma cells. The induction of the antigen-specific IgG-producing cells occurred only upon the expansion.co.7.1 (P3-U1) were used as the fusion partners for the murine B cells. GE Healthcare).

Culture for two days Antigen. IL-4 and IL-5 are produced from Th2 cells. These cytokines were not produced by either the single antigen stimulation. 1. / Journal of Immunological Methods 386 (2012) 60–69 63 Splenocytes from BALB/c mouse The first antigen stimulation Expansion The second antigen stimulation Culture for two days Antigen. IL-4. Fig. their expression induced the differentiation of B cells into plasma cells during IVI and increased the affinity of the antibody. CpG oligodeoxynucleotide. 3. the cells were suspended in fresh media. The expression of Blimp-1 and AID showed that the B cells in this system were activated through a similar activation pathway as in vivo.and 14-folder higher. than that of naïve splenocytes. while IFN-γ and GM-CSF are produced from Th1 cells. was produced from Th2 (IL-4. which induces hypermutation in the antibody gene and leads to affinity maturation. Hence. The production of IL-2 and IL-10 was induced by antigen stimulation but minimally or not at all by the expansion step. The number of spots likewise increased with this increasing cell density.” the concentration after the expansion with the cocktail of cytokines.4. Thus. When the CD4-positive T cells were eliminated prior to the IVI. The expression of Blimp-1 at 4 × 10 6 and 8 × 10 6 cells/mL was approximately 5. Afterward. such as macrophages. Isolated splenocytes were activated with a 3-step stimulation. The number of positive spots at cell densities of less than 2 × 10 6 cells/mL was very low. is produced from accessory cells.and IL-10-producing) and Tr1 (IL-10-producing and IL-4-non-producing) cells. Meanwhile. Cytokine expression during IVI We measured the cytokine expression profile of splenocytes during IVI to study activation signaling in our system (Fig. IFN-γ.05 for stimulated vs. respectively. or their combination. 3B shows the effect of CD4-positive T cells on the production of antigen-specific IgG. which displayed increased expression during the expansion step. Gene expression during IVI The expression of genes related to B-cell activation was studied during the in vitro stimulation by real-time PCR (Fig. than that of naïve splenocytes. its expression significantly increased as the cell density increased from 4 × 106 cells/mL to 8 × 10 6 cells/mL. 1 × 10 6 cells/mL to 8 × 10 6 cells/mL (Fig. 4). the 3-step stimulation was critical for the production of these cytokines. However. The error bars represent the standard deviation (SD). IL-5. the activated vital cells were collected by centrifugation. 3 successive stimulations induced cell population changes important for this response. However. IL-10. the production of antigen-specific IgG remained at the basal level. *. Three expression pattern profiles were observed. the single expansion step. muramyl dipeptide. The concentration of total IgG and IgM in the culture medium after various combinations of stimulation steps were measured with ELISA.3. Procedure for in vitro immunization (IVI). CpG ODN. the number of spots at cell densities of 4 × 106 cells/mL were greater than 5 times that at cell densities of 2 × 10 6 cells/mL. IL: interleukin. IL-12 and TNF-α production were induced by the expansion step but only slightly by antigen stimulation. pb 0. “1st” denotes the concentration after the first antigen stimulation. MDP. These results show that cell density has significant impact on the induction of antigen-specific IgG and thereby indicate the importance of cell-cell interactions during the incubation. The expression of AID. IL-4. 3. and “2nd. Meanwhile. unstimulated cells. 3B). and the next stimulation was applied. Meanwhile. and eventually enhances IFN-γ in Th1 cells. The expression of these genes was known to correlate with antigen-specific antibody expression. and GM-CSF showed a remarkable increase in expression only after the 3-step stimulation. was ~80 and ~200 times higher at 4 × 10 6 and 8 × 106 cells/mL. CpG ODN and MDP Fig. the expression of AID increased as the cell density increased from 1 ×106 cells/mL to 8 × 106 cells/mL. The stimulants in our system may have directly stimulated the splenocytes and caused them to produce these cytokines. Kato et al. CpG ODN and MDP Culture for two days IL IL-2. . which showed increased expression upon antigen stimulation. Thus CD4-positive helper T cells were effectively stimulated in our system and * μg / m L 1 st Exp 2 nd Fig. 2. and IL-21 4. “Exp. Total IgG and IgM production during IVI. 5). Moreover. After each stimulation. indicating that antigen-specific IgG production is dependent on the interaction with CD4-positive helper T cells. The expression of Blimp-1 showed no increase as the culture density changed from 1 × 106 cells/mL to 2 × 106 cells/mL. IL-12. respectively. Among these cytokines.” the concentration after the second antigen stimulation. The maximum number of spots were observed at cell densities of 8 × 10 6 cells/mL. The columns represent the average concentration from 3 independent experiments.M.

/ Journal of Immunological Methods 386 (2012) 60–69 A * B * * Spots / 1x 06 x1 Spots / 5x105 * * * * 1st Exp 2nd 3rd - + - + + - + + - + + + - + + + 1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL 4x106/mL Splenocyte CD4- Fig.05 for stimulated vs. 6A. The ratio of Th1 cells (IFN-γ-producing and IL-4-non-producing) to CD4-positive helper T cells decreased with increasing cell density. The intracellular cytokines IL-4. the CD4-positive helper T cell is indispensable for the induction of antigen-specific IgG antibodies for this in vitro system. which presumably work in concert to promote IgG production. the CD4-positive helper T cells from splenocytes stimulated at various cell densities were characterized. . The fusion efficiency was determined as in B. *. unstimulated cells. A dot analysis of the flow cytometry values for cultures at a density of 4×106 cells/mL is shown in Fig.5.5% at a cell density of 2 × 106 cells/mL (Fig. Role of helper T cells in the immunization As shown above.64 M.05 for stimulated vs. The columns represent the average concentration from 3 independent experiments. p b 0. However. The number of antigen-specific IgG-producing cells was counted by ELISPOT after various combinations of stimulation steps and cell densities.2%) at a cell density of 16 Blimp1 * 250 AID * Gene expression Naïve 1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL Naïve 1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL Fig. “CD4” denotes the experiment in which CD4-positive cells were removed by negative selection from the splenocytes prior to stimulation. for the Th2 cells (IL-4-producing and IFN-γ-non-producing).” and “2nd” denote the same steps as in Fig. The maximum ratio was 7. differentiated into Th1 and Th2 cells to produce the cytokines. 3. Further. The error bars represent the standard deviation (SD). Expression of Blimp-1 and AID was measured by real-time polymerase chain reaction analysis. The error bars represent the SD. showing a maximum value (26. p b 0. *. and IFN-γ were stained with fluorescein-labeled antibodies and measured by flow cytometry. Expression of genes after IVI. 3. A. IL-10. glyceraldehyde3-phosphate dehydrogenase expression in naïve splenocytes was used for the normalization. 6B). Induction of antigen-specific IgG-producing cells in IVI. “1st. 6B). Hence. the ratio increased with the cell density and showed a maximum (7. unstimulated cells. Kato et al. The ratio of IL-4 and IL-10-producing CD4-positive helper T cells to IL-4-producing CD4-positive helper T cells increased with increasing cell density (Fig. The helper T cells were stained using anti-CD4 antibody. 2.” “Exp. “3rd” denotes the third antigen stimulation. 4.5%) at a density of 4 × 10 6 cells/mL.

IL-4 cells / C + cells % -γ CD4+ Th1(IFN-γ +. Cytokine production during in vitro immunization. tumor necrosis factor [TNF6-α.+. interferon [IFN]-γ. “1st. IFN-γ) / CD4 + 1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL 1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL Fig. IL-10. 5. IL-4. The data represent the mean of 3 replicates.” and “2nd” denote the same steps as in Fig.” “Exp.M. The error bars represent the SD. and cytokine production (as indicated by IL-2. The columns represent the values from 3 independent experiments. 2. The supernatants were collected. FACS profiles from intracellular cytokine staining for a culture density of 4 × 106 cells/mL is shown. Splenocytes were activated with various combinations of stimulation steps. 105 104 IL-4 103 102 102 103 104 105 A 102 103 104 105 102 103 104 105 IFN-γ - IL-10 IL-10 + /IL-4 + IL + cells / IL-4+ cells % L-10+ + B IFN. IFN. A. IL-5. B. . IL-12. IL-4 -) / CD4 + IL-4+. / Journal of Immunological Methods 386 (2012) 60–69 65 IL-2 IL-12 IFN-γ TNF-α pg/mL L IL-4 IL-5 IL-10 GM-CSF pg/mL 1st + Exp 2nd - + + - + + + + - + + - + + + + - + + - + + + + - + + - + + + Fig. The ratio of cells producing each cytokine to CD4-positive cells was measured with the changing cell density. Intracellular staining of cytokines in CD4-positive cells in IVI. and granulocyte macrophage colony-stimulating factor [GM-CSF]] was evaluated by Bio-Plex. 6. Kato et al.cells / CD4 cells % -γ 4+ 1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL Th2 (IL-4 +. The error bars represent the SD.

The KD of the antibody was calculated as 5. IL-12.66 M.3± 1. limits the number of positive cells and the insufficient maturation of antibody-producing cells. KLH was injected over the biosensor surface twice at the indicated dilutions. IL-5. while an average of 12.8. MDP. Cytokine production after IVI. Thus.66× 10−2 s−1. Kato et al. 4.1 (n= 3) clones produced the antigen-specific IgG antibody. / Journal of Immunological Methods 386 (2012) 60–69 8×106 cells/mL. The flow rate was 30 μL/min.47 × 10 5 M−1 s−1 and a dissociation rate constant of 7. The effect of CD4-positive cells on cytokine production was analyzed by Bio-Plex. TNF-α. IFN-γ. This difficulty results from the lack of effective immune stimulation and is inherent to the procedure. 3.21 10-7 (M) 1. 2008.07 nM. The affinity of the purified anti-KLH monoclonal IgG antibody was measured by Biacore (Fig. Stimulating B cells to differentiate into plasma cells is necessary for obtaining IgG antibodies with high affinity and thus imperative for an effective IVI protocol.. Saito et al. Hence. our in vitro method could be used to obtain a monoclonal antigen-specific IgG antibody.7 ± 4. These results show that these different cytokines. with an association rate constant of 1. The ratio of antigen-specific IgG-producing clones to antigen-specific IgM-producing clones was 3. 7.. Moreover. As a result. and some immune stimulants (Zafiropoulos et al.6.1 (n= 3) clones produced the antigen-specific IgM antibody. 2007) have been used as immunomodulators during IVI. The data represent the mean of 3 replicates. IL-2. However. For this purpose.769 nM. Tamura et al. 1997).537 nM. 1995. are produced either directly from CD4-positive helper T cells or from cells activated by them. PWM. 1995). To study the effect of CD4-positive helper T cells on signal transduction during IVI. its low efficiency in B-cell activation 40 35 Response (RU) 30 25 20 15 10 5 0 0 50 3. and anti-CD40 antibody (Chin et al.769 M 100 150 200 Time (sec) Fig. Establishment of an monoclonal antibody with IVI Next.07 M KD=5..5 × 108 myelomas. The purified antibody was immobilized onto a CM5 sensor tip. an average of 3. 7). 8). Schilizzi et al. The production of IL-4. LPS. and 0. . Positive hybridomas were cloned by the limiting dilution method.21× 10−7 M. Discussion IVI possesses a number of advantages over conventional immunization. Th1 cells. Increasing the number of positive clones is a crucially important step in obtaining the desired antibody. considerable cell death tends to occur. we measured cytokine production with and without CD4-positive helper T cells at a cell density of 4 × 10 6 cells/mL (Fig. we attempted to establish a monoclonal antibody by using our IVI method.. After immunization with KLH. CD4 positive-cells were removed from the splenocytes prior to the stimulation. and GM-CSF was dramatically reduced when the CD4-positive helper T cells were depleted. 5 × 10 7 splenocytes were fused with 2. Th2 cells were preferentially produced when the cell density was above 4×106 cells/mL. an antigen. (1992) 500 6000 5000 300 pg/mL 3000 Fig. IL-2 production was increased with their absence. However. Kinetic analysis of the binding of keyhole limpet hemocyanin (KLH) to the monoclonal antibody by using the Biacore system. 8.537 M 0. IVI and fusions were independently performed. Among these colonies. The error bars represent the SD. By contrast. which involves mixing cells.. The culture density was 4 × 106 cells/mL. such as 19 days for PMBCs (Chin et al. An average of 356 ± 94 (n= 3) hybridoma colonies were obtained from each fusion experiment. The monoclonal antibody was shown to bind to the antigen in a concentration-dependent manner. the effects of these stimulators are still restrictive and insufficient. 1. and methods employing these stimulators tend to require relatively long stimulation periods. KLH was injected at concentrations of 3. The experimental curves (thin lines) were fitted globally (thick lines) using the mass transfer-limiting model. when the cell density was low. establishing an effective IVI protocol for inducing cells that produce antigenspecific IgG antibodies with a high affinity is essential. with the exception of IL-2. IL-10.

The pattern of production during the procedure was different between total IgG and antigen-specific IgG. we report an efficient procedure for IVI. the number of antigen-specific IgG-producing cells increased more than 3-fold... the cell death worsened. we directly examined the presence of cells producing antigen-specific IgG antibodies by ELISPOT. 2003). This loss of cells during culture decreased the total number of positive cells. Cell density was one of the important factors for the induction of antigen-specific IgG antibodies. and 3) a second antigen stimulation. 2) cell expansion with a cocktail of cytokines. These cytokines would have the effect of increasing and decreasing the production of total IgG and IgM. class switching.. Kato et al.M. IFN-γ and GM-CSF activate macrophages and induce colony formation (Metcalf et al.. real-time PCR analysis showed that Blimp-1 (Nutt et al.. 2). Thus. However. To improve the culture conditions. However. 2007) and AID (Pavri and Nussenzweig. antigen-specific IgG-producing cells can be obtained in the short period of 6 days. An expansion after the second antigen stimulation also increased the ratio of positive cells. while no antigen-specific IgG antibody was induced at a density less than 2 × 10 6 cells/mL. respectively. This procedure enabled the selection of activated vial cells and resulted in a cell culture with better conditions.. but this method is only applicable when T cell clones against the antigen do not already exist.. These cytokines activate the antibody-producing cells and induce IgG production.. antigen-specific IgG was not produced by a single antigen stimulation in our procedure (Fig. 3A). . With our procedure. with the resultant increase in cell density potentially strengthening cell-cell interactions (Haniuda et al. 2008) were crucial for inducing antigenspecific IgG in the IVI system. and antigen-specific IgG production was not induced. as in the vivo immune response. The ELISPOT analysis showed that the 3-step stimulation not only induced antigen-specific IgG antibodies but also facilitated the affinity maturation of resultant antibodies. When the CD4-positive helper T cells were eliminated from the system. While this has never been explored before in an IVI protocol. After the 3-step stimulation. Antigen-specific IgG antibodies were effectively induced at a cell density greater than 4 × 10 6 cells/mL. Moreover. This expression pattern of total IgG and IgM indicate that class switching occurred after the second antigen stimulation. 2). Kato et al. We have established procedures for the cell preparations and culture and succeeded in the induction of antigen-specific IgG antibody production. 2011). CpG ODN (Bal et al. which shows that class switching had occurred but not efficiently. Okazawa et al. the increase in the production of antigen-specific IgG antibody with increasing cell density (Fig. the T and B cells are stimulated directly. The second antigen stimulation after the expansion increased the production of total IgG but reduced that of IgM. especially the antibody-producing ones. 2011. this increase with the second antigen stimulation was not observed if the expansion step was omitted (Fig. The production of antigen-specific IgG antibodies was not observed with this preparation. 2008) had occurred during the IVI. the production of cytokines. interactions between CD4-positive cells and B cells (Kouyama et al. / Journal of Immunological Methods 386 (2012) 60–69 67 added T cell clones to the culture and succeeded in obtaining antigen-specific IgG. our results indicate that the in vivo interactions responsible for IgG induction were at least partly reproduced in our in vitro system. and the total cell number decreased (Zafiropoulos et al. For the first antigen stimulation. IL-10. 1997). Further. but this increase was not effective for the efficient induction of antigen-specific IgG-producing cells.. The single antigen stimulation resulted in IgG production that was 40% of that of the 3-step stimulation. However. The expression of Blimp-1 shows that the B cells differentiated into plasma cells (Diehl et al. By contrast. 2011) is applied with the antigen and effectively stimulates the B cells. Second.. Here. The highest production of total IgG in our experiments so far was induced by the 3-step stimulation (Fig. we collected the activated cells with low centrifugation after each stimulation and restarted the culture at a fixed cell density. This expansion causes cell proliferation. These results show that our system worked well for inducing an antigen-specific response. and IFN-γ. these researchers counted the number of cells producing IgM but not IgG. 2011) were expressed during the IVI. This step is essential for the increase of antigen-specific IgG-producing cells at the end of the culture (Fig. 2007. 2011. was increased. In the expansion step. 1986) and MHC-II expression (Giroux et al. Tamura et al. Hence. (1997) estimated the number of cells producing antigen-specific IgG antibody indirectly from ELISA methods. after the 3-step stimulation (Fig. The production of total IgG showed little change between the expansion and the second antigen steps.. and affinity maturation. When CD8-positive helper T cells and NK cells were not removed prior to the IVI. The second antigen stimulation activates the antigen-specific antibody-producing cells and may effectively induce IgG production. 2002) and class switching (Stavnezer et al. 3) suggests that the interactions between CD4-positive cells and B cells were strengthened in the high density cultures. the expression of AID showed that somatic hypermutation (Cumbers et al. the removal of the cytotoxic lymphocytes was indispensable for the induction of antigen-specific IgG production. The expression of these genes was markedly increased when the cell density of the culture was high. Zafiropoulos et al.. (2007) also induced the antibody from PMBCs and counted the number of antigenspecific antibody-producing cells by ELISPOT. Our procedure consists of 3 steps for inducing antibody production in splenocytes: 1) an initial antigen stimulation. Nojima et al.. indicating that the B cells in our system are activated in the same way as in vivo. the third stimulation is specific to the antigen-specific IgG-producing cells. 2011). this high density was not good for the cell culture. The order of these steps appears to be important for obtaining good results. IL-5. antigen-specific IgG production was remarkably high after the 3-step stimulation. The cytokine milieu is another critical factor for successful IVI.. 3A). The importance of cell density in the culture was further supported by 2 observations made during the course of this study. Nutt et al. cytokine production was suppressed. since the total number of splenocytes was decreased drastically after the second expansion. First. When the second antigen stimulation was applied to the antigen-stimulated splenocytes following the expansion step. However. such as IL-4. Thus. The increased expression of these genes correlated well with an increase in antigen-specific antibody expression. with the third step showing a marked increase in production. Total IgG production was not increased over IgM production in their IVI method. 3A). GM-CSF. 2008.

The expression pattern of total IgG and IgM indicate that class switching occurred after the second antigen stimulation. With improved efficiency. while only 6 days were required for our method. was greater at the higher cell densities.. (1997) induced monoclonal IgG antibody from PMBCs with IVI and showed that the ratio of positive clones was in the order of 10−6. respectively. In our experiment. Coencapsulation of antigen and Toll-like receptor ligand in cationic .. Hence.A.. However. The association rate constant of our antibody was less than that of their antibody by approximately 2 orders of magnitude. In essence. with an association rate constant of 8. 2007. 2011). The results show that our in vitro method can induce a sufficient number of B cells to produce IgG-antibody specific to the antigen. P. 5. E. For further improving the number of positive clones and IgG antibody affinity of IVI. or functional modulations. Conversely. 2012) should be reproduced in vitro during immunization. The differentiation of helper T cells into Th2 cells is desirable for the induction of antigen-specific IgG. the ratio of positive cells should be high. more efficient activation is required. J. Nonetheless. Z. Acknowledgement This work was partially supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry. One worthy future experiment is to collect more clones to examine whether higher affinity clones are in fact present and obtainable by IVI. This number was not sufficient to obtain antibodies with high affinities. and the activation of cytokineproducing CD4-positive helper cells. (1995) established a monoclonal antibody from PMBCs using IVI.. Lane. Hence. Rev. Conclusion Our method not only induces total IgG production but also produces an antigen-specific IgG relative to other methods. The co-culture of splenocytes with follicular dendritic cells (Nishikawa et al. G. This monoclonal antibody had a KD of 2. obtaining a monoclonal antigen-specific IgG antibody is possible with our in vitro method. Intracellular staining of the cytokines showed that IL-4 and IL-10 production from CD4-positive helper T cells. the ELISPOT analysis showed that the ratio of positive clones was in the order of 10−5.47 × 10 5 M − 1 s − 1 and a dissociation rate constant of 7.. Kato et al. The number of antibody-producing cells is also important for establishing hybridomas. Zafiropoulos et al. we used the cell density at 4 × 106 cells/mL for IVI because these conditions resulted in the predominance of Th2 and were thus suitable for the production of antigen-specific IgG antibody.68 M. S.. Th1 is predominant when the cell density is low during IVI. 2002). To establish a hybridoma.0×10−4 s−1. with an association rate constant of 1. again explaining why cell densities above 4 × 106 cells/mL resulted in the excellent formation of spots representing the production of antigen-specific antibodies. 954. helper T cells in our system differentiated into both Th1 and Th2 cells (Anderson et al. Murphy and Reiner. The cytokines produced after the 3-step stimulation are from both Th1 and Th2 cells. the dissociation rate constant of our antibody was greater by approximately the same order of magnitude. culture conditions that are closer to the in vivo situation should be applied to the splenocytes. We succeeded in establishing an IgG monoclonal antibody against KLH with IVI. These results show that Th2 is predominant when the cell density is high and that higher cell densities are thus preferable to antigen-specific IgG production. Generating intrathymic microenvironments to establish T-cell tolerance. we obtained an average of 3. W. but differentiation into Th1 was also observed in the system. Nat. 2007. this method is also expected to be applicable to the production of human monoclonal antibodies.4×10−8 M. and the monoclonal antibody was purified and shown to have a KD of 5. The Th2 cells induced the differentiation of B cells into plasma cells. For the IVI. For this purpose. since the fusion rate is as high as 10−4. Thus. / Journal of Immunological Methods 386 (2012) 60–69 respectively. Hence.. Bal.. The combination of short experimental cycle of 6 days relative to 19 days of conventional methods and the ability to handle highly toxic antigens makes IVI highly attractive. This would increase the antigen presentation to the CD4-positive helper cells and induce the differentiation and proliferation of antibody-producing cells.. This value was not sufficient for obtaining enough hybridomas after the cell fusion. Jiskoot. Ding. 2006) and T helper cell clones (Uthoff and Boldicke. which activate antibody production by B cells. the 3-step stimulation appears to mimic important processes that occur in vivo. One of these clones was subcloned. 2011.J. Affinity maturation that results in a reduced dissociation of the antibody from the antigen would be necessary in our system for the further improvement of affinity.66×10−2 s−1. The other monoclonal antibodies from our IVI have the same characteristics. Jenkinson. which produced the antigen-specific antibody. References Anderson. This procedure induced the expression of B cell activators including Blimp-1 and AID. 1993) are likely to activate B cells more efficiently. S. These stimulations would induce class switching and affinity maturation more effectively. Bouwstra. Hortensius. Immunol. with smaller association rate constants and larger dissociation rate constants. the positive ratio from our IVI method was 1 order of magnitude higher. This would be related to the affinity maturation process in our IVI. The application of our method is expected to increase the probability of obtaining a desired antibody against a challenging antigen.3 clones from 1 fusion. In our protocol... further potent stimulation should be applied to differentiate helper T cells into Th2 cells. Chin et al. the KD of the antibody was not as high as the one from a normal in vivo method (Sasamori et al.5×103 M−1 s−1 and a dissociation rate constant of 2. ELISPOT analysis showed that our 3-step stimulation procedure increased the number of antigen-specific IgG-producing cells and induced antigen-specific IgG antibody production. the ratio of positive cells should be higher than 10−5 to obtain enough positive cells. which are involved in cell-mediated immunity and humoral immunity. 10–12 days were needed for their immunization.21 × 10 − 7 M.M. If we use 108 cells for the fusion. Hence. Further. our results also show either that the affinities of these antibodies have not been sufficiently increased for practical use or that the number of B cells producing an IgG antibody with a high affinity remains small. 7. the conditions in the germinal center (Victora and Nussenzweig. selectivity.

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