Journal of Immunological Methods 386 (2012) 6069

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Journal of Immunological Methods

journal homepage: www.elsevier.com/locate/jim

Research paper

A method for inducing antigen-specic 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, 3058566, Japan b Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, 3058572, 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 107 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-, Interferon-; TNF-, Tumor necrosis factor-; 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 nave 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.

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To date, IVI methods have been established by treating the PBMCs with L-leucyl-L-leucine methyl ester (Matsumoto et al., 2006) and applying potent stimulators, including MDP, IL-2, and IL-4. Nevertheless, IVI has not been used widely until now, because its procedures are complicated and have yielded unsatisfactory results. In particular, the majority of clones from IVI produce IgMs, which show suboptimal affinities to be broadly useful. Consequently, useful antibodies with high affinity are rarely obtained with the conventional IVI protocols. In addition, most protocols still fail to deliver sufficient stimuli to antigen-specific B cells for their expansion and/or differentiation into antibody-producing cells. Therefore, it has been argued that an improved IVI protocol with conditions for inducing class-switching (Geha et al., 2003) and affinitymaturation, processes that are essential for obtaining antibodyproducing cells in vivo, would be a powerful tool for creating useful monoclonal antibodies (Peled et al., 2008). In this study, we developed an IVI protocol that effectively activates the immune cells. By improving the cell preparation methods and the culture conditions, as well as selecting the most suitable immune stimulants, we succeeded in establishing an efficient method for IVI. This protocol enables the induction of antigen-specific IgG antibody production. 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. 2. Materials and methods 2.1. Mice and IVI Six-week-old female BALB/c mice were obtained from SLC (Japan). The mice were sacrificed, and their spleens were removed aseptically. The spleens were squeezed, and singlecell suspensions were prepared. The cells were washed once in RPMI-1640 (Sigma Aldrich, St. Louis, MO), and re-suspended in 10 mL of RPMI-1640 containing 10% fetal bovine serum (FBS). The red blood cells and the granule cells were removed by using Lympholyte-M (Cedarlane Laboratories, Canada). 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, CA), according to the manufacturer's instructions. For the IVI, the cells were then washed twice and incubated individually at 37 C for 2 days in RPMI-1640 containing 20% FBS, with 10 g of keyhole limpet hemocyanin (KLH, Thermo Fisher Scientific, Waltham, MA) as the antigen, and the following stimulants: 0.25 M CpG ODN (5-tccatgacgttcctgacgtt-3, Hokkaido System Science, Japan) and 0.25 M MDP (Sigma Aldrich). The stimulated cells were collected by centrifugation and then expanded in the culture media with IL-2 at 10 ng/mL, IL-4 at 2.5 ng/mL, and IL-21 at 10 ng/mL (PeproTech Inc., Rocky Hill, NJ) for 2 days. For the secondary antigen stimulation, these expanded cells were collected by centrifugation and stimulated with 0.25 M KLH, 0.08 M CpG ODN, and 0.25 M MDP. 2.2. ELISA A 96-well enzyme-linked immunosorbent assay (ELISA) plate was coated with 50 L of 5 g/mL KLH per well. A

blocking solution (Blocking Reagent for ELISA; Roche) was applied, and the plate was incubated for 2 h. The plate was subsequently washed with PBS containing 0.05% Tween-20 (PBS-T). Afterwards, 50 L of PBS containing the supernatant of stimulated splenocytes was added to each well. After the wells were washed, alkaline-phosphatase-labeled anti-mouse IgG or IgM antibody (Chemicon, MA) was added. The amount of the antigen-specific antibody was measured using the alkalinephosphatase substrate kit (Sigma Aldrich), and the plates were read using a microplate reader (Model 680; Bio-Rad Laboratories) at a wavelength of 405 nm. All experiments were conducted twice, and the average signal intensity was used in the analysis. 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, Montgomery, TX), according to the manufacturer's instructions. 2.3. ELISPOT assay The frequency of B cell-producing antigen-specific IgG was determined using an enzyme-linked immunospot (ELISPOT) assay. Multiscreen HA filtration plates (Millipore, Billerica, MA) were coated with KLH at a concentration of 10 g/mL (50 L/well) and incubated overnight at 4 C. The plates were then blocked for 2 h at 37 C with RPMI 1640 containing 10% FBS. After the plates were washed with PBS, cells were added to the plates at a density of 5 105 cells/well. The cells were cultured for 24 h at 37 C and 5% CO2. After the culture period, the plates were washed with PBS containing 0.05% Tween 20 (PBS-T) and incubated with diluted goat anti-mouse IgG antibody conjugated with alkaline phosphatase (Southern Biotech, Birmingham, AL) for 2 h at 37 C. After the plates were washed with PBS-T, Sigma Fast BCIP/NBT solution (Sigma) was added, and the plates were incubated at room temperature for 10 min. When the development was complete, the number of spots was scored. 2.4. Cytokine measurements Cytokine levels in the culture supernatants of stimulated splenocytes were measured. Murine IL-2, IL-4, IL-5, IL-10, IL-12, GM-CSF, IFN-, and TNF- levels were measured using a Bio-Plex assay with a mouse cytokine group 1 Th1/Th2 assay kit (Bio-Rad Laboratories, Minneapolis, MN,) according to the manufacturer's instructions. 2.5. Flow cytometric analysis Splenocytes were restimulated with PMA (50 ng/ml; Sigma-Aldrich) and Ionomycin (200 ng/ml; Sigma-Aldrich) in the presence of GolgiStop. After staining with PE-Cy7conjugated anti-CD4 mAb, cells were fixed and permeabilized with the Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences). Intracellular cytokines were stained with PE-conjugated anti-IL-4, APC-conjugated anti-IL-10, and FITCconjugated anti-IFN- mAbs. Flow cytometry was performed using FACSAria (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR).

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2.6. Gene expression After RNA from stimulated cells was purified, the corresponding cDNA was synthesized with TAKARA reverse transcriptase. Using this cDNA as a template, real-time PCR was carried out with a Terminal Cycle Dice TP800 (Takara Bio Inc., Japan) using SYBR Green for the detection of the PCR products. The reaction mixture (RT-PCR kit, Takara Bio) contained 12.5 L of SYBR Premix Ex Taq (2 ) (Takara Bio), 10 M PCR forward primer (0.5 L), 10 M PCR reverse primer (0.5 L), and cDNA (2 L) to give a final reaction volume of 25 L. The sequences of the primers for Blimp-1, AID, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were obtained using the Perfect Real Time support system (http://www.takara-bio.co.jp/prt/imtro.htm). GAPDH expression was used for the normalization. 2.7. Establishment of monoclonal antibodies by IVI For establishing mouse monoclonal antibodies against KLH by IVI, splenocytes from BALB/c mice immunized with KLH in vitro were isolated and mixed with an equal number of myeloma cells. Murine myeloma cells P3X63Ag8U.1 (P3-U1) were used as the fusion partners for the murine B cells. 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. The cells were fused by PEG methods. Next, 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, San Diego, CA). The fused cells were suspended in a 96-well plate and grown at 37 C under a 5% CO2-enriched atmosphere. After 24 h, hypoxanthine (HAT; Sigma) medium was added to each well. The hybridomas generated were incubated in a 96-well plate for 7 days. The supernatants from the individual wells were screened by ELISA. The hybridomas that produced the monoclonal antibodies were cloned by a limiting dilution method, and the secreted antibody was purified with a protein G column (GE Healthcare, MA, USA). 2.8. Kinetic analysis by surface plasmon resonance Kinetic analysis was performed using a Biacore J system (GE Healthcare). The KLH was amine coupled to the CM5 sensor chip as instructed by the manufacturer (NHS/EDC coupling kit, GE Healthcare). Purified monoclonal antibodies were dissolved in HBS-EP buffer (0.01 M HEPES [pH 7.4], 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) and injected as the analyte solution. All measurements were performed at a flow rate of 30 L/min at 25 C, and the interaction surface was regenerated with glycine-HCl (pH 1.5). Data were evaluated using the Biaevaluation software (version 4.1; GE Healthcare). 3. Results 3.1. Procedure for IVI and total IgG and IgM production Fig. 1 shows the experimental procedure used for inducing the production of antigen-specific IgG antibodies in vitro. First, splenocytes isolated from 6-week-old BALB/c mice were

treated to eliminate cytotoxic lymphocytes, such as CD8positive T cells and NK cells, through negative selection with antibody-coated magnetic beads. Next, the first antigen stimulation, which used CpG ODN and MDP as stimulants, was applied to these cells for 2 days. Afterwards, 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. 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. We measured the production of total IgG and IgM production from the stimulated splenocytes during the IVI (Fig. 2). Neither IgG nor IgM was produced without the stimulation. In contrast, a single antigen stimulation evoked the production of both immunoglobulins. Moreover, 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, and induced the highest production of IgM. Antigen-stimulated splenocytes that had undergone expansion and the second antigen stimulation showed the highest production of IgG but showed decreased IgM production. The 3-step stimulation induced the highest production of IgG but reduced the production of IgM compared to the single and 2-step stimulations. 3.2. The induction of antigen-specic IgG antibody by in vitro immunization After the stimulation, the number of antigen-specific IgG-producing cells among the activated splenocytes was determined with ELISPOT (Fig. 3A). Antigen-specific IgGproducing cells could not be induced by a single stimulation with the antigen. The induction of the antigen-specific IgG-producing cells occurred only upon the expansion. Further, the 2nd stimulation after the expansion of the antigenstimulated splenocytes induced more antigen-specific IgGproducing cells. The number of positive cells increased more than 3-fold by the 2nd stimulation. The size and color of the spots on the cells after the expansion were smaller and lighter, respectively, than those of the spots on the cells after the 2nd stimulation. The spots of the cells after the 3-step stimulation were large and dark in color, indicating that antibodies were being produced in large amounts and that the affinities against the antigen were higher than those of the previous steps. Thus, the only splenocytes after the 3-step stimulation produced higher affinity antigen-specific IgG antibodies. The repeated antigen stimulations did not increase antigen-specific IgGproducing cells. Further, the employment of an expansion step between the antigen stimulations appeared to be critical for the production of antigen-specific IgG. Omitting any one of these 3 steps either substantially reduced or occasionally diminished the production of antigen-specific IgG. Expansion after the 2nd stimulation led to substantial cell death during culture (data not shown). Thus, the second expansion is not effective for the induction of antigen-specific IgG-producing cells. Next, 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. As shown by ELISPOT, the number of cells producing antigen-specific IgG antibody increased from

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Splenocytes from BALB/c mouse

The first antigen stimulation

Expansion

The second antigen stimulation

Culture for two days Antigen, CpG ODN and MDP

Culture for two days IL IL-2, IL-4, and IL-21 4,

Culture for two days Antigen, CpG ODN and MDP

Fig. 1. Procedure for in vitro immunization (IVI). Isolated splenocytes were activated with a 3-step stimulation. After each stimulation, the activated vital cells were collected by centrifugation. Afterward, the cells were suspended in fresh media, and the next stimulation was applied. CpG ODN, CpG oligodeoxynucleotide; IL: interleukin; MDP, muramyl dipeptide.

1 10 6 cells/mL to 8 10 6 cells/mL (Fig. 3B). The number of spots likewise increased with this increasing cell density. The number of positive spots at cell densities of less than 2 10 6 cells/mL was very low. Meanwhile, 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. The maximum number of spots were observed at cell densities of 8 10 6 cells/mL. 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. Fig. 3B shows the effect of CD4-positive T cells on the production of antigen-specific IgG. When the CD4-positive T cells were eliminated prior to the IVI, the production of antigen-specific IgG remained at the basal level, indicating that antigen-specific IgG production is dependent on the interaction with CD4-positive helper T cells. 3.3. 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. 4).

The expression of Blimp-1 showed no increase as the culture density changed from 1 106 cells/mL to 2 106 cells/mL. However, its expression significantly increased as the cell density increased from 4 106 cells/mL to 8 10 6 cells/mL. Meanwhile, the expression of AID increased as the cell density increased from 1 106 cells/mL to 8 106 cells/mL. The expression of AID, which induces hypermutation in the antibody gene and leads to affinity maturation, was ~80 and ~200 times higher at 4 10 6 and 8 106 cells/mL, respectively, than that of nave splenocytes. The expression of Blimp-1 at 4 10 6 and 8 10 6 cells/mL was approximately 5- and 14-folder higher, respectively, than that of nave splenocytes. The expression of these genes was known to correlate with antigen-specific antibody expression. 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. Moreover, their expression induced the differentiation of B cells into plasma cells during IVI and increased the affinity of the antibody. 3.4. Cytokine expression during IVI We measured the cytokine expression profile of splenocytes during IVI to study activation signaling in our system (Fig. 5). Three expression pattern profiles were observed. The production of IL-2 and IL-10 was induced by antigen stimulation but minimally or not at all by the expansion step. Meanwhile, IL-12 and TNF- production were induced by the expansion step but only slightly by antigen stimulation. The stimulants in our system may have directly stimulated the splenocytes and caused them to produce these cytokines. However, IL-4, IL-5, IFN-, and GM-CSF showed a remarkable increase in expression only after the 3-step stimulation. These cytokines were not produced by either the single antigen stimulation, the single expansion step, or their combination. Hence, the 3-step stimulation was critical for the production of these cytokines. Thus, 3 successive stimulations induced cell population changes important for this response. Among these cytokines, IL-4 and IL-5 are produced from Th2 cells, while IFN- and GM-CSF are produced from Th1 cells. IL-10, which showed increased expression upon antigen stimulation, was produced from Th2 (IL-4- and IL-10-producing) and Tr1 (IL-10-producing and IL-4-non-producing) cells. IL-12, which displayed increased expression during the expansion step, is produced from accessory cells, such as macrophages, and eventually enhances IFN- in Th1 cells. Thus CD4-positive helper T cells were effectively stimulated in our system and

g / m L
1 st Exp 2 nd
Fig. 2. Total IgG and IgM production during IVI. The concentration of total IgG and IgM in the culture medium after various combinations of stimulation steps were measured with ELISA. 1st denotes the concentration after the first antigen stimulation; Exp, the concentration after the expansion with the cocktail of cytokines; and 2nd, the concentration after the second antigen stimulation. The columns represent the average concentration from 3 independent experiments. The error bars represent the standard deviation (SD). *, pb 0.05 for stimulated vs. unstimulated cells.

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* *
Spots / 1x 06 x1

Spots / 5x105

1st Exp 2nd 3rd

+ -

+ + -

+ + -

+ + + -

+ + +

1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL 4x106/mL

Splenocyte

CD4-

Fig. 3. Induction of antigen-specific IgG-producing cells in IVI. A. The number of antigen-specific IgG-producing cells was counted by ELISPOT after various combinations of stimulation steps and cell densities. 1st, Exp, and 2nd denote the same steps as in Fig. 2. 3rd denotes the third antigen stimulation. The fusion efficiency was determined as in B. CD4 denotes the experiment in which CD4-positive cells were removed by negative selection from the splenocytes prior to stimulation. The columns represent the average concentration from 3 independent experiments. The error bars represent the standard deviation (SD). *, p b 0.05 for stimulated vs. unstimulated cells.

differentiated into Th1 and Th2 cells to produce the cytokines, which presumably work in concert to promote IgG production. 3.5. Role of helper T cells in the immunization As shown above, the CD4-positive helper T cell is indispensable for the induction of antigen-specific IgG antibodies for this in vitro system. Hence, the CD4-positive helper T cells from splenocytes stimulated at various cell densities were characterized. The intracellular cytokines IL-4, IL-10, and IFN- were stained with fluorescein-labeled antibodies and measured by flow cytometry. The helper T cells

were stained using anti-CD4 antibody. A dot analysis of the flow cytometry values for cultures at a density of 4106 cells/mL is shown in Fig. 6A. The ratio of Th1 cells (IFN--producing and IL-4-non-producing) to CD4-positive helper T cells decreased with increasing cell density. The maximum ratio was 7.5% at a cell density of 2 106 cells/mL (Fig. 6B). However, for the Th2 cells (IL-4-producing and IFN--non-producing), the ratio increased with the cell density and showed a maximum (7.5%) at a density of 4 10 6 cells/mL. 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. 6B), showing a maximum value (26.2%) at a cell density of

16

Blimp1

250

AID

Gene expression

Nave

1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL

Nave

1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL

Fig. 4. Expression of genes after IVI. Expression of Blimp-1 and AID was measured by real-time polymerase chain reaction analysis. Further, glyceraldehyde3-phosphate dehydrogenase expression in nave splenocytes was used for the normalization. The error bars represent the SD. *, p b 0.05 for stimulated vs. unstimulated cells.

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IL-2

IL-12

IFN-

TNF-

pg/mL L

IL-4

IL-5

IL-10

GM-CSF

pg/mL

1st + Exp 2nd -

+ + -

+ + +

+ -

+ + -

+ + +

+ -

+ + -

+ + +

+ -

+ + -

+ + +

Fig. 5. Cytokine production during in vitro immunization. Splenocytes were activated with various combinations of stimulation steps. The supernatants were collected, and cytokine production (as indicated by IL-2, IL-4, IL-5, IL-10, IL-12, tumor necrosis factor [TNF6-, interferon [IFN]-, and granulocyte macrophage colony-stimulating factor [GM-CSF]] was evaluated by Bio-Plex. 1st, Exp, and 2nd denote the same steps as in Fig. 2. The data represent the mean of 3 replicates. The error bars represent the SD.

105

104

IL-4

103

102

102

103

104

105

102

103

104

105

102

103

104

105

IFN- -

IL-10 IL-10 + /IL-4 + IL + cells / IL-4+ cells % L-10+ +

B
IFN- +, IL-4 cells / C + cells % - CD4+

Th1(IFN- +, IL-4 -) / CD4 +

IL-4+, IFN- cells / CD4 cells % - 4+
1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL

Th2 (IL-4 +, IFN-) / CD4 +

1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL

1x106 /mL 2x106 /mL 4x106 /mL 8x106 /mL

Fig. 6. Intracellular staining of cytokines in CD4-positive cells in IVI. A. FACS profiles from intracellular cytokine staining for a culture density of 4 106 cells/mL is shown. B. The ratio of cells producing each cytokine to CD4-positive cells was measured with the changing cell density. The columns represent the values from 3 independent experiments. The error bars represent the SD.

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8106 cells/mL. Thus, Th2 cells were preferentially produced when the cell density was above 4106 cells/mL; Th1 cells, when the cell density was low. To study the effect of CD4-positive helper T cells on signal transduction during IVI, we measured cytokine production with and without CD4-positive helper T cells at a cell density of 4 10 6 cells/mL (Fig. 7). The production of IL-4, IL-5, IL-10, IL-12, IFN-, TNF-, and GM-CSF was dramatically reduced when the CD4-positive helper T cells were depleted. By contrast, IL-2 production was increased with their absence. These results show that these different cytokines, with the exception of IL-2, are produced either directly from CD4-positive helper T cells or from cells activated by them. 3.6. Establishment of an monoclonal antibody with IVI Next, we attempted to establish a monoclonal antibody by using our IVI method. After immunization with KLH, 5 10 7 splenocytes were fused with 2.5 108 myelomas. IVI and fusions were independently performed. An average of 356 94 (n= 3) hybridoma colonies were obtained from each fusion experiment. Among these colonies, an average of 3.3 1.1 (n= 3) clones produced the antigen-specific IgG antibody, while an average of 12.7 4.1 (n= 3) clones produced the antigen-specific IgM antibody. The ratio of antigen-specific IgG-producing clones to antigen-specific IgM-producing clones was 3.8. Positive hybridomas were cloned by the limiting dilution method. The affinity of the purified anti-KLH monoclonal IgG antibody was measured by Biacore (Fig. 8). The monoclonal antibody was shown to bind to the antigen in a concentration-dependent manner. The KD of the antibody was calculated as 5.21 107 M, with an association rate constant of 1.47 10 5 M1 s1 and a dissociation rate constant of 7.66 102 s1. Hence, our in vitro method could be used to obtain a monoclonal antigen-specific IgG antibody. 4. Discussion IVI possesses a number of advantages over conventional immunization. However, its low efficiency in B-cell activation

40 35

Response (RU)

30 25 20 15 10 5 0 0 50

3.07 M
KD=5.21 10-7 (M)

1.537 M 0.769 M

100

150

200

Time (sec)
Fig. 8. Kinetic analysis of the binding of keyhole limpet hemocyanin (KLH) to the monoclonal antibody by using the Biacore system. The purified antibody was immobilized onto a CM5 sensor tip. KLH was injected over the biosensor surface twice at the indicated dilutions. The flow rate was 30 L/min. The experimental curves (thin lines) were fitted globally (thick lines) using the mass transfer-limiting model. KLH was injected at concentrations of 3.07 nM, 1.537 nM, and 0.769 nM.

limits the number of positive cells and the insufficient maturation of antibody-producing cells. This difficulty results from the lack of effective immune stimulation and is inherent to the procedure, which involves mixing cells, an antigen, and some immune stimulants (Zafiropoulos et al., 1997). Increasing the number of positive clones is a crucially important step in obtaining the desired antibody. Moreover, establishing an effective IVI protocol for inducing cells that produce antigenspecific IgG antibodies with a high affinity is essential. 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. For this purpose, MDP, LPS, PWM, IL-2, and anti-CD40 antibody (Chin et al., 1995; Saito et al., 2008; Tamura et al., 2007) have been used as immunomodulators during IVI. However, the effects of these stimulators are still restrictive and insufficient, and methods employing these stimulators tend to require relatively long stimulation periods, such as 19 days for PMBCs (Chin et al., 1995). As a result, considerable cell death tends to occur. Schilizzi et al. (1992)

500

6000

5000

300

pg/mL

3000

Fig. 7. Cytokine production after IVI. The effect of CD4-positive cells on cytokine production was analyzed by Bio-Plex. CD4 positive-cells were removed from the splenocytes prior to the stimulation. The culture density was 4 106 cells/mL. The data represent the mean of 3 replicates. The error bars represent the SD.

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added T cell clones to the culture and succeeded in obtaining antigen-specific IgG, but this method is only applicable when T cell clones against the antigen do not already exist. Here, we report an efficient procedure for IVI. We have established procedures for the cell preparations and culture and succeeded in the induction of antigen-specific IgG antibody production. Our procedure consists of 3 steps for inducing antibody production in splenocytes: 1) an initial antigen stimulation, 2) cell expansion with a cocktail of cytokines, and 3) a second antigen stimulation. With our procedure, antigen-specific IgG-producing cells can be obtained in the short period of 6 days. 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. The order of these steps appears to be important for obtaining good results. For the first antigen stimulation, CpG ODN (Bal et al., 2011; Kato et al., 2011) is applied with the antigen and effectively stimulates the B cells, especially the antibody-producing ones. In the expansion step, the T and B cells are stimulated directly. This expansion causes cell proliferation, with the resultant increase in cell density potentially strengthening cell-cell interactions (Haniuda et al., 2011; Nojima et al., 2011). This step is essential for the increase of antigen-specific IgG-producing cells at the end of the culture (Fig. 3A). When the second antigen stimulation was applied to the antigen-stimulated splenocytes following the expansion step, the number of antigen-specific IgG-producing cells increased more than 3-fold. However, this increase with the second antigen stimulation was not observed if the expansion step was omitted (Fig. 3A). The second antigen stimulation activates the antigen-specific antibody-producing cells and may effectively induce IgG production. An expansion after the second antigen stimulation also increased the ratio of positive cells, but this increase was not effective for the efficient induction of antigen-specific IgG-producing cells, since the total number of splenocytes was decreased drastically after the second expansion. This loss of cells during culture decreased the total number of positive cells. When CD8-positive helper T cells and NK cells were not removed prior to the IVI, the cell death worsened, and the total cell number decreased (Zafiropoulos et al., 1997). The production of antigen-specific IgG antibodies was not observed with this preparation. Thus, the removal of the cytotoxic lymphocytes was indispensable for the induction of antigen-specific IgG production. The highest production of total IgG in our experiments so far was induced by the 3-step stimulation (Fig. 2). The single antigen stimulation resulted in IgG production that was 40% of that of the 3-step stimulation. The second antigen stimulation after the expansion increased the production of total IgG but reduced that of IgM. This expression pattern of total IgG and IgM indicate that class switching occurred after the second antigen stimulation. However, antigen-specific IgG was not produced by a single antigen stimulation in our procedure (Fig. 3A). By contrast, antigen-specific IgG production was remarkably high after the 3-step stimulation, with the third step showing a marked increase in production. The pattern of production during the procedure was different between total IgG and antigen-specific IgG. The production of total IgG showed little change between the expansion and the second antigen steps. Hence, the third stimulation is specific to the antigen-specific IgG-producing cells. These

results show that our system worked well for inducing an antigen-specific response. While this has never been explored before in an IVI protocol, we directly examined the presence of cells producing antigen-specific IgG antibodies by ELISPOT. Zafiropoulos et al. (1997) estimated the number of cells producing antigen-specific IgG antibody indirectly from ELISA methods. Tamura et al. (2007) also induced the antibody from PMBCs and counted the number of antigenspecific antibody-producing cells by ELISPOT. However, these researchers counted the number of cells producing IgM but not IgG. Total IgG production was not increased over IgM production in their IVI method, which shows that class switching had occurred but not efficiently. Cell density was one of the important factors for the induction of antigen-specific IgG antibodies. Antigen-specific IgG antibodies were effectively induced at a cell density greater than 4 10 6 cells/mL, while no antigen-specific IgG antibody was induced at a density less than 2 10 6 cells/mL. However, this high density was not good for the cell culture. To improve the culture conditions, we collected the activated cells with low centrifugation after each stimulation and restarted the culture at a fixed cell density. This procedure enabled the selection of activated vial cells and resulted in a cell culture with better conditions. The importance of cell density in the culture was further supported by 2 observations made during the course of this study. First, as in the vivo immune response, interactions between CD4-positive cells and B cells (Kouyama et al., 2007; Okazawa et al., 2008) were crucial for inducing antigenspecific IgG in the IVI system. When the CD4-positive helper T cells were eliminated from the system, cytokine production was suppressed, and antigen-specific IgG production was not induced. Thus, our results indicate that the in vivo interactions responsible for IgG induction were at least partly reproduced in our in vitro system. Moreover, the increase in the production of antigen-specific IgG antibody with increasing cell density (Fig. 3) suggests that the interactions between CD4-positive cells and B cells were strengthened in the high density cultures. Second, real-time PCR analysis showed that Blimp-1 (Nutt et al., 2007) and AID (Pavri and Nussenzweig, 2011) were expressed during the IVI, indicating that the B cells in our system are activated in the same way as in vivo. Further, the expression of AID showed that somatic hypermutation (Cumbers et al., 2002) and class switching (Stavnezer et al., 2008) had occurred during the IVI. The expression of Blimp-1 shows that the B cells differentiated into plasma cells (Diehl et al., 2008; Nutt et al., 2011). The expression of these genes was markedly increased when the cell density of the culture was high. The increased expression of these genes correlated well with an increase in antigen-specific antibody expression. The cytokine milieu is another critical factor for successful IVI. After the 3-step stimulation, the production of cytokines, such as IL-4, IL-5, IL-10, GM-CSF, and IFN-, was increased. These cytokines activate the antibody-producing cells and induce IgG production, class switching, and affinity maturation. These cytokines would have the effect of increasing and decreasing the production of total IgG and IgM, respectively, after the 3-step stimulation (Fig. 2). IFN- and GM-CSF activate macrophages and induce colony formation (Metcalf et al., 1986) and MHC-II expression (Giroux et al., 2003),

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respectively. This would increase the antigen presentation to the CD4-positive helper cells and induce the differentiation and proliferation of antibody-producing cells. The cytokines produced after the 3-step stimulation are from both Th1 and Th2 cells. Hence, helper T cells in our system differentiated into both Th1 and Th2 cells (Anderson et al., 2007; Murphy and Reiner, 2002), which are involved in cell-mediated immunity and humoral immunity, respectively. The Th2 cells induced the differentiation of B cells into plasma cells, which produced the antigen-specific antibody. The differentiation of helper T cells into Th2 cells is desirable for the induction of antigen-specific IgG, but differentiation into Th1 was also observed in the system. Hence, further potent stimulation should be applied to differentiate helper T cells into Th2 cells. Intracellular staining of the cytokines showed that IL-4 and IL-10 production from CD4-positive helper T cells, which activate antibody production by B cells, was greater at the higher cell densities. 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, again explaining why cell densities above 4 106 cells/mL resulted in the excellent formation of spots representing the production of antigen-specific antibodies. Th1 is predominant when the cell density is low during IVI. In our protocol, 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. We succeeded in establishing an IgG monoclonal antibody against KLH with IVI. One of these clones was subcloned, and the monoclonal antibody was purified and shown to have a KD of 5.21 10 7 M, with an association rate constant of 1.47 10 5 M 1 s 1 and a dissociation rate constant of 7.66102 s1. Hence, obtaining a monoclonal antigen-specific IgG antibody is possible with our in vitro method. However, the KD of the antibody was not as high as the one from a normal in vivo method (Sasamori et al., 2011). Chin et al. (1995) established a monoclonal antibody from PMBCs using IVI. This monoclonal antibody had a KD of 2.4108 M, with an association rate constant of 8.5103 M1 s1 and a dissociation rate constant of 2.0104 s1. The association rate constant of our antibody was less than that of their antibody by approximately 2 orders of magnitude. Conversely, the dissociation rate constant of our antibody was greater by approximately the same order of magnitude. One worthy future experiment is to collect more clones to examine whether higher affinity clones are in fact present and obtainable by IVI. 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. The other monoclonal antibodies from our IVI have the same characteristics, with smaller association rate constants and larger dissociation rate constants. This would be related to the affinity maturation process in our IVI. 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. The results show that our in vitro method can induce a sufficient number of B cells to produce IgG-antibody specific to the antigen. For the IVI, the ELISPOT analysis showed that the ratio of positive clones was in the order of 105. Zafiropoulos et al. (1997) induced monoclonal IgG antibody from PMBCs with

IVI and showed that the ratio of positive clones was in the order of 106. Hence, the positive ratio from our IVI method was 1 order of magnitude higher. Further, 1012 days were needed for their immunization, while only 6 days were required for our method. The number of antibody-producing cells is also important for establishing hybridomas. To establish a hybridoma, the ratio of positive cells should be high, since the fusion rate is as high as 104. If we use 108 cells for the fusion, the ratio of positive cells should be higher than 105 to obtain enough positive cells. This value was not sufficient for obtaining enough hybridomas after the cell fusion. In our experiment, we obtained an average of 3.3 clones from 1 fusion. This number was not sufficient to obtain antibodies with high affinities, selectivity, or functional modulations. Nonetheless, 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. For further improving the number of positive clones and IgG antibody affinity of IVI, more efficient activation is required. For this purpose, culture conditions that are closer to the in vivo situation should be applied to the splenocytes. In essence, the conditions in the germinal center (Victora and Nussenzweig, 2012) should be reproduced in vitro during immunization. The co-culture of splenocytes with follicular dendritic cells (Nishikawa et al., 2006) and T helper cell clones (Uthoff and Boldicke, 1993) are likely to activate B cells more efficiently. These stimulations would induce class switching and affinity maturation more effectively. 5. Conclusion Our method not only induces total IgG production but also produces an antigen-specific IgG relative to other methods. 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. This procedure induced the expression of B cell activators including Blimp-1 and AID; and the activation of cytokineproducing CD4-positive helper cells. The expression pattern of total IgG and IgM indicate that class switching occurred after the second antigen stimulation. Thus, the 3-step stimulation appears to mimic important processes that occur in vivo. The application of our method is expected to increase the probability of obtaining a desired antibody against a challenging antigen. With improved efficiency, this method is also expected to be applicable to the production of human monoclonal antibodies. Acknowledgement This work was partially supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry. References
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