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Analysis of cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulin
Journal of Bioscience and Bioengineering VOL. 115 No. 3, 339e345, 2013 www.elsevier.com/locate/jbiosc
Analysis of cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulin Takashi Inagaki,1, 2 Tatsunari Yoshimi,1 Satoshi Kobayashi,1 Masahiro Kawahara,3 and Teruyuki Nagamune2, 3, * Innovative Antibody Engineering Laboratory, Advance Co. Ltd., 5-7 Kobunacho, Chuo-ku, Tokyo 103-8354, Japan,1 Department of Bioengineering, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan,2 and Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan3 Received 10 April 2012; accepted 9 October 2012 Available online 22 November 2012
Although various in vitro immunization methods to generate antigen-specific antibodies have been described, a highly effective method that can generate high-affinity immunoglobulins has not yet been reported. Herein, we analyzed a cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulins. We identified a combination of T cell-dependent stimuli (IL-4, IL-5, anti-CD38 and anti-CD40 antibodies) plus lipopolysaccharides (LPS) that stimulates antigen-exposed splenocytes in vitro, followed by induction of the cells phenotypically equivalent to germinal center B cells. We also observed that LPS induced high expression levels of mRNA for activation-induced cytidine deaminase. We stimulated antigen-exposed splenocytes, followed by the accumulation of mutations in immunoglobulin genes. From the immunized splenocytes, hybridoma clones secreting antigen-specific immunoglobulins were obtained. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: In vitro immunization; Activation-induced cytidine deaminase; Germinal center; Somatic hypermutation; T cell dependent stimuli]
Monoclonal antibodies are now commonly used for the detection and quantification of antigens as well as for the detection and isolation of particular cell types, such as cancer cells. Recently, they have been used as therapeutics for various diseases such as rheumatoid arthritis (1) and cancer (2). The so-called antibody drugs are expected to have maximal therapeutic effect with minimal side effects. Monoclonal antibodies are typically produced by the immunization of animals with a target antigen, followed by hybridoma cell culture. However, this method has various problems. It takes a long time (3e6 months) and is expensive because over 1 mg of antigen is usually required. It is difficult to prepare sufficient quantity of antigen, especially in the case of membrane proteins. Recent advances allow us to use in vitro selection methods such as phage display (3), ribosome display (4) and cell surface display (3,5e7) to select the desired antibody fragments. These display methods have the advantage that we could handle large libraries of antibody fragments. However, these methods still take a few weeks to carry out because it is necessary to repeat the selection step. Therefore, a novel approach in immunotechnology for producing monoclonal antibodies, in vitro immunization, was proposed. B lymphocytes are exposed to antigens in vitro and are further * Corresponding author at: Department of Bioengineering, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Tel.: þ81 3 5841 7328; fax: þ81 3 5841 8657. E-mail address: [email protected] (T. Nagamune).
stimulated by thymocyte-derived lymphokines. Usually, these thymocyte-derived lymphokines are produced by young thymocytes (8) or by mixed lymphocyte cultures (9) that have been used as cell culture supplements. Also a co-culture system using an antigenactivated T helper cell clone has been used (10). However, they are poorly reproducible methods due to lot-to-lot variation of thymocyte-derived lymphokines and clonal variation of antigen-activated T helper cells. Other supportive media including mitogens (11), adjuvant peptides (12) and a combination of cytokines and mitogens (13,14) have been used. However, these studies did not analyze whether such stimuli could trigger various immune responses such as somatic hypermutation (SHM). The germinal center (GC) was recognized as an important region in the B-cell humoral immune response. The GC consists of activated B cells that exhibit rapid proliferation and mutation through SHM (15), and class switch recombination (CSR) (16). A key factor for SHM and CSR is activation-induced cytidine deaminase (AID), whose expression is induced by IL-4 and IL-5 in CD38-stimulated B lymphocytes (17). According to these findings, we thought that artificial induction of SHM and antibody affinity maturation would be possible by in vitro immunization. In this paper, we have carried out a systematic comparison of various stimuli to attain AID induction, induction of GC-like cells and accumulation of mutations in antibody genes, during in vitro immunization of murine splenocytes. As a result, we successfully obtained high-affinity immunoglobulins specific for hen egg lysozyme (HEL).
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.10.008
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Preparation of murine splenocytes A cell suspension of splenocytes was prepared from 4e6-week-old female BALB/c mice. The splenocytes were dispersed through a sterilized nylon mesh (70 mm; Becton Dickinson & Co., Franklin Lakes, NJ, USA) into a single-cell suspension in phosphate buffered saline (PBS). Following lysis of the red blood cells with ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4), the remaining splenocytes were washed and re-suspended in warm (37 C) RPMI1640 containing 25 mM HEPES (SigmaeAldrich, St Louis, MO, USA). In vitro immunization of murine splenocytes For in vitro immunization, splenocytes were re-suspended in RPMI1640 medium without FBS (1 107 cells in 1 mL). The splenocytes were placed in a 15 mL tube and exposed to 1 mM HEL (SigmaeAldrich) and 50 mg/mL N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate (also known as muramyl dipeptide; SigmaeAldrich) for 15 min at room temperature (RT). The following stimuli were added to antigen-activated splenocytes in various combinations: 50 ng/mL of IL-4 (SigmaeAldrich), 50 ng/mL of IL-5 (SigmaeAldrich), 200 mg/mL of LPS (Escherichia coli 0111:B4; SigmaeAldrich), 5 mg/mL of anti-CD38 antibody (aCD38, NIMR-5; Southern Biotechnology Associates, Birmingham, AL, USA), 5 mg/mL of anti-CD40 antibody (aCD40, 1C10; R&D Systems, Minneapolis, MN, USA). RPMI1640 (4 mL) supplemented with 25 mM HEPES, 2 mM L-glutamine, nonessential amino acids (Invitrogen, Carlsbad, CA, USA), 1 mM sodium pyruvate, 50 U/mL penicillin, 50 mg/mL streptomycin, 55 mM 2-mercaptoethanol and 40% FCS was added to stimulated splenocytes immediately without any washing step, and the cells were seeded into a 60-mm dish. Thus, the final concentration of stimuli is 10 ng/mL IL-4, 10 ng/mL IL-5, 40 mg/mL LPS, 1 mg/mL anti-CD38 antibody and 1 mg/mL anti-CD40 antibody. The splenocytes (1 107 cells/5 mL) were cultured for several days without refreshing the culture medium. Real-time PCR analysis Total RNA was isolated from immunized cells using an Isogen kit (Nippon Gene, Tokyo, Japan) and solubilized in sterile water. Complementary DNA was synthesized from 0.5 to 2 mg of total RNA using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan) and an oligo-dT primer in a total volume of 20 ml, of which 1 ml cDNA was used for each sample in a real-time PCR assay. The primers used to amplify AID, Bcl-6 and b-actin were: AID (F), 50 -GGA GCC CGT GCT ATG ACT GT-30 ; AID (R), 50 -GGC TGA GGT TAG GGT TCC ATC T-30 ; Bcl-6 (F), 50 -TCA TTT GCG CCA GAA GCA-30 ; Bcl-6 (R), 50 -GAC ACG CGG TATT GCA CCT T-30 ; b-actin (F), 50 -CCA GTT CGC CAT GGA TGA-30 ; and b-actin (R), 50 -ATG CCG GAG CCG TTG TC-30 , respectively. The reference gene, b-actin, was used to control sample variation in RNA isolation and integrity, RNA input, and reverse transcription. Reactions using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) were set up according to the manufacturer’s instructions, and carried out using a 7500 Fast Real-Time PCR System (Applied Biosystems). Amplification conditions were as follows: 95 C for 10 min; 40 cycles of 95 C for 15 s and 60 C for 30 s. The expression level of mRNA was analyzed by DDCt method. For each sample, the differences in threshold cycles between AID or Bcl-6 and b-actin genes (DCt) were detected, and a calibrated DCt value (DDCt, DCtAID or Bcl-6, DCtbactin) was analyzed. The expression level of mRNA was calculated using the 2DDCt method and normalized against that of unimmunized splenocytes on day 0 to give the relative expression level value of mRNA. The mRNA expression level was calculated from at least three assays for each immunization treatment. The mean SEM is shown in the figures. Flow cytometric analysis Cells were harvested at various time points and labeled using combinations of PE anti-CD45R (RA3-6B2; Miltenyi Biotec, Bergisch Gladbach, Germany), FITC anti-CD38 (NIMR-5; Beckman Coulter, Miami, FL, USA), FITC anti-CD45R (RA3-6B2; Beckman Coulter), anti-mouse Syndecan-1 (CD138; R&D Systems), PE anti-goat IgG (R&D Systems) and polyclonal FITC goat IgG (Beckman Coulter) antibodies. The cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson and Company), and the data were analyzed with FlowJo (Treestar Inc., Palo Alto, CA, USA). Total cell numbers were obtained by counting live lymphocytes after PI staining. The percentages and numbers of each cell population were calculated from at least triplicate assays for each immunization. Mean SEM is shown in the figures. The scFv library was constructed using PCR as Cloning of VH and VL genes described previously (Pluckthun et al, 1996). Briefly, VH and VL were amplified from a cDNA library that was constructed from total RNA of immunized splenocytes and were assembled by PCR. The amplified fragment was digested with EcoRI and HindIII restriction enzymes and ligated into an EcoRI- and HindIII-digested plasmid (pMalc2E; New England Biolabs Inc., Beverly, MA). The DNA sequences of VH and VL were Sequence analysis of VH and VL determined by MegaBACE500 (GE Healthcare, Waukesha, WI). The closest germ line VL and VH for each clone were assigned using IgBLAST (http://www.ncbi.nlm.nih. gov/igblast/). CDRs and FRs were defined by the rules of Kabat et al (18). Because there was no sequence list corresponding to a CDR3 in the IgBLAST database, we analyzed the sequences except for CDR3. Hybridoma formation and selection of HEL-specific immunoglobulin HELimmunized cells were harvested after 5-day culture and then fused with SP2/0eAg14 myeloma cells. Fusion, single step selection and cloning of hybridomas were performed using the ClonaCell-HYÔ system (StemCell Technologies Inc.,
J. BIOSCI. BIOENG., Vancouver, BC, Canada) following the instructions of the manufacturer. To select antigen-specific hybridomas, we performed antigen ELISA. Separated hybridomas were cultured in RPMI1640 medium containing 10% FBS and HT supplement (Invitrogen), and immunoglobulin was purified by using IgG Purification kit-G (Dojindo Molecular technologies, inc., Gaithersburg, MD, USA). Antigen ELISA ELISAs were performed using 96-well MaxisorbÔ MTPs (Thermo Scientific Inc., Bremen, Germany). Each well was coated with 1 mg HEL in 50 ml PBS overnight at 4 C. The antigen-coated wells were then blocked with Protein Free Blocking solution (Thermo Scientific Inc.) for 1 h at RT followed by three washes with PBS. The antibody-containing supernatant (150 ml) was added to the wells for 1 h at 37 C followed by three washes with PBS. The amount of antigen-bound antibody was detected using HRP-labeled anti-mouse IgG (R&D Systems, 1:2000 dilution in Protein Free Blocking solution). HRP activity was measured using 3,30 ,5,50 -tetramethylbenzidine (TMB) as a substrate and the reaction was stopped by adding 100 ml of 1 M HCl. Absorbance at 450 nm was measured using a microtiter plate reader (Bio-Rad, Richmond, CA, USA). Surface plasmon resonance (SPR) analysis Binding kinetics of immunoglobulin was determined by SPR analysis using a BIAcoreÔ X-100 system (GE Healthcare). Immunoglobulins were immobilized on research grade CM5 sensor chips (GE Healthcare) in 10 mM sodium acetate (pH 4.5), using the amine coupling kit supplied by the manufacturer. Unreacted moieties on the surface were blocked with ethanolamine. All measurements were carried out in HBS-EP buffer that contained 10 mM HEPES pH 7.4, 150 mM NaCl, 3.3 mM EDTA and 0.005% Surfactant P-20 (GE Healthcare). Analyses were performed at 25 C and at a flow rate of 3 ml/min. Interaction between antibody and HEL was monitored in real-time and analyzed with BIAevaluation software (GE Healthcare) to determine the kinetic parameters of interaction.
RESULTS Real-time PCR analysis of stimulated splenocytes by T celldependent stimuli and LPS To achieve high-affinity antibody generation, we first planned to induce AID, a key factor for SHM, using a combination of various cytokines and agonist antibodies known as T cell-dependent stimuli. We modified previously described protocols to develop our in vitro immunization methods (Fig. 1A). We used various stimulants at commonly used concentrations to stimulate splenocytes or B cells (19,20). Splenocytes were immunized with HEL (1 mM) as an antigen and cultured for 4 days. The stimulated cells were harvested and their total RNAs were extracted to measure AID mRNA expression level. The expression level of AID is shown in Fig. 1B. The expression level was decreased by antigen stimulation in most conditions. However, the combination of IL-4 þ anti-CD38 antibody, IL-5 þ anti-CD38 antibody, IL-4 þ anti-CD38 antibody þ anti-CD40 antibody and IL-4 þ IL-5 þ anti-CD38 antibody þ anti-CD40 antibody exhibited over 3-fold higher expression level than unstimulated control (Fig. 1B, closed bar). Among these conditions, the expression level was elevated by costimulation with antigen and IL-4 þ IL-5 þ anti-CD38 antibody þ anti-CD40 antibody compared to the absence of antigen stimulation. Therefore, we decided to use the mixture of all components of T cell-dependent stimuli (IL-4, IL-5, anti-CD38 and anti-CD40 antibodies), named as TDS, for further study. Furthermore, B lymphocytes are also activated by T cellindependent stimuli, like LPS, through Toll-like receptors, but the effect of LPS remained to be determined. Therefore, we measured the expression level of AID after stimulation with LPS and TDS. Although LPS strongly induced AID mRNA (17-fold increase compared with unstimulated control, Fig. 1C), these induction was independent of antigen stimulation The elevated expression of AID mRNA dependent on antigen stimulation and higher expression level of AID mRNA was observed by LPS þ TDS stimulation. To maximize the AID mRNA induction dependent on antigen stimulation, we chose LPS þ TDS stimulation for further studies. Time-course analysis of AID mRNA expression levels To examine the optimal period for cell culture, we measured mRNA expression levels at various time points. AID mRNA expression levels gradually increased after 2 days in culture and reached a peak
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FIG. 1. Induction of AID with multiple stimuli. (A) Schematic representation of our in vitro immunization method. (B) The effects of T cell-dependent cytokines and agonist antibodies on the expression level of AID mRNA. Open and closed bars indicate the mRNA expression levels without and with antigen stimulation, respectively. The mRNA expression level was calculated from at least triplicate assays for each immunization. Mean SEM is shown. (C) The effects of LPS and TDS on the expression level of AID mRNA. Open and closed bars indicate the mRNA expression levels without and with LPS stimulation, respectively. The mRNA expression level was calculated from at least triplicate assays for each immunization. Mean SEM is shown.
after 5 days (Fig. 2A), exhibiting up to 1000-fold higher expression levels than before immunization. On the other hand, Bcl-6 mRNA, a genetic marker of the GC (21), gradually increased in a timedependent manner (Fig. 2B). These results indicate that LPS with TDS stimulation increase the content of cells phenotypically equivalent to GC B cells.
Flow cytometric analysis of immunized splenocytes Although mRNA expression levels of AID and Bcl-6 were induced by LPS þ TDS stimulation, we measured them using total RNA of stimulated cells as a template. Therefore, whether we achieved to induce the cells phenotypically equivalent to GC B cells is still unknown. To investigate phenotypic distribution of the
FIG. 2. Time-course analysis of mRNA expression levels of AID and Bcl-6. The mRNA expression level was calculated from at least triplicate assays for each immunization. Mean SEM is shown. Effects of HEL with LPS and TDS (closed square) or LPS with TDS only (open circle) on the mRNA expression profiles of AID (A), Bcl-6 (B).
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FIG. 3. Flow cytometric analysis of immunized splenocytes. (AeF) Time-course analysis of the pre-GC-like, GC-like and pre-PB-like B-cell contents by flow cytometry. Effects of HEL only (A, D), LPS with TDS only (B, E) or HEL with LPS and TDS (C, F) on the number (AeC) and percentage (DeF) of pre-GC-like B cells (open circle), GC-like B cells (closed circle) and pre-PB-like B cells (closed triangle). Splenocytes (1 107 cells; 2 106 cells/mL) were cultured for several days without refreshing culture medium and harvested at various time points for flow cytometric analyses. The number and percentage of each cell population were calculated from at least triplicate assays for each immunization. Mean SEM is shown.
immunized splenocytes, the cells were stained using combinations of antibodies against CD45R (known as a B-cell lineage marker), CD38 (known as a surface marker of pre-GC B cells and downregulated in the GC) (22) and CD138 (known as a surface marker of plasmablasts) (23), and subjected to flow cytometric analysis. Both the splenocytes phenotypically equivalent to GC B cells (defined as GC-like B cells; CD45RþCD38low; 2.5 105 cells, 2.21% of living immunized splenocytes, Fig. 3C and F) and plasmablasts (defined as PB-like B cells; CD45Rlow CD138þ; 1.1 105 cells, 0.94%, data not shown) were detected after 5 days in culture. Pre-PB-like B cells (CD45RþCD138þ) were also detected (1.1 106 cells, 9.93%).
Also, pre-GC-like B cells (CD45RþCD38þ) gradually increased and reached 6.7 106 cells (58.0%) after 5 days in culture. When splenocytes were stimulated by antigen only, pre-GClike B cells and pre-PB-like B cells were observed (Fig. 3A and D), but the population of these cells reached only 13.4% (1.5 105 cells) and 8.0% (8.9 104 cells), respectively. On the other hand, when splenocytes were stimulated by LPS þ TDS, the population and number (Fig. 3B and E) of B-cell subsets were dramatically increased irrespective of antigen addition. These results suggested that LPS þ TDS stimulation could efficiently induce GC-like B cells in vitro.
TABLE 1. Mutation frequency of obtained anti-HEL scFv clones. Culture period
Day Day Day Day a
4 5 6 7
VH
VL
scFv
Mutation (bp)
Totala (bp)
Mutation frequency (%)
Mutation (bp)
Totala (bp)
Mutation frequency (%)
Mutation (bp)
Totala (bp)
Mutation frequency (%)
26 78 74 56
2355 2627 2611 2336
1.1% 3.0% 2.8% 2.4%
55 49 51 61
2433 2105 2535 2118
2.3% 2.3% 2.0% 2.9%
81 127 125 117
4788 4732 5146 4454
1.69% 2.68% 2.43% 2.63%
Total number of analyzed nucleotides derived from 6 to 8 selected clones.
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Sequence analysis of VH and VL from immunized splenocytes We selected eight clones from splenocytes at each culture period with different affinities and analyzed the number of point mutations (Table 1). The number of mutations ranged from 0 to 37 in the VL sequence, and 0e30 in the VH sequence. Although mutations were observed not only in complementarity determining region (CDR) but also in framework region (FR), the mutations that caused replacement of amino acids were highly observed in CDR (at least 3-fold higher than those observed in FR). Overall, 62.5% of the selected clones (20 clones) had more than 2% difference from the most similar germ line gene, and 8 clones out of 20 clones had more than 5% difference. The average rate of point mutations in the VL and VH sequences was 1.1% and 2.3% from a 4-day culture, 3.0% and 2.3% from a 5-day culture, 2.8% and 2.0% from a 6-day culture, and 2.4% and 2.9% from a 7day culture, respectively. Because a commonly used threshold for considering a case “mutated” was proposed as 2% difference (24), these results suggest that we could successfully induce the accumulation of point mutations after 5-day culture of splenocytes. CDR amino acid sequences of VH were analyzed (Table 2). As a result, we obtained very similar clones which are thought to be derived from the same germ line gene such as day 4e4, 4e7, 5e2 and 5e3. However, they had different CDR3 amino acid sequences and point mutations in CDR1 or CDR2. These results suggest that specific binders for HEL existed in naïve splenocytes and these clones accumulated point mutations. According to these results, we use 5-day culture for further hybridoma formation.
TABLE 3. Kinetic analysis of the obtained anti-HEL immunoglobulin.
Selection of anti-HEL immunoglobulin We fused immunized splenocytes with myeloma cells to generate hybridomas. Hybridomas were separated by ClonaCell HY kit and 196 clones were screened by antigen ELISA. Four clones which exhibited higher absorbance were subjected to SPR analysis to determine their affinity (Table 3). The analysis revealed that the immunoglobulin clone no. 189 demonstrated a Kd of 0.5 nM, a kon
Clone no. 189 135 110 114
kon (M1 s1)
Kd (nM) 0.5 62 72 75
9.9 1.1 3.7 7.1
5
10 104 104 103
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koff (s1) 5.5 6.7 2.7 5.3
104 104 103 104
of 9.9 105 M1 s1 and a koff of 5.5 104 s1 (Fig. 4A) and no. 135 demonstrated a Kd of 62 nM, a kon of 1.1 104 M1 s1 and a koff of 6.7 104 s1 (Fig. 4B). These results indicated that we could immunize splenocytes by our in vitro immunization method. DISCUSSION In this paper, we were able to induce AID mRNA expression and GC-like B cells using our in vitro immunization protocol. We also obtained anti-HEL antibody and detected the accumulation of mutation in their variable regions. Previous reports have shown that the chicken DT40 B-cell line (25,26), Ramos cells (27) and mouse IgGþ/AIDþ B-cell lines derived from GC B cells (28) can induce SHM in immunoglobulin genes through the induction of AID in vitro. These methods attained easy and large-scale variation of immunoglobulin genes; however, they did not immunize antigen to produce antigen-specific antibody. Therefore, one has to deal with a large library of cells (about 108 cells) and utilize magnetic sorting or flow cytometry to sort and separate the positive clones. Even if such separation methods were utilized, the resulting positive cell rate was still low (10.3% after screening once) (26). Therefore, it is important to develop a new in vitro immunization protocol that can efficiently induce antigen-specific immunoglobulins. Until now, many in vitro immunization protocols using peripheral blood mononuclear cells (29,30), murine splenocytes (31) and murine popliteal lymph nodes (32) have been developed to successfully generate antigen-specific antibodies. Thymus-conditioned medium (TCM), mixed lymphokines, such as IL-4, IL-2 and
TABLE 2. VH CDR amino acid sequence of obtained anti-HEL scFv clones. Culture period Day 4
Day 5
Day 6
Day 7
Clone no. 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Closest germ line e VH36-60.a2.90 VOx1 VHQ52.a3.8 VH7183.a7.10 VHQ52.a9.26 VHQ52.a3.8 J558.45 VOx1 VHQ52.a3.8 VHQ52.a3.8 VH7183.a30.50 J558.45 J558.44 e e VHSM7.a3.93 VH81X VH7183.a47.76 VHQ52.a9.26 VHQ52.a27.79 H13-3 J558.23 J558.12.102 e VHA1 J558.45 VH7183.a15.24 e VHQ52.a7.18 J558.47 J558.17
CDR1 sequence
CDR2 sequence
CDR3 sequence
e SDYAWN SYGVH SYGVS SYGMS SYGVH SYGVS SYWMH SYGVH SYGVS SYGVS SYGMS SYWMH SYTMH e e DTYMH e SFGMH SYGVH SYGVH SYTMH DYYIN DYEMH e SYDIN SYWMH SYAMS e SYGVH SYVMH SYWIE
e YISYSGSTSYNPSLKS VIWAGGSTNYNSALMS VIWGDGSTNYHSALIS TISSGGSYTYYPDSVKG VIWSDGSTTYNSALKS VIWGDGSTNYHSALIS YINPSTGYTEYNQKFKD VIWAGGSTNYNSALMS VIWGDGSTNYHSALIS VIWGDGSTNYHSALIS TIMSNGGSTYYPDSVKG YINPSTGYTEYNQKFKD YINPSSGYTNYNQKFKD e e RIDPANGNTKYDPKFQG e YISSGSSTIYYADTVKG VIWSDGSTTYNSALKS VIWAGGSTNYNSALMS YINPSSGYTEYNQKFKD EIYPGSGNTYYNEKFKG AIDPETGGTAYNQKFKG e WIYPGDGSTKYNEKFKG YINPSTGYTEYNQKFKD TISSGGSYTYYPDSVKG e VIWRGGSTDYNAAFMS YINPYNDGTKYNEKFKG EILPGSGSTNYNEKFKG
e CARYDWYFDVW CASIGTTSYW CAKLGHYYAMDYW CARYGTDEDW CAREYDYAWFAYW CAREVRQAWFAYW CARGGYAMDYW CASYGYDWAWFAYW CARDWMDGLDYW CAKNERYDAMDYW CARGTARAGAMDYW CARSGRYDAMDYW CARQ e e CARRQLGLPFAYW e CARWGRYDVEGFAYW CARGPMDYW CARGYGDGYW CARGLTTALYYYAMDYW CARGPDYDGYW CARGGTGTRVFAYW e CARDYGRSMDYW CARSEYGSSYGYFDVW CARGNEDW e CAKIVGGDAMDYW CARDYRYSWFAYW CARLEYYYGSSYGYW
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FIG. 4. SPR analyses of anti-HEL IgG. (A) The SPR sensorgram of anti-HEL IgG (clone no. 189). The overlay plot shows the binding of HEL at different concentrations (8.6, 17.1, 34.2, 68.5, 137 nM) to immobilized anti-HEL IgG. (B) The SPR sensorgram of anti-HEL IgG (clone no. 135). The overlay plot shows the binding of HEL at different concentrations (16.7, 33.3, 66.7, 133, 267 nM) to immobilized anti-HEL IgG.
IL-10, and mitogens such as LPS were used for activation of B cells in these methods. Although TCM included many factors that could stimulate antigen-activated B lymphocytes such as B-cell differentiation factor and B-cell growth factor (33,34), TCM could not induce CSR (28). On the other hand, IL-4 with LPS was able to induce CSR (IgM, IgG1 or IgE) (13,14), exhibiting a 5e10% increase in the number of IgG1 cells (14). Because CSR needs both AID expression and induction of GC-like B cells (16), these previous reports are consistent with our data that IL-4 with LPS could not induce Bcl-6 mRNA, a hallmark of GC-like B cells. Therefore, we considered that they were unable to induce GC formation because they did not include the molecules that stimulate the CD38 signaling pathway. Taking our findings in conjunction with previous reports together, IL-5 and anti-CD38 would appear to be important in the induction of GC formation. In this report, we demonstrated that we could immunize murine splenocytes in vitro and obtain an antigen-specific highaffinity immunoglobulin. If we can differentiate induced pluripotent stem (iPS) cells into splenic B cells, we could construct a complete in vitro antibody generation system. Moreover, we could immunize human B cells by means of modifying stimulation conditions. Although our method could not achieve efficient selection of high-affinity antibody, our method can generate immunized splenocytes rapidly. Therefore, our method has an advantage to adapt various selection methods for high-affinity antibody generation using immunized cells.
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8. Luben, R. A. and Mohler, M. A.: In vitro immunization as an adjunct to the production of hybridomas producing antibodies against the lymphokine osteoclast activating factor, Mol. Immunol., 17, 635e639 (1980). 9. Borrebaeck, C. A. and Moller, S. A.: In vitro immunization. Effect of growth and differentiation factors on antigen-specific B cell activation and production of monoclonal antibodies to autologous antigens and weak immunogens, J. Immunol., 136, 3710e3715 (1986). 10. Uthoff, S. and Böldicke, T.: In vitro immunization of mouse spleen cells for the production of monoclonal IgG1 antibodies using an antigen-specific T helper cell clone (D.10.G4.1), J. Immunol. Methods, 166, 165e175 (1993). 11. Strike, L. E., Devens, B. H., and Lundak, R. L.: Production of human-human hybridomas secreting antibody to sheep erythrocytes after in vitro immunization, J. Immunol., 132, 1798e1803 (1984). 12. Boss, B. D.: An improved in vitro immunization procedure for the production of monoclonal antibodies against neural and other antigens, Brain Res., 291, 193e196 (1984). 13. Snapper, C. M. and Paul, W. E.: B cell stimulatory factor-1 (interleukin 4) prepares resting murine B cells to secrete IgG1 upon subsequent stimulation with bacterial lipopolysaccharide, J. Immunol., 139, 10e17 (1987). 14. Purkerson, J. M. and Isakson, P. C.: Isotype switching in anti-immunoglobulin-activated B lymphoblasts: differential requirements for interleukin 4 and other lymphokines to elicit membrane vs. secreted IgG1, Eur. J. Immunol., 21, 707e714 (1991). 15. Martin, A., Bardwell, P. D., Woo, C. J., Fan, M., Shulman, M. J., and Scharff, M. D.: Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas, Nature, 415, 802e806 (2002). 16. Oppezzo, P., Vuillier, F., Vasconcelos, Y., Dumas, G., Magnac, C., PayelleBrogard, B., Pritsch, O., and Dighiero, G.: Chronic lymphocytic leukemia B cells expressing AID display dissociation between class switch recombination and somatic hypermutation, Blood, 101, 4029e4032 (2003). 17. Horikawa, K. and Takatsu, K.: Interleukin-5 regulates genes involved in B-cell terminal maturation, Immunology, 118, 497e508 (2006). 18. Kabat, E. A., Wu, T. T., Perry, H. M., Gottesmann, K. S., and Foeller, C.: Sequences of proteins of immunological interest. NIH publication No. 91-3242, 103-646, 5th ed. U.S. Department of Health and Human Services, Washington, D.C. (1991). 19. Baba, M., Kikuchi, Y., Mori, S., Kimoto, H., Inui, S., Sakaguchi, N., Inoue, J., Yamamoto, T., Takemori, T., Howard, M., and Takatsu, K.: Mouse germinal center B cells with the xid mutation retain responsiveness to antimouse CD40 antibodies but diminish IL-5 responsiveness, Int. Immunol., 9, 1463e1473 (1997). 20. Nakanishi, K., Matsui, K., Kashiwamura, S., Nishioka, Y., Nomura, J., Nishimura, Y., Sakaguchi, N., Yonehara, S., Higashino, K., and Shinka, S.: IL-4 and anti-CD40 protect against Fas-mediated B cell apoptosis and induce B cell growth and differentiation, Int. Immunol., 8, 791e798 (1996). 21. Allman, D., Jain, A., Dent, A., Maile, R. R., Selvaggi, T., Kehry, M. R., and Staudt, L. M.: BCL-6 expression during B-cell activation, Blood, 87, 5257e5268 (1996). 22. Shinall, S. M., Gonzalez-Fernandez, M., Noelle, R. J., and Waldschmidt, T. J.: Identification of murine germinal center B cell subsets defined by the expression of surface isotypes and differentiation antigens, J. Immunol., 164, 5729e5738 (2000). 23. Wehrli, N., Legler, D. F., Finke, D., Toellner, K. M., Loetscher, P., Baggiolini, M., MacLennan, I. C., and Acha-Orbea, H.: Changing responsiveness to chemokines allows medullary plasmablasts to leave lymph nodes, Eur. J. Immunol., 31, 609e616 (2001). 24. Messmer, B. T., Albesiano, E., Messmer, D., and Chiorazzi, N.: The pattern and distribution of immunoglobulin VH gene mutations in chronic lymphocytic
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leukemia B cells are consistent with the canonical somatic hypermutation process, Blood, 103, 3490e3495 (2004). Seo, H., Masuoka, M., Murofushi, H., Takeda, S., Shibata, T., and Ohta, K.: Rapid generation of specific antibodies by enhanced homologous recombination, Nat. Biotechnol., 23, 731e735 (2005). Todo, K., Miyake, K., Magari, M., Kanayama, N., and Ohmori, H.: Novel in vitro screening system for monoclonal antibodies using hypermutating chicken B cell library, J. Biosci. Bioeng., 102, 478e481 (2006). Cumbers, S. J., Williams, G. T., Davies, S. L., Grenfell, R. L., Takeda, S., Batista, F. D., Sale, J. E., and Neuberger, M. S.: Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines, Nat. Biotechnol., 20, 1129e1134 (2002). Bhattacharya, P., Grigera, F., Rogozin, I. B., McCarty, T., Morse, H. C., 3rd, and Kenter, A. L.: Identification of murine B cell lines that undergo somatic hypermutation focused to A: T and G: C residues, Eur. J. Immunol., 38, 227e239 (2008). Tamura, T., Tomimatsu, K., Katakura, Y., Yamashita, M., Matsumoto, S. E., Aiba, Y., Jung, Y. S., Abe, Y., Fujiki, T., Teruya, K., and Shirahata, S.: Antipeptide antibody production elicited by in vitro immunization of human peripheral blood mononuclear cells, Biosci. Biotechnol. Biochem., 71, 2871e2875 (2007).
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30. Tomimatsu, K., Matsumoto, S. E., Yamashita, M., Teruya, K., Katakura, Y., Kabayama, S., and Shirahata, S.: Production of human monoclonal antibodies against Fc(epsilon)RI(alpha) by a method combining in vitro immunization with phage display, Biosci. Biotechnol. Biochem., 73, 1465e1469 (2009). 31. Ravi, M., Sundar, S. S., Kumar, K. K., Parvathi, D., and Paul, S. F.: Hybridoma generation by in vitro immunization of murine splenocytes with cytosolic proteins of Chinese hamster ovary (CHO) mitotic cells, Hybridoma (Larchmt), 26, 311e315 (2007). 32. Belo, E. F., Ferraz, A. S., Coutinho, L. M., Oliveira, A. P., Carmo, A. M., Tunes, C. F., Ferreira, T., Ito, A. Y., Machado, M. S., De, L. F. D., and De Gaspari, E. N.: Production of monoclonal antibodies against Neisseria meningitidis using popliteal lymph nodes and in vivo/in vitro immunization: prevalence study of new monoclonal antibodies in greater Sao Paulo, Brazil, Hybridoma (Larchmt), 26, 302e310 (2007). 33. Farrar, J. J., Howard, M., Fuller-Farrar, J., and Paul, W. E.: Biochemical and physicochemical characterization of mouse B cell growth factor: a lymphokine distinct from interleukin 2, J. Immunol., 131, 1838e1842 (1983). 34. Kishimoto, T., Yoshizaki, K., Kimoto, M., Okada, M., Kuritani, T., Kikutani, H., Shimizu, K., Nakagawa, T., Nakagawa, N., Miki, Y., and other 4 authors: B cell growth and differentiation factors and mechanism of B cell activation, Immunol. Rev., 78, 97e118 (1984).
Analysis of cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulin Takashi Inagaki,1, 2 Tatsunari Yoshimi,1 Satoshi Kobayashi,1 Masahiro Kawahara,3 and Teruyuki Nagamune2, 3, * Innovative Antibody Engineering Laboratory, Advance Co. Ltd., 5-7 Kobunacho, Chuo-ku, Tokyo 103-8354, Japan,1 Department of Bioengineering, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan,2 and Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan3 Received 10 April 2012; accepted 9 October 2012 Available online 22 November 2012
Although various in vitro immunization methods to generate antigen-specific antibodies have been described, a highly effective method that can generate high-affinity immunoglobulins has not yet been reported. Herein, we analyzed a cellular phenotype during in vitro immunization of murine splenocytes for generating antigen-specific immunoglobulins. We identified a combination of T cell-dependent stimuli (IL-4, IL-5, anti-CD38 and anti-CD40 antibodies) plus lipopolysaccharides (LPS) that stimulates antigen-exposed splenocytes in vitro, followed by induction of the cells phenotypically equivalent to germinal center B cells. We also observed that LPS induced high expression levels of mRNA for activation-induced cytidine deaminase. We stimulated antigen-exposed splenocytes, followed by the accumulation of mutations in immunoglobulin genes. From the immunized splenocytes, hybridoma clones secreting antigen-specific immunoglobulins were obtained. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: In vitro immunization; Activation-induced cytidine deaminase; Germinal center; Somatic hypermutation; T cell dependent stimuli]
Monoclonal antibodies are now commonly used for the detection and quantification of antigens as well as for the detection and isolation of particular cell types, such as cancer cells. Recently, they have been used as therapeutics for various diseases such as rheumatoid arthritis (1) and cancer (2). The so-called antibody drugs are expected to have maximal therapeutic effect with minimal side effects. Monoclonal antibodies are typically produced by the immunization of animals with a target antigen, followed by hybridoma cell culture. However, this method has various problems. It takes a long time (3e6 months) and is expensive because over 1 mg of antigen is usually required. It is difficult to prepare sufficient quantity of antigen, especially in the case of membrane proteins. Recent advances allow us to use in vitro selection methods such as phage display (3), ribosome display (4) and cell surface display (3,5e7) to select the desired antibody fragments. These display methods have the advantage that we could handle large libraries of antibody fragments. However, these methods still take a few weeks to carry out because it is necessary to repeat the selection step. Therefore, a novel approach in immunotechnology for producing monoclonal antibodies, in vitro immunization, was proposed. B lymphocytes are exposed to antigens in vitro and are further * Corresponding author at: Department of Bioengineering, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. Tel.: þ81 3 5841 7328; fax: þ81 3 5841 8657. E-mail address: [email protected] (T. Nagamune).
stimulated by thymocyte-derived lymphokines. Usually, these thymocyte-derived lymphokines are produced by young thymocytes (8) or by mixed lymphocyte cultures (9) that have been used as cell culture supplements. Also a co-culture system using an antigenactivated T helper cell clone has been used (10). However, they are poorly reproducible methods due to lot-to-lot variation of thymocyte-derived lymphokines and clonal variation of antigen-activated T helper cells. Other supportive media including mitogens (11), adjuvant peptides (12) and a combination of cytokines and mitogens (13,14) have been used. However, these studies did not analyze whether such stimuli could trigger various immune responses such as somatic hypermutation (SHM). The germinal center (GC) was recognized as an important region in the B-cell humoral immune response. The GC consists of activated B cells that exhibit rapid proliferation and mutation through SHM (15), and class switch recombination (CSR) (16). A key factor for SHM and CSR is activation-induced cytidine deaminase (AID), whose expression is induced by IL-4 and IL-5 in CD38-stimulated B lymphocytes (17). According to these findings, we thought that artificial induction of SHM and antibody affinity maturation would be possible by in vitro immunization. In this paper, we have carried out a systematic comparison of various stimuli to attain AID induction, induction of GC-like cells and accumulation of mutations in antibody genes, during in vitro immunization of murine splenocytes. As a result, we successfully obtained high-affinity immunoglobulins specific for hen egg lysozyme (HEL).
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.10.008
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Preparation of murine splenocytes A cell suspension of splenocytes was prepared from 4e6-week-old female BALB/c mice. The splenocytes were dispersed through a sterilized nylon mesh (70 mm; Becton Dickinson & Co., Franklin Lakes, NJ, USA) into a single-cell suspension in phosphate buffered saline (PBS). Following lysis of the red blood cells with ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4), the remaining splenocytes were washed and re-suspended in warm (37 C) RPMI1640 containing 25 mM HEPES (SigmaeAldrich, St Louis, MO, USA). In vitro immunization of murine splenocytes For in vitro immunization, splenocytes were re-suspended in RPMI1640 medium without FBS (1 107 cells in 1 mL). The splenocytes were placed in a 15 mL tube and exposed to 1 mM HEL (SigmaeAldrich) and 50 mg/mL N-acetylmuramyl-L-alanyl-D-isoglutamine hydrate (also known as muramyl dipeptide; SigmaeAldrich) for 15 min at room temperature (RT). The following stimuli were added to antigen-activated splenocytes in various combinations: 50 ng/mL of IL-4 (SigmaeAldrich), 50 ng/mL of IL-5 (SigmaeAldrich), 200 mg/mL of LPS (Escherichia coli 0111:B4; SigmaeAldrich), 5 mg/mL of anti-CD38 antibody (aCD38, NIMR-5; Southern Biotechnology Associates, Birmingham, AL, USA), 5 mg/mL of anti-CD40 antibody (aCD40, 1C10; R&D Systems, Minneapolis, MN, USA). RPMI1640 (4 mL) supplemented with 25 mM HEPES, 2 mM L-glutamine, nonessential amino acids (Invitrogen, Carlsbad, CA, USA), 1 mM sodium pyruvate, 50 U/mL penicillin, 50 mg/mL streptomycin, 55 mM 2-mercaptoethanol and 40% FCS was added to stimulated splenocytes immediately without any washing step, and the cells were seeded into a 60-mm dish. Thus, the final concentration of stimuli is 10 ng/mL IL-4, 10 ng/mL IL-5, 40 mg/mL LPS, 1 mg/mL anti-CD38 antibody and 1 mg/mL anti-CD40 antibody. The splenocytes (1 107 cells/5 mL) were cultured for several days without refreshing the culture medium. Real-time PCR analysis Total RNA was isolated from immunized cells using an Isogen kit (Nippon Gene, Tokyo, Japan) and solubilized in sterile water. Complementary DNA was synthesized from 0.5 to 2 mg of total RNA using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan) and an oligo-dT primer in a total volume of 20 ml, of which 1 ml cDNA was used for each sample in a real-time PCR assay. The primers used to amplify AID, Bcl-6 and b-actin were: AID (F), 50 -GGA GCC CGT GCT ATG ACT GT-30 ; AID (R), 50 -GGC TGA GGT TAG GGT TCC ATC T-30 ; Bcl-6 (F), 50 -TCA TTT GCG CCA GAA GCA-30 ; Bcl-6 (R), 50 -GAC ACG CGG TATT GCA CCT T-30 ; b-actin (F), 50 -CCA GTT CGC CAT GGA TGA-30 ; and b-actin (R), 50 -ATG CCG GAG CCG TTG TC-30 , respectively. The reference gene, b-actin, was used to control sample variation in RNA isolation and integrity, RNA input, and reverse transcription. Reactions using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) were set up according to the manufacturer’s instructions, and carried out using a 7500 Fast Real-Time PCR System (Applied Biosystems). Amplification conditions were as follows: 95 C for 10 min; 40 cycles of 95 C for 15 s and 60 C for 30 s. The expression level of mRNA was analyzed by DDCt method. For each sample, the differences in threshold cycles between AID or Bcl-6 and b-actin genes (DCt) were detected, and a calibrated DCt value (DDCt, DCtAID or Bcl-6, DCtbactin) was analyzed. The expression level of mRNA was calculated using the 2DDCt method and normalized against that of unimmunized splenocytes on day 0 to give the relative expression level value of mRNA. The mRNA expression level was calculated from at least three assays for each immunization treatment. The mean SEM is shown in the figures. Flow cytometric analysis Cells were harvested at various time points and labeled using combinations of PE anti-CD45R (RA3-6B2; Miltenyi Biotec, Bergisch Gladbach, Germany), FITC anti-CD38 (NIMR-5; Beckman Coulter, Miami, FL, USA), FITC anti-CD45R (RA3-6B2; Beckman Coulter), anti-mouse Syndecan-1 (CD138; R&D Systems), PE anti-goat IgG (R&D Systems) and polyclonal FITC goat IgG (Beckman Coulter) antibodies. The cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson and Company), and the data were analyzed with FlowJo (Treestar Inc., Palo Alto, CA, USA). Total cell numbers were obtained by counting live lymphocytes after PI staining. The percentages and numbers of each cell population were calculated from at least triplicate assays for each immunization. Mean SEM is shown in the figures. The scFv library was constructed using PCR as Cloning of VH and VL genes described previously (Pluckthun et al, 1996). Briefly, VH and VL were amplified from a cDNA library that was constructed from total RNA of immunized splenocytes and were assembled by PCR. The amplified fragment was digested with EcoRI and HindIII restriction enzymes and ligated into an EcoRI- and HindIII-digested plasmid (pMalc2E; New England Biolabs Inc., Beverly, MA). The DNA sequences of VH and VL were Sequence analysis of VH and VL determined by MegaBACE500 (GE Healthcare, Waukesha, WI). The closest germ line VL and VH for each clone were assigned using IgBLAST (http://www.ncbi.nlm.nih. gov/igblast/). CDRs and FRs were defined by the rules of Kabat et al (18). Because there was no sequence list corresponding to a CDR3 in the IgBLAST database, we analyzed the sequences except for CDR3. Hybridoma formation and selection of HEL-specific immunoglobulin HELimmunized cells were harvested after 5-day culture and then fused with SP2/0eAg14 myeloma cells. Fusion, single step selection and cloning of hybridomas were performed using the ClonaCell-HYÔ system (StemCell Technologies Inc.,
J. BIOSCI. BIOENG., Vancouver, BC, Canada) following the instructions of the manufacturer. To select antigen-specific hybridomas, we performed antigen ELISA. Separated hybridomas were cultured in RPMI1640 medium containing 10% FBS and HT supplement (Invitrogen), and immunoglobulin was purified by using IgG Purification kit-G (Dojindo Molecular technologies, inc., Gaithersburg, MD, USA). Antigen ELISA ELISAs were performed using 96-well MaxisorbÔ MTPs (Thermo Scientific Inc., Bremen, Germany). Each well was coated with 1 mg HEL in 50 ml PBS overnight at 4 C. The antigen-coated wells were then blocked with Protein Free Blocking solution (Thermo Scientific Inc.) for 1 h at RT followed by three washes with PBS. The antibody-containing supernatant (150 ml) was added to the wells for 1 h at 37 C followed by three washes with PBS. The amount of antigen-bound antibody was detected using HRP-labeled anti-mouse IgG (R&D Systems, 1:2000 dilution in Protein Free Blocking solution). HRP activity was measured using 3,30 ,5,50 -tetramethylbenzidine (TMB) as a substrate and the reaction was stopped by adding 100 ml of 1 M HCl. Absorbance at 450 nm was measured using a microtiter plate reader (Bio-Rad, Richmond, CA, USA). Surface plasmon resonance (SPR) analysis Binding kinetics of immunoglobulin was determined by SPR analysis using a BIAcoreÔ X-100 system (GE Healthcare). Immunoglobulins were immobilized on research grade CM5 sensor chips (GE Healthcare) in 10 mM sodium acetate (pH 4.5), using the amine coupling kit supplied by the manufacturer. Unreacted moieties on the surface were blocked with ethanolamine. All measurements were carried out in HBS-EP buffer that contained 10 mM HEPES pH 7.4, 150 mM NaCl, 3.3 mM EDTA and 0.005% Surfactant P-20 (GE Healthcare). Analyses were performed at 25 C and at a flow rate of 3 ml/min. Interaction between antibody and HEL was monitored in real-time and analyzed with BIAevaluation software (GE Healthcare) to determine the kinetic parameters of interaction.
RESULTS Real-time PCR analysis of stimulated splenocytes by T celldependent stimuli and LPS To achieve high-affinity antibody generation, we first planned to induce AID, a key factor for SHM, using a combination of various cytokines and agonist antibodies known as T cell-dependent stimuli. We modified previously described protocols to develop our in vitro immunization methods (Fig. 1A). We used various stimulants at commonly used concentrations to stimulate splenocytes or B cells (19,20). Splenocytes were immunized with HEL (1 mM) as an antigen and cultured for 4 days. The stimulated cells were harvested and their total RNAs were extracted to measure AID mRNA expression level. The expression level of AID is shown in Fig. 1B. The expression level was decreased by antigen stimulation in most conditions. However, the combination of IL-4 þ anti-CD38 antibody, IL-5 þ anti-CD38 antibody, IL-4 þ anti-CD38 antibody þ anti-CD40 antibody and IL-4 þ IL-5 þ anti-CD38 antibody þ anti-CD40 antibody exhibited over 3-fold higher expression level than unstimulated control (Fig. 1B, closed bar). Among these conditions, the expression level was elevated by costimulation with antigen and IL-4 þ IL-5 þ anti-CD38 antibody þ anti-CD40 antibody compared to the absence of antigen stimulation. Therefore, we decided to use the mixture of all components of T cell-dependent stimuli (IL-4, IL-5, anti-CD38 and anti-CD40 antibodies), named as TDS, for further study. Furthermore, B lymphocytes are also activated by T cellindependent stimuli, like LPS, through Toll-like receptors, but the effect of LPS remained to be determined. Therefore, we measured the expression level of AID after stimulation with LPS and TDS. Although LPS strongly induced AID mRNA (17-fold increase compared with unstimulated control, Fig. 1C), these induction was independent of antigen stimulation The elevated expression of AID mRNA dependent on antigen stimulation and higher expression level of AID mRNA was observed by LPS þ TDS stimulation. To maximize the AID mRNA induction dependent on antigen stimulation, we chose LPS þ TDS stimulation for further studies. Time-course analysis of AID mRNA expression levels To examine the optimal period for cell culture, we measured mRNA expression levels at various time points. AID mRNA expression levels gradually increased after 2 days in culture and reached a peak
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FIG. 1. Induction of AID with multiple stimuli. (A) Schematic representation of our in vitro immunization method. (B) The effects of T cell-dependent cytokines and agonist antibodies on the expression level of AID mRNA. Open and closed bars indicate the mRNA expression levels without and with antigen stimulation, respectively. The mRNA expression level was calculated from at least triplicate assays for each immunization. Mean SEM is shown. (C) The effects of LPS and TDS on the expression level of AID mRNA. Open and closed bars indicate the mRNA expression levels without and with LPS stimulation, respectively. The mRNA expression level was calculated from at least triplicate assays for each immunization. Mean SEM is shown.
after 5 days (Fig. 2A), exhibiting up to 1000-fold higher expression levels than before immunization. On the other hand, Bcl-6 mRNA, a genetic marker of the GC (21), gradually increased in a timedependent manner (Fig. 2B). These results indicate that LPS with TDS stimulation increase the content of cells phenotypically equivalent to GC B cells.
Flow cytometric analysis of immunized splenocytes Although mRNA expression levels of AID and Bcl-6 were induced by LPS þ TDS stimulation, we measured them using total RNA of stimulated cells as a template. Therefore, whether we achieved to induce the cells phenotypically equivalent to GC B cells is still unknown. To investigate phenotypic distribution of the
FIG. 2. Time-course analysis of mRNA expression levels of AID and Bcl-6. The mRNA expression level was calculated from at least triplicate assays for each immunization. Mean SEM is shown. Effects of HEL with LPS and TDS (closed square) or LPS with TDS only (open circle) on the mRNA expression profiles of AID (A), Bcl-6 (B).
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FIG. 3. Flow cytometric analysis of immunized splenocytes. (AeF) Time-course analysis of the pre-GC-like, GC-like and pre-PB-like B-cell contents by flow cytometry. Effects of HEL only (A, D), LPS with TDS only (B, E) or HEL with LPS and TDS (C, F) on the number (AeC) and percentage (DeF) of pre-GC-like B cells (open circle), GC-like B cells (closed circle) and pre-PB-like B cells (closed triangle). Splenocytes (1 107 cells; 2 106 cells/mL) were cultured for several days without refreshing culture medium and harvested at various time points for flow cytometric analyses. The number and percentage of each cell population were calculated from at least triplicate assays for each immunization. Mean SEM is shown.
immunized splenocytes, the cells were stained using combinations of antibodies against CD45R (known as a B-cell lineage marker), CD38 (known as a surface marker of pre-GC B cells and downregulated in the GC) (22) and CD138 (known as a surface marker of plasmablasts) (23), and subjected to flow cytometric analysis. Both the splenocytes phenotypically equivalent to GC B cells (defined as GC-like B cells; CD45RþCD38low; 2.5 105 cells, 2.21% of living immunized splenocytes, Fig. 3C and F) and plasmablasts (defined as PB-like B cells; CD45Rlow CD138þ; 1.1 105 cells, 0.94%, data not shown) were detected after 5 days in culture. Pre-PB-like B cells (CD45RþCD138þ) were also detected (1.1 106 cells, 9.93%).
Also, pre-GC-like B cells (CD45RþCD38þ) gradually increased and reached 6.7 106 cells (58.0%) after 5 days in culture. When splenocytes were stimulated by antigen only, pre-GClike B cells and pre-PB-like B cells were observed (Fig. 3A and D), but the population of these cells reached only 13.4% (1.5 105 cells) and 8.0% (8.9 104 cells), respectively. On the other hand, when splenocytes were stimulated by LPS þ TDS, the population and number (Fig. 3B and E) of B-cell subsets were dramatically increased irrespective of antigen addition. These results suggested that LPS þ TDS stimulation could efficiently induce GC-like B cells in vitro.
TABLE 1. Mutation frequency of obtained anti-HEL scFv clones. Culture period
Day Day Day Day a
4 5 6 7
VH
VL
scFv
Mutation (bp)
Totala (bp)
Mutation frequency (%)
Mutation (bp)
Totala (bp)
Mutation frequency (%)
Mutation (bp)
Totala (bp)
Mutation frequency (%)
26 78 74 56
2355 2627 2611 2336
1.1% 3.0% 2.8% 2.4%
55 49 51 61
2433 2105 2535 2118
2.3% 2.3% 2.0% 2.9%
81 127 125 117
4788 4732 5146 4454
1.69% 2.68% 2.43% 2.63%
Total number of analyzed nucleotides derived from 6 to 8 selected clones.
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Sequence analysis of VH and VL from immunized splenocytes We selected eight clones from splenocytes at each culture period with different affinities and analyzed the number of point mutations (Table 1). The number of mutations ranged from 0 to 37 in the VL sequence, and 0e30 in the VH sequence. Although mutations were observed not only in complementarity determining region (CDR) but also in framework region (FR), the mutations that caused replacement of amino acids were highly observed in CDR (at least 3-fold higher than those observed in FR). Overall, 62.5% of the selected clones (20 clones) had more than 2% difference from the most similar germ line gene, and 8 clones out of 20 clones had more than 5% difference. The average rate of point mutations in the VL and VH sequences was 1.1% and 2.3% from a 4-day culture, 3.0% and 2.3% from a 5-day culture, 2.8% and 2.0% from a 6-day culture, and 2.4% and 2.9% from a 7day culture, respectively. Because a commonly used threshold for considering a case “mutated” was proposed as 2% difference (24), these results suggest that we could successfully induce the accumulation of point mutations after 5-day culture of splenocytes. CDR amino acid sequences of VH were analyzed (Table 2). As a result, we obtained very similar clones which are thought to be derived from the same germ line gene such as day 4e4, 4e7, 5e2 and 5e3. However, they had different CDR3 amino acid sequences and point mutations in CDR1 or CDR2. These results suggest that specific binders for HEL existed in naïve splenocytes and these clones accumulated point mutations. According to these results, we use 5-day culture for further hybridoma formation.
TABLE 3. Kinetic analysis of the obtained anti-HEL immunoglobulin.
Selection of anti-HEL immunoglobulin We fused immunized splenocytes with myeloma cells to generate hybridomas. Hybridomas were separated by ClonaCell HY kit and 196 clones were screened by antigen ELISA. Four clones which exhibited higher absorbance were subjected to SPR analysis to determine their affinity (Table 3). The analysis revealed that the immunoglobulin clone no. 189 demonstrated a Kd of 0.5 nM, a kon
Clone no. 189 135 110 114
kon (M1 s1)
Kd (nM) 0.5 62 72 75
9.9 1.1 3.7 7.1
5
10 104 104 103
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koff (s1) 5.5 6.7 2.7 5.3
104 104 103 104
of 9.9 105 M1 s1 and a koff of 5.5 104 s1 (Fig. 4A) and no. 135 demonstrated a Kd of 62 nM, a kon of 1.1 104 M1 s1 and a koff of 6.7 104 s1 (Fig. 4B). These results indicated that we could immunize splenocytes by our in vitro immunization method. DISCUSSION In this paper, we were able to induce AID mRNA expression and GC-like B cells using our in vitro immunization protocol. We also obtained anti-HEL antibody and detected the accumulation of mutation in their variable regions. Previous reports have shown that the chicken DT40 B-cell line (25,26), Ramos cells (27) and mouse IgGþ/AIDþ B-cell lines derived from GC B cells (28) can induce SHM in immunoglobulin genes through the induction of AID in vitro. These methods attained easy and large-scale variation of immunoglobulin genes; however, they did not immunize antigen to produce antigen-specific antibody. Therefore, one has to deal with a large library of cells (about 108 cells) and utilize magnetic sorting or flow cytometry to sort and separate the positive clones. Even if such separation methods were utilized, the resulting positive cell rate was still low (10.3% after screening once) (26). Therefore, it is important to develop a new in vitro immunization protocol that can efficiently induce antigen-specific immunoglobulins. Until now, many in vitro immunization protocols using peripheral blood mononuclear cells (29,30), murine splenocytes (31) and murine popliteal lymph nodes (32) have been developed to successfully generate antigen-specific antibodies. Thymus-conditioned medium (TCM), mixed lymphokines, such as IL-4, IL-2 and
TABLE 2. VH CDR amino acid sequence of obtained anti-HEL scFv clones. Culture period Day 4
Day 5
Day 6
Day 7
Clone no. 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Closest germ line e VH36-60.a2.90 VOx1 VHQ52.a3.8 VH7183.a7.10 VHQ52.a9.26 VHQ52.a3.8 J558.45 VOx1 VHQ52.a3.8 VHQ52.a3.8 VH7183.a30.50 J558.45 J558.44 e e VHSM7.a3.93 VH81X VH7183.a47.76 VHQ52.a9.26 VHQ52.a27.79 H13-3 J558.23 J558.12.102 e VHA1 J558.45 VH7183.a15.24 e VHQ52.a7.18 J558.47 J558.17
CDR1 sequence
CDR2 sequence
CDR3 sequence
e SDYAWN SYGVH SYGVS SYGMS SYGVH SYGVS SYWMH SYGVH SYGVS SYGVS SYGMS SYWMH SYTMH e e DTYMH e SFGMH SYGVH SYGVH SYTMH DYYIN DYEMH e SYDIN SYWMH SYAMS e SYGVH SYVMH SYWIE
e YISYSGSTSYNPSLKS VIWAGGSTNYNSALMS VIWGDGSTNYHSALIS TISSGGSYTYYPDSVKG VIWSDGSTTYNSALKS VIWGDGSTNYHSALIS YINPSTGYTEYNQKFKD VIWAGGSTNYNSALMS VIWGDGSTNYHSALIS VIWGDGSTNYHSALIS TIMSNGGSTYYPDSVKG YINPSTGYTEYNQKFKD YINPSSGYTNYNQKFKD e e RIDPANGNTKYDPKFQG e YISSGSSTIYYADTVKG VIWSDGSTTYNSALKS VIWAGGSTNYNSALMS YINPSSGYTEYNQKFKD EIYPGSGNTYYNEKFKG AIDPETGGTAYNQKFKG e WIYPGDGSTKYNEKFKG YINPSTGYTEYNQKFKD TISSGGSYTYYPDSVKG e VIWRGGSTDYNAAFMS YINPYNDGTKYNEKFKG EILPGSGSTNYNEKFKG
e CARYDWYFDVW CASIGTTSYW CAKLGHYYAMDYW CARYGTDEDW CAREYDYAWFAYW CAREVRQAWFAYW CARGGYAMDYW CASYGYDWAWFAYW CARDWMDGLDYW CAKNERYDAMDYW CARGTARAGAMDYW CARSGRYDAMDYW CARQ e e CARRQLGLPFAYW e CARWGRYDVEGFAYW CARGPMDYW CARGYGDGYW CARGLTTALYYYAMDYW CARGPDYDGYW CARGGTGTRVFAYW e CARDYGRSMDYW CARSEYGSSYGYFDVW CARGNEDW e CAKIVGGDAMDYW CARDYRYSWFAYW CARLEYYYGSSYGYW
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FIG. 4. SPR analyses of anti-HEL IgG. (A) The SPR sensorgram of anti-HEL IgG (clone no. 189). The overlay plot shows the binding of HEL at different concentrations (8.6, 17.1, 34.2, 68.5, 137 nM) to immobilized anti-HEL IgG. (B) The SPR sensorgram of anti-HEL IgG (clone no. 135). The overlay plot shows the binding of HEL at different concentrations (16.7, 33.3, 66.7, 133, 267 nM) to immobilized anti-HEL IgG.
IL-10, and mitogens such as LPS were used for activation of B cells in these methods. Although TCM included many factors that could stimulate antigen-activated B lymphocytes such as B-cell differentiation factor and B-cell growth factor (33,34), TCM could not induce CSR (28). On the other hand, IL-4 with LPS was able to induce CSR (IgM, IgG1 or IgE) (13,14), exhibiting a 5e10% increase in the number of IgG1 cells (14). Because CSR needs both AID expression and induction of GC-like B cells (16), these previous reports are consistent with our data that IL-4 with LPS could not induce Bcl-6 mRNA, a hallmark of GC-like B cells. Therefore, we considered that they were unable to induce GC formation because they did not include the molecules that stimulate the CD38 signaling pathway. Taking our findings in conjunction with previous reports together, IL-5 and anti-CD38 would appear to be important in the induction of GC formation. In this report, we demonstrated that we could immunize murine splenocytes in vitro and obtain an antigen-specific highaffinity immunoglobulin. If we can differentiate induced pluripotent stem (iPS) cells into splenic B cells, we could construct a complete in vitro antibody generation system. Moreover, we could immunize human B cells by means of modifying stimulation conditions. Although our method could not achieve efficient selection of high-affinity antibody, our method can generate immunized splenocytes rapidly. Therefore, our method has an advantage to adapt various selection methods for high-affinity antibody generation using immunized cells.
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