CD40L EXPRESSING MAMMALIAN CELLS AND THEIR USE
20230399396 · 2023-12-14
Assignee
Inventors
- Josef Endl (Weilheim, DE)
- Jens FISCHER (Weilheim, DE)
- Peter Kern (Penzberg, DE)
- Sonja Offner (Penzberg, DE)
- Josef Platzer (Geretsried, DE)
- Stefan Seeber (Sindelsdorf, DE)
Cpc classification
C07K16/28
CHEMISTRY; METALLURGY
C07K2317/14
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
International classification
C07K16/28
CHEMISTRY; METALLURGY
C07K16/00
CHEMISTRY; METALLURGY
Abstract
Herein is reported a co-cultivation system for co-cultivating a pool of rabbit B-cells or single deposited rabbit B-cells wherein cells CD40L expressing CHO cells are used as feeder in the presence of TL-2 and TL-21.
Claims
1. A method for producing an antibody comprising the step of co-cultivating a rabbit B-cell with a rabbit CD40L expressing BHK or CHO cell in the presence of IL-2 and IL-21.
2. The method according to claim 1, characterized in that the B-cell is a non-mature B-cell.
3. The method according to claim 1, characterized in that the IL-2 is human IL-2 and the IL-21 is murine IL-21.
4. The method according to claim 1, characterized in that the B-cell is a single deposited B-cell.
5. The method according to claim 1, characterized in that the B-cells are IgG positive B-cells (IgG.sup.+ B-cells).
6-7. (canceled)
8. A method for cultivating a rabbit B-cell secreting an antibody that specifically binds to a T-cell surface antigen and that mediates a negative stimulus to T-cells comprising the co-cultivation of the B-cell and a rabbit CD40L expressing mammalian cell in the presence of IL-2 and IL-21.
9. A method for the cultivation of an antibody secreting rabbit B-cell comprising in the following order i) a first co-cultivation of the rabbit B-cell and a rabbit CD40L expressing mammalian cell in the presence of a mitogenic stimulant, and ii) a subsequent second co-cultivation of the rabbit B-cell and the rabbit CD40L expressing mammalian cell in the presence of an antibody production stimulant.
10. The method according to claim 9, characterized in that the mitogenic stimulant is selected from the group comprising CD40- and CD40L-interacting compounds, ICOS- and ICOS-L-interacting compounds, APRIL, BAFF, CR2, CXCL9, CXCL12 (SDF-1), CXCL13, CXCL16, Flt-3L, Interleukin-1 (α/β), Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-7, Interleukin-10, Interleukin-14, Interleukin-21, SAP (signaling lymphocyte activation molecule [SLAM] associated protein), Staphylococcus A strain Cowan 1 particles (SAC; heat-killed, formalin-fixed), TLR Ligands such as LPS, different CpG ODNs or Resiquimod (R-848), TSLP, Tumor necrosis factor (TNF) alpha, type I Interferons (e.g. IFN W/p), and type II interferon (e.g. IFNγ).
11. The method according to any one of claims 9 or 10, characterized in that the antibody production stimulant is selected from the group comprising CD40- and CD40L-interacting compounds, ICOS- and:ICOS-L-interacting compounds, APRIL, BAFF, CR2, CXCL9, CXCL12 (SDF-1), CXCL13, CXCL16, Flt-3L, Interleukin-1 ((α/β), Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-9, Interleukin-10, Interleukin-13, Interleukin-21, Interleukin-33, SAP (signaling lymphocyte activation molecule [SLAM] associated protein), Staphylococcus A strain Cowan 1 particles (SAC; heat-killed, formalin-fixed), TLR Ligands such as LPS, different CpG ODNs or Resiquimod (R-848), TSLP, Tumor necrosis factor (TNF) alpha, type I Interferons (e.g. IFN α/β), and type II interferon (e.g. IFNγ).
12. A method for producing an antibody, which specifically binds to an antigen, comprising the following steps: a) co-cultivating a pool of antibody secreting rabbit B-cells or a single antibody secreting rabbit B-cell with rabbit CD40L expressing mammalian cells in the presence of IL-2 and IL-21, b) cultivating a cell comprising a nucleic acid encoding the variable regions or a humanized variant thereof of the antibody secreted by the rabbit B-cell co-cultivated in step a) within one or more expression cassettes, c) recovering the antibody from the cell or the cultivation medium and thereby producing an antibody, which specifically binds to an antigen.
13. The method according to claim 12, characterized in that the method comprises the following steps: a) providing a population of antibody secreting (mature) rabbit B-cells (obtained from the blood of an experimental animal or a human), b) staining the cells of the population of rabbit B-cells with at least one fluorescence dye (in one embodiment with one to three, or two to three fluorescence dyes), c) depositing single cells of the stained population of B-cells in individual containers (in one embodiment is the container a well of a multi well plate), d) cultivating the deposited individual rabbit B-cells in the presence of rabbit CD40L expressing mammalian cells and IL-2 and IL21, e) determining the binding specificity of the antibodies secreted in the cultivation of the individual rabbit B-cells, f) determining the amino acid sequence of the variable light and heavy chain domain of specifically binding antibodies by a reverse transcriptase PCR and nucleotide sequencing, and thereby obtaining a monoclonal antibody variable light and heavy chain domain encoding nucleic acid, g) introducing the monoclonal antibody light and heavy chain variable domain encoding nucleic acid or a variant thereof encoding a humanized version of the light and/or heavy chain variable domain in an expression cassette for the expression of an antibody, h) introducing the nucleic acid in a cell, i) cultivating the cell and recovering the antibody from the cell or the cell culture supernatant and thereby producing an antibody, which specifically binds to an antigen.
14-15. (canceled)
16. A method for producing an antibody comprising the step of co-cultivating a human B-cell with a human CD40L expressing mammalian cell in the presence of IL-2 or IL-21 or IL-6 or a combination thereof.
17. The method according to claim 16, characterized in that the B-cell is a non-mature B-cell.
18. The method according to claim 16, characterized in that the co-cultivation is in the presence of IL-2, or IL-2 and IL-21, or IL-2 and IL-21 and IL-6.
19. The method according to claim 16, characterized in that the B-cell is a single deposited B-cell.
20. The method according to claim 16, characterized in that the B-cells are IgG positive B-cells (IgG.sup.+ B-cells).
21-23. (canceled)
24. A method for cultivating a human B-cell secreting an antibody that specifically binds to a T-cell surface antigen and that mediates a negative stimulus to T-cells comprising the co-cultivation of the human B-cell and a human CD40L expressing mammalian cell in the presence of IL-2 and/or IL-21 and/or IL-6.
25. A method for producing an antibody, which specifically binds to an antigen, comprising the following steps: a) co-cultivating a pool of antibody secreting human B-cells or a single antibody secreting human B-cell with human CD40L expressing mammalian cells in the presence of IL-2 and/or IL-21 and/or IL-6, b) cultivating a cell comprising a nucleic acid encoding the variable regions or a variant thereof of the antibody secreted by the human B-cell co-cultivated in step a) within one or more expression cassettes, c) recovering the antibody from the cell or the cultivation medium and thereby producing an antibody, which specifically binds to an antigen.
26. The method according to claim 25, characterized in that the method comprises the following steps: a) providing a population of antibody secreting (mature) human B-cells, b) staining the cells of the population of human B-cells with at least one fluorescence dye (in one embodiment with one to three, or two to three fluorescence dyes), c) depositing single cells of the stained population of B-cells in individual containers (in one embodiment is the container a well of a multi well plate), d) cultivating the deposited individual human B-cells in the presence of human CD40L expressing mammalian cells and IL-2 and/or IL21 and/or IL-6, e) determining the binding specificity of the antibodies secreted in the cultivation of the individual human B-cells, f) determining the amino acid sequence of the variable light and heavy chain domain of specifically binding antibodies by a reverse transcriptase PCR and nucleotide sequencing, and thereby obtaining a monoclonal antibody variable light and heavy chain domain encoding nucleic acid, g) introducing the monoclonal antibody light and heavy chain variable domain encoding nucleic acid in an expression cassette for the expression of an antibody, h) introducing the nucleic acid in a cell, i) cultivating the cell and recovering the antibody from the cell or the cell culture supernatant and thereby producing an antibody, which specifically binds to an antigen.
27. The method according to any one of claims 25 to 26, characterized in that the co-cultivation is in the presence of IL-2, or IL-2 and IL-21, or IL-2 and IL-21 and IL-6.
28-29. (canceled)
Description
DESCRIPTION OF THE FIGURES
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DESCRIPTION OF THE SEQUENCES
[0398] SEQ ID NO: 01 amino acid sequence of rabbit CD40L [0399] SEQ ID NO: 02 amino acid sequence of human CD40L [0400] SEQ ID NO: 03 amino acid sequence of mouse CD40L
EXAMPLES
Material & Methods
Recombinant DNA Techniques
[0401] Standard methods were used to manipulate DNA as described in Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). The molecular biological reagents were used according to the manufacturer's instructions.
DNA Sequence Determination
[0402] DNA sequences were determined by double strand sequencing performed at SequiServe GmbH (Vaterstetten, Germany).
DNA and Protein Sequence Analysis and Sequence Data Management
[0403] The GCG's (Genetics Computer Group, Madison, Wisconsin) software package variant 10.2 and Infomax's VECTOR NTI ADVANCE@ suite variant 8.0 was used for sequence creation, mapping, analysis, annotation and illustration.
Gene Synthesis
[0404] Desired gene segments encoding cDNA of rabbit CD40L were prepared by Geneart GmbH (Regensburg, Germany). The gene segments are flanked by singular restriction endonuclease cleavage sites to facilitate expression construct cloning as described below. The DNA sequence of the subcloned gene fragments were confirmed by DNA sequencing.
Cytokines
[0405] Zubler Mix: 2 ng/ml mouse IL-1β, 50 ng/ml mouse IL-2, 10 ng/ml mouse IL-10, and 2 ng/ml mouse TNFα(final concentration) Cytokines tested for establishment of a defined cytokine cocktail (given as final concentration in case not stated otherwise):
TABLE-US-00001 cytokine final concentration supplier Catnr. huIL-2 50 U/ml Roche Dia. GmbH 11147528001 huIL-21 25 ng/ml eBioscience ® 14-8219 muIL-21 100 ng/ml R&Dsystems ® 594-ML huIL-6 300 U/ml Roche Dia. GmbH 11138600001 huIL-10 25 ng/ml BD 554611 huIL-1β 12.5 ng/ml R&Dsystems ® 201-LB huIL-33 100 ng/ml Peprotech ® 200-33 TNFα 25 ng/ml R&Dsystems ® 210-TA
CHO rbCD40L Medium
[0406]
TABLE-US-00002 500 ml HyQSFM4CHO ™ # SH30549 Perbio ® 50 ml FCS # A15-512 PAA 1 ml Pen/Strep # 11074440001 Roche Dia. GmbH 5 ml Hygromycin # 10843555-001 Roche Dia. GmbH
Rabbit B-Cell Medium
[0407]
TABLE-US-00003 500 ml RPMI 1640 #P04-17500 PAN Biotech ® 50 ml FCS #A15-512 PAA 5 ml L-Gln #25030-024 Invitrogen ® 5 ml potassium Pyruvate #P04-43100 PAN Biotech ® 5 ml HEPES #15630-056 Invitrogen ® 500 μl β-Mercaptoethanol # 31350010 Invitrogen ® 1 ml Pen/Strep #11074440001 Roche Dia. GmbH
Additives to Rabbit B-Cell Medium
[0408]
TABLE-US-00004 SAC #507858 Calbiochem ® IL-21 #14-8219 eBioscience ® IL-2 #1114752800 Roche ® 96er U-plate #3799 Corning ®
Phenotyping/Sorting of Antibodies
[0409]
TABLE-US-00005 goat anti-rabbit IgG Fc-antibody AbDSerotec ® STAR121F rat anti-rabbit CD138-antibody Roche GlycArt AG anti-human CD40 mAb (clone Beckman Coulter ® IM1374 Mab89) anti human/murine (rabbit cross- reactive) anti CD40L antibody: anti-muCD40L antibody R&D systems ® AF1163 anti-huCD40L antibody &D systems ® AF617 R donkey anti-goat IgG antibody Molecular Probes ® A11055 Alexa 488
Miscellaneous
[0410]
TABLE-US-00006 anti-FITC antibody-coupled Miltenyi Biotec ® #130-048-701 microbeads human B-cell negative isolation kit Invitrogen ® #113.13D Nucleofector Kit T Lonza ® VCA-1002 CBA for total IgG BD Biosciences ® #558679
Animals
[0411] Wild-type New Zealand White Rabbits were used as a source of blood. They were housed and maintained according to the Institutional Animal Care and Use committee guidelines and Association for Assessment and Accreditation of Laboratory Animal Care (Germany, Europe).
Coating of Plates
[0412] Sterile streptavidin-coated 6-well plates (cell culture grade) were incubated with biotinylated antigens in a concentration at 0.5-1 μg/ml in PBS at room temperature for one hour. Sterile cell culture 6-well plates were coated with 2 μg/ml non-biotinylated protein antigen in carbonate buffer (0.1 M sodium bicarbonate, 34 mM Disodiumhydrogencarbonate, pH 9.55) over night at 4° C.
[0413] Plates were washed in sterile PBS three times before use.
Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMC)
[0414] EDTA containing whole blood was diluted twofold with 1xPBS before density centrifugation on lympholyte mammal (Cedarlane Laboratories) or FICOLL® Paque Plus (GE Healthcare, cat. #17-1440-03), which was performed to isolate rabbit PBMCs. PBMCs were washed twice before staining with antibodies.
EL4-B5 Medium
[0415] RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2 mM Glutamine, 1% penicillin/streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05 mM β-mercaptoethanol (Gibco, Paisley, Scotland)
Depletion of Macrophages/Monocytes
[0416] Sterile 6-well plates (cell culture grade) were used to deplete macrophages and monocytes through unspecific adhesion. Each well was filled at maximum with 4 ml media and up to 6×10.sup.6 peripheral blood mononuclear cells from the immunized rabbit and allowed to bind for one hour at 37° C. in the incubator. 50% of the cells in the supernatant were used for the panning step; the remaining 50% of cells were kept on ice until the immune fluorescence staining.
Enrichment of B-Cells on the Protein Antigen
[0417] Six-well tissue culture plates coated with the protein antigen were seeded with up to 6×10.sup.6 cells per 4 ml medium and allowed to bind for one hour at 37° C. in the incubator. After the enrichment step on the protein antigen non-adherent cells were removed by carefully washing the wells 1-2 times with 1xPBS. The remaining sticky cells were detached by trypsin for 10 min at 37° C. in the incubator and then washed twice in media. The cells were kept on ice until the immune fluorescence staining.
Immune Fluorescent Staining and Flow Cytometry (Sorting and Analysis)
[0418] Anti-rabbit IgG FITC used for single cell sorting was from AbD Serotec (STAR121F, DUsseldorf, Germany).
[0419] For surface staining, cells were incubated with the optimally diluted anti-rabbit IgG FITC antibody in PBS for 30 min. with rolling at 4° C. in the dark. Following centrifugation, the supernatants were removed by aspiration. The PBMCs were subjected to two cycles of centrifugation and washing with ice cold PBS. Finally the PBMCs were resuspended in ice cold PBS and immediately subjected to the FACS analyses. Propidium iodide in a concentration of 5 μg/ml (BD Pharmingen, San Diego, CA, USA) was added prior to the FACS analyses to discriminate between dead and live cells. In other experiments the stained cells were single deposited by FACS.
[0420] A Becton Dickinson FACSARIA™ equipped with a computer and the FACSDIVA™ software (BD Biosciences, USA) were used to collect and analyze the data.
Proliferation Assays
[0421] a) Cell Titer Glo (CTG) viability assay
[0422] The CTG viability assay (Promega; #G7571) was used according to the instructions of the manufacturer. [0423] b).sup.3H Thymidine Assay
[0424] After 6 days of incubation .sup.3H-Thymidin was added (0.5 μCi/well) and incubated for further 16 hours. The incorporation of .sup.3H-Thymidine during cell proliferation was determined with a microplate scintillation counter (Wallac). [0425] c) Microscopic analysis
[0426] For the acquisition of microscopic images, a phase contrast microscope from Leica (Leica DM IL) combined with a high resolution camera (Leica DFC290 HD) was used. [0427] d) Analysis of B-cell activation via CFSE-labeling.
[0428] Isolated B-cells were washed with sterile phosphate buffer saline solution (PBS). Up to 1×10.sup.7 cells were resuspended in 1 ml protein-free PBS and incubated with CFSE (#C34554, Invitrogen/Molecular Probes) for 3 to 10 minutes at a final concentration of 2.5 μM at 37° C. CFSE loading was stopped by addition of an excess of FCS-supplemented medium. After extensive washing with FCS-containing medium, B-cells were used in co-culture experiments. Proliferation of CD19.sup.+ gated (B-)cells as a consequence of CFSE dilution was confirmed by flow cytometric analysis (FL-1 channel) after indicated time points.
B-Cell Culture
[0429] B-cell cultures were prepared by a method similar to that described by Zubler, et al. (see e.g. Eur. J. Immunol. 14 (1984) 357-363; J. Exp. Med. 160 (1984) 1170-1183). Briefly, single sorted B-cells were cultured in 96-well plates with 210 l/well EL4 B5 medium with PANSORBIN® Cells (1:20000) (Calbiochem (Merck), Darmstadt, Deutschland), 5% rabbit thymocyte supernatant and gamma-irradiated EL4-B5 murine thymoma cells (2×10.sup.4/well) for 7 days at 37° C. in an atmosphere of 5% CO.sub.2 in the incubator. B-cell culture supernatants were removed for screening and the cells harvested immediately for variable region gene recovery or frozen at −80° C. in 100 μl RLT buffer (Qiagen, Hilden, Germany).
[0430] Human B-cells were cultured accordingly. Human CD40L expressing feeder cells (BHK cells) were used for co-cultures and the indicated feeder mixes.
Example 1
[0431] Generation of the expression plasmids for full-length rabbit CD40L The amino acid sequence for the rabbit CD40L gene was taken from GenBank, Accession number XP_002720374 (SEQ ID NO: 01).
[0432] Rabbit CD40L CDNA sequence XP_002720374 —SEQ ID NO: 01
TABLE-US-00007 1 MIETYSQPTP RSVATGPSVS MKIFMYLLTV FLITQMIGSA LFAVYLHRRL 51 DKIEDERNLH EDFVEMKTIQ RCNKGEGSLS LLNCKEIRSQ FEGFVKDIML 101 NKEEPKKEIN FEMQKGDQDP QIAAHLISEA SSKSSSVLQW AKKGYYTMSN 151 TLVTLENGKQ LKVKRQGFYY IYAQVTFCSN QEPSSQAPFI ASLCLKSSGG 201 SERILLRAAN ARSSSKTCEQ QSIHLGGVFE LQADASVFVN VIDASQVNHG 251 TGFTSFGLLK L
[0433] The amino-acid sequence was back-translated into an encoding nucleic acid sequence (DNA).
Plasmid Number 7111
[0434] The gene segment encoding the synthesized rabbit CD40L cDNA was cloned into a cDNA-expression vector. In this expression vector its expression is controlled by a shortened intron A-deleted immediate early enhancer and promoter from the human cytomegalovirus (HCMV) including a human heavy chain immunoglobulin 5′-untranslated region (UTR), where the intron A with splice donor and acceptor sites are included, the full-length rabbit CD40L gene with its signal anchor sequence, and the strong polyadenylation signal from bovine growth hormone. The expression plasmid contains also an origin of replication and a β-lactamase gene from the vector pUC18 for plasmid amplification in Escherichia coli.
Plasmid Number 7112
[0435] Plasmid number 7112 was constructed in the same way as plasmid number 7111 but contains an additional hygromycin resistance gene for the generation/selection of stably transfected mammalian cell lines.
Example 2
[0436] Generation of rabbit CD40L expressing CHO-K1 cells According to the manufacturer's instructions CHO-K1 (ATCC CCL-61) cells were transfected with a rabbit CD40L coding plasmid (#7112) (e.g. using lipofection method or the Nucleofector kit T (Lonza formerly Amaxa, VCA-1002) with the Nucleofector program U-17). After transfection cells were cultured in 6-well plates for 24 hours until 0.5 mg/ml Hygromycin was added to establish selection pressure. Two weeks after transfection medium was supplemented with 10% FCS (v/v). After an expansion and selection phase of five weeks the transfected cells were analyzed on a FACS Aria using a cross-reactive polyclonal goat anti-murine CD40L primary antibody (R&D Systems; AF1163) and an anti-goat IgG secondary antibody (Alexa488-labeled; Molecular Probes; #A11055). Cells with highest mean fluorescence intensity (top 5%) were used for a single cell sort in U-bottom 96-well plates. After additional two weeks of expansion, clones were analyzed by FACS for rabbit CD40L expression.
[0437] Likewise human CD40L expressing BHK cells were obtained.
Example 3
Irradiation of Feeder Cells for Co-Culture
[0438] CHO clones stably expressing rabbit CD40L were grown under selection pressure (0.5 mg/ml Hygromycin) in CHO rbCD40L medium and stored in liquid nitrogen until further usage.
[0439] Two passages before using the cells as stimulators for sorted rabbit B-cells Hygromycin was removed from the medium and one day before co-culture, medium was switched to rabbit B-cell medium. Cells were harvested and adjusted to a cell concentration of 1×10.sup.6/ml before 7-irradiation at 50 Gy. After this, 1×10.sup.4 cells were plated in 100 μl per well in a U-bottom 96 well plate, centrifuged for 1 min. at 500 xg and incubated overnight at 37° C.
Example 4
[0440] Isolation procedure of primary antibody-secreting cells (ASCs) derived from rabbit peripheral blood
[0441] For the isolation of rabbit B-cells whole blood of non-immunized New Zealand White rabbits was used.
[0442] In some approaches, in a first step the lymphocyte population was isolated by lympholyte density centrifugation (Biozol; #CL5120) or Ficoll Paque Plus (GE Healthcare, cat. #17-1440-03). In a second step rabbit lymphocytes were incubated with a FITC-labeled anti-rabbit IgG specific antibody (final concentration=10 μg/ml; STAR121F; Serotec). FITC.sup.+ cells (rbIgG.sup.+ cells) were then purified via anti FITC Micro beads (#130-048-701; Miltenyi Biotec) (pool deposition).
[0443] In other approaches (“single cell deposition”), FITC.sup.+ cells were selected by FACS technology (see Material & Methods).
Example 5
Establishment of a Co-Culture System
[0444] In preliminary experiments stimulator cells and rabbit B-cells were mainly co-cultured at a ratio of 2:1.
[0445] Single rabbit B-cells were sorted on the plates prepared with the stimulator cells by using a FACS Aria as described in Example 4. For single B-cell co-culture, one rabbit B-cell was deposited on a monolayer of 10,000 irradiated feeder cells. If indicated a feeder-mix was added (e.g. standard supplements were 25 ng/ml IL-21, 50 U/ml IL-2, and 1:10,000 SAC) in 100 μl B-cell medium—and co-cultures were grown for 6 days at 37° C. and 5% CO.sub.2.
Example 6
Antibody Production Visualized by IgG-Specific ELISA
[0446] After co-culture 150 μl cultivation supernatant was transferred for subsequent rabbit IgG determination. Expanded rabbit B-cells were stored in liquid nitrogen in 96-well format by adding FCS and DMSO.
[0447] Optionally, incubation length was prolonged up to 11-14 days. Therefore 150 μl freshly prepared rabbit B-cell medium with feeder mix and SAC was added and supernatant was again assessed for rabbit IgG concentration.
Example 7
Cultivation of IgG.SUP.+ Rabbit B-Cells
[0448] Single-sorted IgG.sup.+ rabbit B-cells were cultured either in combination with the murine thymoma cell line EL4-B5 and rabbit thymocyte supernatant (TSN) and SAC or in the presence of a rabbit CD40L expressing CHO cell line (10,000 cells/well; +/− additional SAC) for one week. TSN was replaced by recombinant cytokines IL-2 and IL-21.
[0449] After 6 days, a rabbit IgG-specific ELISA was performed and the percentage of rabbit IgG.sup.+ wells (of total wells) was determined (determination of rbIgG productivity).
Results
[0450] The percentage of rabbit IgG.sup.+ wells was higher when a co-culture system comprising rabbit CD40L expressing CHO cells and a mixture of IL-2 and IL-21 was used (7.1% vs. 6.7% (with EL4-B5 cells)). The difference was increased if SAC was also added to the rabbit CD40L expressing CHO cells (17.9% vs. 6.7%). Similar results were obtained by using rabbit B-cells from different immunization campaigns against different antigens as shown in the following Table.
TABLE-US-00008 TABLES B-cell co-cultures comprising different feeder mixes. Shown is the percentage of IgG.sup.+ wells. feeder EL4-B5 rbCD40L IgG.sup.+ cells cells cells rbCD40L wells feeder TSN IL-2/21 cells [%] mix SAC SAC IL-2/21 campaign 1 36.1 16.7 6.0 2 6.7 17.9 7.1 feeder EL4-B5 rbCD40L IgG.sup.+ cells cells cells wells feeder TSN IL-2/21 [%] mix SAC SAC campaign 3 13.1 16.2 4 5.9 53.8 5 0 6.8 6 5.6 5.8 mix of EL4- B5 cells and feeder EL4-B5 rbCD40L rbCD40L IgG.sup.+ cells cells cells cells wells feeder TSN IL-2/21 IL-2/21 [%] mix SAC SAC SAC campaign 7 5.9 10.7 8.9
[0451] In the co-cultivation of IgG.sup.+CD138+ double-positive sorted B-cells the rbCD40L system with added cytokines and SAC did not result in IgG-producing B-cell clones. The co-culture with the EL4-B5 cells (incl. TSN and SAC) resulted in B-cell clones and IgG production as shown in the following Table (22.2% vs. no significant proliferation observable vs. 6.0%).
TABLE-US-00009 TABLE Co-cultivation of IgG.sup.+CD138.sup.+ sorted B-cells. feeder IgG.sup.+ cells EL4-B5 cells rbCD40L cells wells feeder TSN IL-2/21 rbCD40L cells [%] mix SAC SAC IL-2/21 campaign 8 22.2 (not 6.0 detectable/BDL*) (*no significant proliferation observed)
Example 8
Synthetic Rabbit B-Cell Culture System
[0452] Two thousand five hundred rabbit IgG.sup.+ B-cells/well were co-cultivated with irradiated rabbit CD40L expressing CHO cells or EL4-B5 cells (5,000 each) without the addition of TSN. In addition to the co-cultivation without any supplements, the impact of the addition of recombinant human IL-2 vs. the Zubler-Mix was tested. After 7 days of co-cultivation the proliferation of the B-cells was determined using the CTG assay as described earlier. The co-cultivations comprising rbCD40L cells resulted in B-cell proliferation (see following Table and
TABLE-US-00010 TABLE Proliferation of rabbit B-cells in the presence of different feeder components (medium, SAC, γ-irradiated EL4-B5 or rbCD40L expressing cells) and additional cytokines. B-cells (2,500 per well) no cytokines Zubler Mix huIL-2 in the presence of [RLU] [RLU] [RLU] medium 807 1697 1225 SAC 954 1565 1805 EL4-B5 cells (5,000) 1432 3901 2098 EL4-B5 cells (5,000) 8318 8413 4848 and SAC rbCD40L cells (5,000) 278750 577021 909641 rbCD40L cells (5,000) 367092 792172 1083221 and SAC
[0453] Five hundred rabbit B-cells per well were cultured at a ratio of 1:10 with indicated feeder cells and stimuli (see Table below). After 7 days of cultivation co-cultures of rabbit B-cells with rbCD4L expressing CHO cells improved cellular growth was observed compared to cells without CD40L expression (determined via CTG assay).
TABLE-US-00011 TABLE B-cell proliferation [RLU/CTG assays]. 500 B-cells/well were co-cultured for 7 days with indicated cellular systems and additional recombinant cytokines/mixes. B-cells (500 per well) in muIL-21 huIL-21 the presence huIL-2 huIL-2 huIL-21 muIL-21 huIL-2 of [RLU] [RLU] [RLU] [RLU] [RLU] only B-cells 874 1170 651 795 552 SAC 2389 2962 1314 1642 1261 wt-CHO cells 41901 38774 39538 37841 34401 wt-CHO cells 41130 48654 34626 33234 35804 and SAC rbCD40L cells 961594 900960 245470 326428 87819 rbCD40L cells 777151 900366 105349 158961 112764 and SAC
[0454] A proliferative effect was detected in the presence of rbCD40L expressing feeder cells. In principle the addition of recombinant IL-2 and IL-21 increased the cellular growth. Under these conditions, addition of SAC showed no improvement. In contrast, in other conditions addition of SAC slightly improved the proliferation. In contrast, neither IL-2, IL-21 nor the combination thereof resulted in an improvement in the EL4-B5 comprising co-culture. Exemplary wells are shown in
TABLE-US-00012 TABLE IgG production by 500 B-cells/well after 7 days [ng/ml]: IL-2 and IL-21 B-cells (500 per well) Addition of recombinant . . . muIL-21 huIL-21 B-cells in the huIL-2 huIL-2 huIL-21 muIL-21 huIL-2 presence of [ng/ml] [ng/ml] [ng/ml] [ng/ml] [ng/ml] only B-cells 109 111 86 84 84 SAC 111 111 84 84 84 wt-CHO cells 115 113 85 87 85 wt-CHO cells 114 109 88 87 83 and SAC rbCD40L cells 4042 9000 2727 2941 89 rbCD40L cells 4244 2845 425 194 80 and SAC
[0455] In two separate experiments single sorted and pools of rabbit B-cells were co-cultured with EL4-B5 cells or rbCD40L expressing cells either in the presence of TSN and SAC with IL-2/IL-6/IL-21 in various combinations. In the following Table the results are shown (the number of wells with detectable rabbit IgG production was counted). The combination of rbCD40L expressing feeder cells and TSN and SAC did not result in an IgG production comparable to the EL4-B5 system. On the other hand, in the co-cultivation of rabbit B-cells with rbCD40L expressing cells and a combination of indicated cytokines such as (hu) IL-2 and IL-21 significantly more IgG was produced (shown by the percentage or number of IgG.sup.+ wells). No IgG.sup.+ wells were detected with addition of SAC alone and rbCD40L stimulator cells. SAC increased the IgG response of the IL-2/21 supplemented rbCD40L feeder system (see following Tables).
TABLE-US-00013 TABLE Rabbit IgG production of single sorted B-cells (percentage of rbIgG.sup.+ wells (left columns) or absolute numbers of IgG.sup.+ wells (right columns)). IgG.sup.+ wells EL4-B5 rbCD40L [number] EL4-B5 [%] cells cells IgG.sup.+ wells cells rbCD40L TSN and 54 0 TSN and 39 0 SAC SAC huIL-2 and 0 12 huIL-2 and 0 8 huIL-21 huIL-21 huIL-2 and 4 17 huIL-2 and 3 12 huIL-21 and huIL-21 SAC and SAC huIL-2 and 1 15 huIL-2 and 1 11 muIL-21 muIL-21 huIL-2 and 11 26 huIL-2 and 8 19 huIL-6 and huIL-6 and huIL-21 huIL-21 huIL-2 and 4 26 huIL-2 and 3 19 huIL-10 and huIL-10 and huIL-21 huIL-21
TABLE-US-00014 TABLE Rabbit IgG production of deposited B-cell pools (numbers or percentage of IgG.sup.+ wells). EL4-B5 cells rbCD40L cells [number] [number (%)] TSN and proliferation 0 (0%) SAC not detectable huIL-21 and proliferation 6 (8.3%) huIL-2 not detectable huIL-21 and proliferation 17 (23.6%) huIL-2 and not detectable SAC muIL-21 and proliferation 1 (1.4%) huIL-2 not detectable huIL-21 and proliferation 0 (0%) huIL-2 and not detectable huIL-6 huIL-21 and proliferation 0 (0%) huIL-2 and not detectable huIL-10
Example 9
System for the Generation of T Cell-Inhibition Antibodies
[0456] After immunization with a T-cell-specific antigen a pool of antibodies is generated, which not only bind to the given antigen but may also exhibit a potential agonistic effect. In case the obtained antibody negatively interferes with T-cell biology and also interferes with the feeder system in case the culture system is based on a T-cell (like the thymoma cell line EL4-B5).
[0457] Single sorted B-cells, e.g. rabbit or human, are co-cultured either with EL4-B5 feeder cells or with CD40L expressing cells (see e.g. Example 5). The single sorted B-cells are, thus, derived from animals immunized with a T-cell antigen.
[0458] Single sorted B-cells are co-cultured with the indicated feeder mix. After about 7 days of co-cultivation the number of B-cell clones is determined and the cultivation supernatant is assessed for IgG content (as described in Materials and Methods and Example 6).
[0459] The co-cultivation can be performed with (single-sorted or pooled) B-cells derived from a human or a wild-type or a transgenic animal after immunization with antigens e.g. known for T-cell suppressive properties.
Example 10
Generation of a Sequential B-Cell Culture System
[0460] Single sorted B-cells derived from an immunized mammal are co-cultured with irradiated CD40L expressing cells. In a first cultivation phase different mitogenic factors (e.g. cytokines) are added (see list below) and the co-cultivation is continued in the presence of the mitogenic factors for 1 to 14 days. After the first cultivation phase the mitogenic factor is removed from the cultivation medium (either by change of the cultivation medium for mitogenic factor free medium and/or washing of the cells or by dialysis or centrifugation/sedimentation) and the cultivation is continued in the presence of an antibody production stimulant (see list below). The IgG concentration in the cultivation supernatant is tested in the second cultivation phase after 3 or 5 or 7 or 21 days of co-cultivation.
[0461] The mitogenic stimulant can be selected from the list comprising CD40- and CD40L-interacting compounds, ICOS- and ICOS-L-interacting compounds, APRIL, BAFF, CR2, CXCL9, CXCL12 (SDF-1), CXCL13, CXCL16, Flt-3L, Interleukin-1 ((a/P), Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-7, Interleukin-10, Interleukin-14, Interleukin-21, SAP (signaling lymphocyte activation molecule [SLAM] associated protein), Staphylococcus A strain Cowan 1 particles (SAC; heat-killed, formalin-fixed), TLR Ligands such as LPS, different CpG ODNs or Resiquimod (R-848), TSLP, Tumor necrosis factor (TNF) alpha, type I Interferons (e.g. IFN W/p), type II interferon (e.g. IFNγ), cross-linking anti-IgG antibody, cross-linking anti-CD20 antibody, and cross-linking anti-CD27 antibody.
[0462] The antibody production stimulant can be selected from the list comprising CD40- and CD40L-interacting compounds, ICOS- and ICOS-L-interacting compounds, APRIL, BAFF, CR2, CXCL9, CXCL12 (SDF-1), CXCL13, CXCL16, Flt-3L, Interleukin-1 (α/β), Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-9, Interleukin-10, Interleukin-13, Interleukin-21, Interleukin-33, SAP (signaling lymphocyte activation molecule [SLAM] associated protein), Staphylococcus A strain Cowan 1 particles (SAC; heat-killed, formalin-fixed), TLR Ligands such as LPS, different CpG ODNs or Resiquimod (R-848), TSLP, Tumor necrosis factor (TNF) alpha, type I Interferons (e.g. IFN (a/P), type II interferon (e.g. IFNγ), cross-linking anti-IgG antibody, cross-linking anti-CD20 antibody, and cross-linking antiCD27 antibody.
Example 11
Co-Cultivation System for Generation of Human Antibodies
[0463] The effect of different cytokines in promoting human B-cell growth in the presence of human CD40L expressing BHK cells was tested as described in the examples above.
[0464] Ficoll-isolated human PBMC (2×10.sup.5) were seeded in a 96-well plate and cultured for 8 days in the presence of the indicated stimuli. Optionally, CD3/CD28 antibodies were added to induce a polyclonal T-cell stimulation. As can be seen from the following Table recombinant human IL-21 induced the highest human IgG production (independent from additional T-cell activation).
TABLE-US-00015 TABLE Human IgG production after 8 days. human PBMC w/o CD3/CD28 plus . . . [μg/ml] [μg/ml] Medium 0.22 0.09 LPS 0.00 0.00 TNFα 0.11 0.00 huIL-6 0.16 0.02 huIL-21 4.31 0.00
[0465] Isolated human peripheral IgG.sup.+ B-cells (750 per well and 2,500 per well, respectively) were co-cultured with γ-irradiated human CD40L-expressing BHK cells in the presence of different stimuli (see Table below and
TABLE-US-00016 TABLE Relative proliferation [%] of human peripheral B-cells after 8 days. human B-cells and human B-cells and huCD40L expressing 750 IgG.sup.+ huCD40L expressing BHK cells in the memory BHK cells in the 2,500 IgG.sup.+ presence of . . . B-cells/well presence of . . . B-cells/well Medium 100 Medium 100 CpG and IL-6 95 CpG and IL-6 113 IL-4 101 IL-4 115 IL-2 89 IL-2 101 TNFα and IL-6 71 TNFα and IL-6 101 IL-21 136 IL-21 145 IL-33 91 IL-33 105 IL-2 and IL-6 101 IL-2 and IL-6 104 IL-2 and IL-21 130 IL-2 and IL-21 150 IL-6 and IL-21 129 IL-6 and IL-21 127 IL-2 and IL-6 and IL-21 141 IL-2 and IL-6 and IL-21 152 SAC and IL-2 and IL-6 136 SAC and IL-2 and IL-6 131 SAC and IL-2 and IL-6 132 SAC and IL-2 and IL-6 101 and IL-21 and IL-21
[0466] It can be seen that isolated IgG.sup.+ memory B-cells (isolated with MACS kit #130-094-350) as well as IgG.sup.+ B-cells (directly isolated via anti-IgG-microbeads (MACS kit #130-047-501) responded similar to the different stimuli. Addition of IL-21 alone (or in combination) resulted in an improved proliferation response after 8 days of co-cultivation. Co-stimulation with IL-2, IL-6 or a combination thereof did not enhance the effect neither did the addition of SAC.
[0467] The supernatants were tested for IgG production. From the following Table it can be seen that the addition of IL-21 and its combinations resulted in an improved IgG production in a co-cultivation with huCD40L expressing BHK cells.
TABLE-US-00017 TABLES Human IgG production after 8 days. IgG.sup.+ memory B-cells (750/well) with human IgG after 8 days huCD40L expressing BHK cells and [ng/ml] TNFα and IL-6 100 IL-6 50 IL-21 3010 IL-2 and IL-21 530 IL-6 and IL-21 2480 IL-2 and IL-6 and IL-21 2390 SAC and IL-2 and IL-21 10 SAC and IL-2 and IL-6 and IL-21 110 total IgG.sup.+ B-cells (2,500/well) with human IgG after 8 days huCD40L expressing BHK cells and [ng/ml] TNFα and IL-6 0 IL-6 60 IL-21 3845 IL-2 and IL-21 750 IL-6 and IL-21 2885 IL-2 and IL-6 and IL-21 2955 SAC and IL-2 and IL-21 285 SAC and IL-2 and IL-6 and IL-21 135
[0468] The proliferation of untouched isolated human B-cells was also assessed. B-cells were separated from PBMC using the DYNAL® Kit 113.13D (Invitrogen), counted and 90,000 CFSE-labeled B-cells per well were co-cultured with γ-irradiated human CD40L expressing BHK cells (CFSE-negative). The results are shown in the following Table and
TABLE-US-00018 TABLE B-cell proliferation (absolute cell numbers counted by FACS analysis of CD19.sup.+ cells). B-cells and huCD40L expressing B-cell proliferation BHK cells and [cell number] medium 132 anti-huCD40 antibody 1995 LPS 240 CpG 263 TNFα 241 SAC 336 IL-1b 84 IL-2 244 IL-4 600 IL-6 256 IL-10 422 IL-21 1542 IL-1b and IL-6 117 IL-2 and IL-6 242 IL-2 and IL-6* 283 (IL-6 added after 48 hours) IL-2 and IL-21 3659 IL-2 and IL-6 and IL-21 4247 SAC and IL-1b and IL-6 281 SAC and IL-2 and IL-6 641 SAC and IL-2 and IL-6* 985 (IL-6 added after 48 hours) SAC and IL-2 and IL-21 3588 SAC and IL-2 and IL-6 and IL-21 3357
[0469] The absolute cell numbers are increased by the addition of IL-21 alone or IL-2/IL-21 or IL-21/IL-6/IL-21.
[0470] Exemplary dotplots show the CFSE-dilution as a degree of the proliferation analyzed by FACS (see
[0471] Untouched human naive B-cells, depleted from activated B-cells, T-cells, NK-cells, monocytes, macrophages, granulocytes, platelets, plasma cells and erythrocytes (Dynal kit, Invitrogen, #113.13D) were used in co-culture with γ-irradiated human CD40L expressing BHK cells and a variety of additional stimuli. The proliferation of 50,000 initially deposited B-cells/well is shown in the Table below.
TABLE-US-00019 TABLE Proliferation of naive B-cells (50,000/well) in co-culture with irradiated huCD40L BHK cells for 7 days (number of proliferated B-cells detected by FACS analysis). B-cells and huCD40L expressing B-cell proliferation BHK cells and [cell number] Medium 0 anti-huCD40 antibody 62 LPS 1324 CpG 1587 TNFα 960 SAC 1147 IL-1b 2103 IL-2 2361 IL-4 8145 IL-6 2619 IL-10 4422 IL-21 16275 IL-1b and IL-6 989 IL-2 and IL-6 1500 IL-2 and IL-6* 2265 (IL-6 added after 48 hours) IL-2 and IL-21 19210 IL-2 and IL-6 and IL-21 19017 SAC and IL-1b and IL-6 927 SAC and IL-2 and IL-6 2509 SAC and IL-2 and IL-6* 2732 (IL-6 added after 48 hours) SAC and IL-2 and IL-21 19057 SAC and IL-2 and IL-6 and IL-21 16607
[0472] The addition of IL-21 (either alone or in combination with IL-2 and/or IL-6) resulted in improved B-cell growth.
Example 12
B-Cell Culture System Using SAC
[0473] Isolated human B-cells (5×10.sup.4 cells/well isolated from naive human PBMC using the negative B-cell isolation kit (#113.13D, Invitrogen)) were cultured in a 96-well round bottom plate and incubated for 6 days in the presence of the indicated supplements before proliferation (CTG assay) or human IgG production (ELISA) was determined. The results are shown in the following Table.
TABLE-US-00020 TABLE total huIgG in human B-cells Proliferation supernatant and [RLU] [ng/ml] — 6453 0 IL-2 22217 3.9 SAC 60817 55.1 SAC and IL-6 69512 46.6 SAC and IL-2 and 200882 273.9 IL-6 SAC and IL-2 295067 546.5
[0474] It can be seen that SAC in combination with IL-2 and to a lesser degree the combination of IL-2 and IL-6 resulted in improved B-cell proliferation and IgG production. Exemplary B-cell clusters after 3 days of incubation are shown in
[0475] Using a pool of purified peripheral rabbit B-cells the addition of SAC resulted in increased proliferation of B-cells (see following Tables).
TABLE-US-00021 TABLE Proliferation of rabbit B-cells in the presence of different feeder components (medium, SAC, γ-irradiated EL4-B5 or rbCD40L expressing cells) and additional cytokines. B-cells (2,500 per well) no cytokines Zubler Mix huIL-2 in the presence of [RLU] [RLU] [RLU] medium 807 1697 1225 SAC 954 1565 1805 EL4-B5 cells (5,000) 1432 3901 2098 EL4-B5 cells (5,000) 8318 8413 4848 and SAC rbCD40L cells (5,000) 278750 577021 909641 rbCD40L cells (5,000) 367092 792172 1083221 and SAC
TABLE-US-00022 TABLE B-cell proliferation [RLU/CTG assays], 500 B-cells/well were co-cultured for 7 days with indicated cellular systems and additional recombinant cytokines/mixes. B-cells (500 per well) in muIL-21 huIL-21 the presence huIL-2 huIL-2 huIL-21 muIL-21 huIL-2 of [RLU] [RLU] [RLU] [RLU] [RLU] only B-cells 874 1170 651 795 552 SAC 2389 2962 1314 1642 1261 wt-CHO cells 41901 38774 39538 37841 34401 wt-CHO cells 41130 48654 34626 33234 35804 and SAC rbCD40L cells 961594 900960 245470 326428 87819 rbCD40L cells 777151 900366 105349 158961 112764 and SAC
[0476] The addition of SAC also resulted in an increase of the IgG production of cultivated B-cells (number of IgG positive wells and/or IgG secretion).
Example 13
Rabbit B-Cell Co-Cultivation
[0477] Five hundred rabbit IgG.sup.+ B-cells/well were co-cultivated with γ-irradiated rabbit CD40L expressing CHO cells or γ-irradiated CHO-K1 cells (10,000 each) without the addition of TSN. In addition to the co-cultivation without any supplements, the impact of the addition of recombinant human IL-2, recombinant murine IL-2, recombinant human IL-21, recombinant murine IL-21 and combinations thereof was tested. After 7 days of co-cultivation the antibody titer in the supernatant of the B-cells was determined using the ELISA as described earlier. The co-cultivations comprising rbCD40L cells in the presence of IL-2 and IL-21 resulted in B-cell proliferation and antibody secretion (see
Example 14
Rabbit B-Cell Co-Cultivation
[0478] Five hundred B-cells isolated from frozen and thawed rabbit IgG.sup.+ PBMCs per well were co-cultivated with γ-irradiated rabbit CD40L expressing CHO cells or 7-irradiated CHO-K1 cells (10,000 each) without the addition of TSN. In addition to the co-cultivation without any supplements, the impact of the addition of recombinant human IL-2, recombinant murine IL-2, recombinant human IL-21, recombinant murine IL-21 and combinations thereof was tested. After 7 days of co-cultivation the antibody titer in the supernatant of the B-cells was determined using the ELISA as described earlier. The co-cultivations comprising rbCD40L cells in the presence of IL-2 and IL-21 resulted in B-cell proliferation and antibody secretion (see
Example 15
Synthetic Rabbit B-Cell Culture System
Low B-Cell Density
[0479] Five hundred rabbit IgG.sup.+ B-cells/well (isolated from an NZW rabbit) were co-cultivated with either γ-irradiated rabbit CD40L expressing CHO cells or γ-irradiated CHO-K1 cells (10,000 each) without the addition of TSN. In addition to the co-cultivation without any supplements, the impact of the addition of recombinant human IL-2, recombinant murine IL-2, recombinant human IL-21, recombinant murine IL-21 and combinations thereof was tested. After 7 days of co-cultivation the antibody titer in the supernatant of the B-cells was determined using the ELISA as described earlier. The co-cultivations comprising rbCD40L cells in the presence of IL-2 and IL-21 resulted in B-cell proliferation and antibody secretion (see
High B-Cell Density
[0480] Two thousand five hundred rabbit IgG.sup.+ B-cells/well (from the same NZW rabbit as in the previous low density experiment) were co-cultivated with γ-irradiated rabbit CD40L expressing CHO cells or γ-irradiated CHO-K1 cells (10,000 each) without the addition of TSN. In addition to the co-cultivation without any supplements, the impact of the addition of recombinant human IL-2, recombinant murine IL-2, recombinant human IL-21, recombinant murine IL-21 and combinations thereof was tested. After 7 days of co-cultivation the antibody titer in the supernatant of the B-cells was determined using the ELISA as described earlier. The co-cultivations comprising rbCD40L cells in the presence of IL-2 and IL-21 resulted in B-cell proliferation and antibody secretion (see
Example 16
Kinetic Analysis of Synthetic B-Cell Culture System
[0481] Rabbit IgG.sup.+ cells are magnetically enriched from PBMC of NZW rabbit whole blood as described in Example 4. Five hundred rabbit B-cells per well are cultured at a ratio of 1:20 with γ-irradiated rabbit CD40L.sup.+ CHO feeder cells or control γ-irradiated CHO-K1 feeder cells (10,000 each; generation as described in examples 1-3) in the absence or presence of IL-2, IL-21 or combination of IL-2 and IL-21 (of the same or different origin, e.g. mouse and human). After 1, 2, 3, 4, 5, 6 and 7 days of cultivation (d1-d7) the co-cultures of rabbit B-cells with rabbit CD40L expressing CHO cells are assessed for rabbit IgG production in the supernatant and cellular growth. These data are compared to cells co-cultivated with feeder cells without CD40L expression and/or in the absence of additional exogenous stimuli (the same single interleukins or combination of interleukins). The growth and proliferation kinetics are determined via CTG assay and by microscopic analysis (see above).
[0482] Interleukins are applied/used in the following final concentrations: [0483] murine IL-2 at 50 U/ml (Roche Diagnostics GmbH, cat #11271164001) [0484] human IL-2 at 50 U/ml (Roche Diagnostics GmbH, cat. #11147528001) [0485] human IL-21 in the range of 1fold to 3fold of the EDso concentration, which varies between 10-100 ng/ml, depending on the IL-21 batch (eBioscience, cat. #14-8219) [0486] murine TL-21 at 100 ng/ml (R&D Systems, cat. #594-ML).