Method for the production of thymocyte supernatant

Abstract

Herein is reported a method for producing a thymocyte supernatant comprising the steps of co-cultivating thymocytes and mononuclear cells at a cell ratio of at least 0.5:1.2 in the presence of phorbol-12-myristate-13-acetate and Phytohemagglutinin M for up to 60 hours, and separating the co-cultivation medium from the cells and thereby producing the thymocyte supernatant.

Claims

1. A method for producing a thymocyte supernatant comprising the following steps: co-cultivating thymocytes and macrophages at a cell ratio of 0.5:1.2 to 0.5:2 in the presence of phorbol-12-myristate-13-acetate and phytohemagglutinin M for up to 60 hours, and separating the co-cultivation medium from the cells and thereby producing the thymocyte supernatant.

2. The method according to claim 1, wherein the ratio is about 0.5:2.

3. The method according to claim 1, wherein the thymocyte cell density is about 510.sup.5 cells/ml in the co-cultivating.

4. The method according to claim 1 wherein prior to the co-cultivating the thymocytes are incubated for up to 60 hours at 37 C. in cultivation medium.

5. The method according to claim 1, wherein the macrophages are isolated from PBMCs by adherence to a solid surface at a cell density of 210.sup.6 cells/ml and the attached macrophages are incubated for about 40 hours in cultivation medium prior to the co-cultivating with the thymocytes.

6. The method according to claim 1, wherein prior to the co-cultivating of the thymocytes and macrophages the cultivation medium of the thymocytes is replaced by fresh medium containing 10 ng/ml phorbol-12-myristate-13-acetate (PMA) and 5 g/ml phytohemagglutinin M (PHA-M).

7. The method according to claim 1, wherein the co-cultivating is started by removing the cultivation medium from the macrophages and adding the thymocyte suspension.

8. The method according to claim 1, wherein the medium is RPMI medium supplemented with 10% (v/v) FCS, 1% (w/v) of a 200 mM glutamine solution that comprises penicillin and streptomycin, 2% (v/v) of a 100 mM sodium pyruvate solution, and 1% (v/v) of a 1 M 2-(4-(2-hydroxyethyl)-1-piperazine)-ethane sulfonic acid (HEPES) buffer, further comprising 0.05 M -mercaptoethanol.

Description

DESCRIPTION OF THE FIGURE

(1) FIG. 1 shows the lot-to-lot variability of TSN lots produced with a method known from the art (left part) and with the inventive method as reported herein (right part). Solid square: ratio of the number IgG-positive wells of the respective TSN lot to the number of IgG-positive wells obtained with a reference TSN lot; dash-point-dash line: average of the ratio of the number of IgG positive wells; solid triangles: ratio of the average IgG concentration in the wells obtained with the respective TSN lot to the average IgG concentration in the wells obtained with a reference TSN lot; dashed line: average of the IgG concentration ratios.

EXAMPLES

(2) Materials and Methods

(3) Recombinant DNA Techniques

(4) 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.

(5) Media and Buffers

(6) Blocking buffer for ELISA comprises 1PBS and 1% BSA.

(7) Coating buffer for ELISA comprises 4.29 g Na2CO3*10 H2O and 2.93 g NaHCO.sub.3 add water to a final volume of 1 liter, pH 9.6 adjusted with 2 N HCl.

(8) Ethanol-solution for RNA isolation comprises 70% Ethanol or 80% Ethanol.

(9) FACS-buffer for immuno fluorescence staining comprises 1PBS and 0.1% BSA.

(10) IMDM-buffer for ELISA comprises 1PBS, 5% IMDM and 0.5% BSA.

(11) Incubation buffer 1 for ELISA comprises 1PBS, 0.5% CroteinC.

(12) Incubation buffer 2 for ELISA comprises 1PBS, 0.5% CroteinC and 0.02% Tween 20.

(13) Incubation buffer 3 for ELISA comprises 1PBS, 0.1% BSA.

(14) Incubation buffer 4 for ELISA comprises 1PBS, 0.5% BSA, 0.05% Tween, PBS (10), 0.01 M KH2PO4, 0.1 M Na2HPO4, 1.37 M NaCl, 0.027 M KCl, pH 7.0.

(15) PCR-buffer comprises 500 mM KCl, 15 mM MgCl2, 100 mM Tris/HCl, pH 9.0.

(16) Wash buffer 1 for ELISA comprises 1PBS, 0.05% Tween 20.

(17) Wash buffer 2 for ELISA comprises 1PBS, 0.1% Tween 20.

(18) Wash buffer 3 for ELISA comprises water, 0.9% NaCl, 0.05% Tween 20.

(19) EL-4 B5 medium comprises 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 -mercaptoethanol (Gibco, Paisley, Scotland).

(20) Animal Care

(21) The experimental animals were held according to the German animal protection law (TierSCHG) as well as according to the respective European guidelines.

(22) Mice and hamster were received at an age of from 6 to 8 weeks and were immunized prior to an age of 12 weeks. The antigen was at first applied together with complete Freud's adjuvant (CFA). Further applications were with incomplete Freud's adjuvant (IFA). The antigen containing emulsion was applied subcutaneously whereby the emulsion comprised an amount of from 50 to 100 g antigen depending on the weight of the receiving experimental animal.

(23) Proliferation assays a) Cell Titer Glo (CTG) viability assay The CTG viability assay (Promega; #G7571) was used according to the instructions of the manufacturer. b) .sup.3H Thymidine Assay After 6 days of incubation .sup.3H-Thymidin was added (0.5 Ci/well) and incubated for further 16 hours. The incorporation of .sup.3H-Thymidine during cell proliferation was determined with a microplate scintillation counter (Wallac). c) Microscopic analysis 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. d) Analysis of B-cell activation via CFSE-labeling. Isolated B-cells were washed with sterile phosphate buffer saline solution (PBS). Up to 110.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 M 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+gated (B)cells as a consequence of CFSE dilution was confirmed by flow cytometric analysis (FL-1 channel) after indicated time points.
Quantification of IgG

(24) The 96-well multi well plate in which the co-cultivation was performed was centrifuged after seven days of co-cultivation at 300g for 5 min. 150 l supernatant was removed and diluted at a ratio of 2:1 with PBS in a second 96-well multi well plate.

(25) The antibody was used at a concentration of 50 ng/ml. If the OD was or exceeded 1 after an incubation time of 5 min. a dilution series of from 0.8 to 108 ng/ml IgG was tested.

(26) Panning on Antigen

(27) a) Coating of Plates

(28) Biotin/Streptavidin: Sterile streptavidin-coated 6-well plates (cell culture grade) were incubated with biotinylated antigen at a concentration of 0.5-1(2) g/ml in PBS at room temperature for one hour. Plates were washed in sterile PBS three times before use.

(29) Covalently bound protein: Sterile cell culture 6-well plates were coated with 2 g/ml protein in carbonate buffer (0.1 M sodium bicarbonate, 34 mM disodium hydrogen carbonate, pH 9.55) over night at 4 C. Plates were washed in sterile PBS three times before use.

(30) b) Panning of B-Cells on Peptides

(31) 6-well tissue culture plates coated with the respective antigen were seeded with up to 610.sup.6 cells per 4 ml medium and allowed to bind for one hour at 37 C. in the incubator. Non-adherent cells were removed by carefully washing the wells 1-2 times with 1PBS. 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.

(32) Gene Synthesis

(33) Desired gene segments encoding cDNA 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.

(34) TABLE-US-00006 Rabbit B-cell medium/EL4-B5 medium: 500 ml RPMI 1640 #P04-17500 PAN Biotech 50 ml FCS #A15-512 PAA 5 ml L-glutamine #25030-024 Invitrogen 5 ml potassium pyruvate #P04-43100 PAN Biotech 5 ml HEPES #15630-056 Invitrogen 500 l -mercaptoethanol # 31350010 Invitrogen 1 ml Pen/Strep #11074440001 Roche Dia. GmbH

(35) TABLE-US-00007 Additives to rabbit B-cell medium SAC #507858 Calbiochem

(36) TABLE-US-00008 multi-well-plates 96er U-plate #3799 Corning

(37) TABLE-US-00009 Phenotyping/sorting of antibodies goat anti-rabbit IgG Fc-antibody AbDSerotec STAR121F 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 Alexa 488 Molecular Probes A11055

(38) TABLE-US-00010 Miscellaneous anti-FITC antibody-coupled microbeads Miltenyi Biotec #130-048-701 human B-cell negative isolation kit Invitrogen #113.13D Nucleofector Kit T Lonza VCA-1002 CBA for total IgG BD Biosciences #558679

Example 1

(39) Immunization of Rabbits

(40) NZW rabbits (Charles River Laboratories International, Inc.) were used for immunization. The antigen was solved in K.sub.3PO.sub.4 buffer pH 7.0 at a concentration of 1 mg/ml and mixed (1:1) with complete Freud's adjuvant (CFA) till generation of stabile emulsion. The rabbits received an intra-dermal (i.d.) injection of 2 ml of emulsion followed by a second intra muscular (i.m.) and third subcutaneous (s.c.) injection each with 1 ml in one week interval. The fourth i.m. injection of 1 ml was performed two weeks later followed by two further s.c. injections of 1 ml in four weeks interval.

(41) During the immunization serum antibody titer was determined with an antigen specific assay. At an antibody titer with an IC.sub.50 of 1:10000 the blood or the spleen of the immunized animal was removed. For reactivation of antigen specific B-cells 30 g to 50 g of the antigen was applied intravenously to the experimental animal three days prior to the removal of the blood or the spleen.

Example 2

(42) Removal of Organs, Blood and Macrophages

(43) Blood from rabbits was obtained by punctuation of the ear vein or, for larger volumes, of the ear artery. Whole blood (10 ml) was collected from rabbits 4-6 days after the third, fourth, fifth and sixth immunization and used for single cell sorting by FACS.

(44) Macrophages were isolated from the obtained blood by attachment to cell culture plastic.

(45) If a larger amount of was required, peritoneal macrophages were isolated. For this the animals have to be at least 3 months of age. For the removal of peritoneal macrophages, animals were sacrificed and 5 ml of EL-4 B5 medium with a temperature of 37 C. was immediately injected into the peritoneal cavity. After kneading the animal's belly for 5 minutes, the solution containing the cells was removed.

(46) EDTA containing whole blood was diluted twofold with 1PBS before density centrifugation on lympholyte mammal (Cedarlane Laboratories) or Ficoll Paque Plus (GE Healthcare, cat. #17-1440-03), which was performed to isolate rabbit PBMC. PBMCs were washed twice before staining with antibodies.

Example 3

(47) Density Gradient Centrifugation

(48) The isolation of peripheral blood mononuclear cells (PBMCs) was effected by density gradient separation with Lympholyte according to manufacturer's instructions A (Lympholyte-mammal, Cedarlane).

(49) Withdrawn blood (optionally supplemented with EDTA) was diluted 1:1 with phosphate buffered saline (PBS), e.g. 60 ml blood plus 60 ml buffer. In a centrifuge vial the same volume of density separation medium was provided and the diluted blood is carefully added via the wall of the vial on top of the density separation medium. The ratio of the density separation medium to the PBS-diluted blood is about 1:1.5. Depending on the total volume of diluted blood a certain number of vials will be required. The vials were centrifuged for 20 min. at 800g without braking. Each individual white interim layer was added to 25 ml PBS, supplemented with PBS to a total of 50 ml and centrifuged at 800g for 10 min. The supernatants were discarded, the pellet were resuspended in 1/120 of the final volume of PBS, pellets were combined on a 1:1 basis and PBS was added to a final volume of 50 ml. Thereafter the vials were centrifuged again. Depending on the number of blood samples the samples are combined on a 1:1 ratio after each centrifugation step until only one sample is left. The final pellet was resuspended in PBS.

Example 4

(50) Hypotonic Lysis of Red Blood Cells

(51) For disruption of red blood cells by hypotonic lysis an ammonium chloride solution (BD Lyse) was diluted 1:10 with water and added at a ratio of 1:16 to whole blood. For lysis of the red blood cells the mixture was incubated for 15 min. in the dark. For separation of cell debris from intact cells the solution was centrifuged for 10 min. at 800g. The supernatant was discarded, the pellet was resuspended in PBS, washed again, centrifuged and the pellet was resuspended in PBS. Example 8

Example 5

(52) Depletion of Macrophages

(53) Sterile 6-well plates (cell culture grade) were used to deplete macrophages and monocytes through unspecific adhesion. Wells were either coated with KLH (keyhole limpet haemocyanine) or with streptavidin and the control peptides. Each well was filled with 3 ml to (at maximum) 4 ml medium and up to 610.sup.6 peripheral blood mononuclear cells from the immunized rabbit and allowed to bind for 60 to 90 min. at 37 C. in the incubator. Thereafter the lymphocyte containing supernatant was transferred to a centrifugation vial and centrifuged at 800g for 10 min. The pellet was resuspended in PBS.

Example 6

(54) Enrichment of Antigen-Specific B-Cells

(55) The respective antigen was diluted with coating buffer to a final concentration of 2 g/ml. 3 ml of this solution were added to the well of a 6-well multi well plate and incubated over night at room temperature. Prior to use the supernatant was removed and the wells were washed twice with PBS. The B-cell solution was adjusted to a cell density of 210.sup.6 cells/ml and 3 ml are added to each well (up to 610.sup.6 cells per 3-4 ml medium) of a 6-well multi well plate. The plate was incubated for 60 to 90 min. at 37 C. The supernatant was removed and non-adherent cells were removed by carefully washing the wells 1-4 times with 1PBS. For recovery of the sticky antigen-specific B-cells 1 ml of a trypsin/EDTA-solution was added to the wells of the multi well plate and incubated for 10 to 15 min. at 37 C. The incubation was stopped by addition of medium and the supernatant was transferred to a centrifugation vial. The wells were washed twice with PBS and the supernatants were combined with the other supernatants. The cells were pelleted by centrifugation for 10 min. at 800g. The cells were kept on ice until the immune fluorescence staining. The pellet was optionally resuspended in PBS.

Example 7

(56) Cultivation of T-cells

(57) The T-cells were isolated from the thymus of 3-4 week old mice and hamsters, or of 4-5 week old rabbits, respectively. The cells were centrifuged and immediately cultivated or frozen in aliquots of 4-510.sup.7 cells. The thymocytes were seeded with a minimum cell density of 510.sup.5 cells/ml of EL-4 B5 medium in 175 cm.sup.2 culture flasks and incubated for up to 48 hours (40-48 hours depending on the TSN production method the macrophages will be used in; see Examples 9 and 10) at 37 C.

Example 8

(58) Cultivation of Macrophages

(59) Macrophages were isolated from the peritoneal cavity of mice and hamsters, respectively, of an age of at least three months. Peritoneal macrophages from mice or hamsters, or blood mononuclear cells from rabbits were cultivated in EL-4 B5 medium at a cell density of at least 110.sup.5 cells/ml in 175 cm.sup.2 culture flasks for 1.5 hours at 37 C. Afterwards the medium was removed and non-attached cells were removed from the attached macrophages by washing with warm EL-4 B5 medium, followed by cultivation for about 48 hours in 35 ml medium.

Example 9

(60) Co-Cultivation of T-Cells and Macrophages According to the Current Invention

(61) T-cells (see Example 7, 40 hours cultivation) and macrophages (see Example 8) were cultivated in separate flasks. Prior to combining both cell populations, the T-cells were centrifuged for 10 min. at 800g. The supernatant was discarded and the cell pellet was resuspended in 10 ml EL-4 B5 medium. The final cultivation medium contained T-cells adjusted to a cell density of 510.sup.5 cells/ml, 10 ng phorbol-12-myristate-13-acetate (PMA) per ml of medium, and 5 g phytohemagglutinin M (PHA-M) per ml of medium (=T-cell suspension). Thereafter, the cultivation medium was removed from the macrophages (=medium-depleted macrophages). An amount/volume of the T-cell suspension was added to the flasks containing the medium-depleted macrophages to obtain a final but defined macrophage cell density of from 1.25-210.sup.6 macrophages/ml. After 30-46 hours of co-cultivation, the cultivation medium was removed and was termed TSN solution. For removal of remaining cells the TSN solution was filtered through a 0.22 m filter. The TSN solution was frozen at 80 C. in aliquots (of 4.2 ml).

Example 10Comparative Example

(62) Co-Cultivation of T-Cells and Macrophages According to the State of the Art

(63) T-cells (see Example 7, 48 hours cultivation) and macrophages (see Example 8) were cultivated in separate flasks. Prior to combining both cell populations, the T-cells were centrifuged for 10 min. at 800g. The supernatant was discarded and the cell pellet was resuspended in 10 ml EL-4 B5 medium. The final cultivation medium contained T-cells adjusted to a cell density of 510.sup.5 cells/ml, 10 ng phorbol-12-myristate-13-acetate (PMA) per ml of medium, and 5 g phytohemagglutinin M (PHA-M) per ml of medium (=T-cell suspension). Thereafter, the cultivation medium was removed from the macrophages (=medium-depleted macrophages). An amount/volume of the T-cell suspension was added to the flasks containing the medium-depleted macrophages to obtain a final macrophage cell density of 110.sup.6 macrophages/ml. After 36 hours of co-cultivation, the cultivation medium was removed and was termed TSN solution. For removal of remaining cells the TSN solution was filtered through a 0.22 m filter. The TSN solution was frozen at 80 C. in aliquots (of 4 ml).

Example 11

(64) Cultivation of EL-4 B5 Cells

(65) The frozen EL-4 B5 cells were thawed rapidly in a water bath at 37 C. and diluted with 10 ml EL-4 B5 medium. After centrifugation at 300g for 10 minutes the supernatant was discarded and the pellet resuspended in 1 ml medium.

(66) The EL-4 B5 cells were inoculated at a cell density of 810 cells/ml in T175 cultivation flasks. Cell density was determined every second day and adjusted to 810.sup.4 cells/ml. The cells have a doubling time of approximately 10 hours. Cells were harvested and adjusted to a cell density of 110.sup.6 cells/ml before -irradiation at 50 Gy.

Example 12

(67) Co-Cultivation of B-Cells and EL-4 B5 Cells

(68) Single sorted B-cells were cultured in 96-well plates with 210 l/well EL-4 B5 medium with Pansorbin Cells (1:20000) (Calbiochem (Merck), Darmstadt, Deutschland), 5% thymocyte supernatant produced according to Example 9 or 10 and gamma-irradiated EL-4-B5 murine thymoma cells (210.sup.4/well) for 7 days at 37 C. in an atmosphere of 5% CO2 in the incubator. B-cell culture supernatants were removed for screening and the cells harvested immediately for variable region gene cloning or frozen at 80 C. in 100 l RLT buffer (Qiagen, Hilden, Germany).

(69) It has been found that depending on the amount of macrophages present in the co-cultivation of thymocytes (T-cells) with mononuclear cells (macrophages), i.e. depending on the ratio of said cells, the produced TSN shows different properties. The respective results are shown in the following Table 5 (total wells=4*84). The reference value was obtained with the comparative state of the art method according to Example 10.

(70) TABLE-US-00011 TABLE 5 average average frequency productivity average of IgG of all ratio IgG positive IgG [*10.sup.6 cells/ml] positive wells positive thymocytes:mono- wells [% of total wells nuclear cells [n] wells] [g/ml] example 0.5:1 (ref.) 33.3 2.3 39.6 2.7 0.35 0.05 10 0.5:1.25 38.8 4.8 46.1 5.7 0.81 0.10 9 0.5:1.5 34.5 5.5 41.1 6.6 1.22 0.12 9 0.5:2 37.3 1.8 44.4 2.1 1.32 0.23 9

Example 13

(71) Co-Cultivation of B-Cells and EL-4 B5 Cells with Different Lots Produced According to Examples 9 and 10

(72) To show the robustness of the herein reported TSN production method compared to the method known from the art multiple lots have been prepared with the methods according to Examples 9 and 10. The results are presented in the following Tables 6 and 7.

(73) TABLE-US-00012 TABLE 6 According to Example 10: ratio IgG positive ratio average productivity TSN lot wells to reference wells to reference reference 1.00 1.00 1 0.80 0.75 2 0.83 0.89 3 0.84 0.65 4 0.78 0.42 5 1.04 0.49 6 0.88 0.56 7 0.96 0.64 8 0.94 0.69 9 1.11 0.79 10 0.83 0.66 11 1.11 0.39 12 1.04 0.78 13 0.95 0.48 average 0.93 0.63

(74) TABLE-US-00013 TABLE 7 According to example 9: ratio IgG positive ratio average productivity TSN lot wells to reference wells to reference reference 1.00 1.00 14 0.83 0.52 15 0.96 0.96 16 0.99 1.55 17 1.04 1.15 18 0.98 1.21 19 1.33 1.53 20 0.81 1.37 21 1.05 1.54 22 1.01 1.09 23 1.14 1.26 24 1.23 1.02 25 1.03 1.38 26 1.03 0.96 27 1.04 1.31 28 1.17 1.52 29 1.10 1.46 30 1.00 1.95 31 0.97 2.60 32 0.94 1.27 33 0.94 1.15 34 0.85 1.05 35 0.96 1.32 average 1.02 1.33