Chapter 2 Materials and Methods
2.2 M ETHODS
2.2.4 Splenocyte isolation
Mice were sacrificed by dislocation and their spleens were quickly harvested in a laminar flow hood. Spleens were placed in a 280 µm-pored mesh and chopped by scissors. 10 ml of RPMI 1640 (Invitrogen Co., USA) supplemented with 10% FBS, 0.2% NaHCO3 and 1% PSA. were slowly added onto the mesh while spleens were being ground until the spleen tissue became white. Single cell suspension was collected in a Petri dish and recovered by centrifugation at 1,200 rpm at 4℃ for 5 min.
Supernatant was discarded and 10 ml 1X ACK lysis buffer was added for 5 min at room temperature. 1X ACK buffer can lyse the red blood cells while leaving the rest of the lymphocytes and leucocytes. The mixture was then diluted by 10 ml of RPMI 1640 and cells were recovered by centrifugation at 1,200 rpm at 4℃ for 5 min. After the supernatant was discarded, the cells were rinsed by 10 ml PBS once more. Finally, cells were resuspended in RPMI 1640 and underwent cell calculation by trypan
10
blue exclusion. For the cell proliferation of splenocyte, cells were plated in a 96-well at 2.5 x 105 per well. For the cytokine profiles of splenocyte, cells were plated in a 24-well plate at 1 x 106 per well.
2.2.5 The cytotoxicity of LPPC to PBMCs or splenocytes
PBMC (1×105 cells per well) or splenocyte (2.5×105 cells per well) were respectively dispensed into 96-well culture plates and then except for control treated with different condition. After 72 hr, the cells were centrifuged at 400g for 15 min. Removed the medium, and added 100 µl MTT working solution per well. And then, the 96-well culture plates were put back incubator with 5% CO2 at 37℃ for 4 hr. The supernatant was removed, and added 100 µl DMSO to dissolve the purple crystal. Put plates on the shaker for 10 min. The optical density was determined by a microplate reader (Tecan) set to 595 nm and the data were analyzed by Magellan5 software.
2.2.6 The activities of monoclonal antibodies adsorbed on LPPC
In this study, anti-CD3 monoclonal antibody (2C11 or OKT3) was utilized as first signal for activation of T cell, and the other monoclonal antibody, anti-CD28 as second signal was for optimal activation of T cell.
PBMC (1×105 cells per well) or splenocyte (2.5×105 cells per well) were respectively dispensed into 96-well culture plates and then except for control treated with different condition. 40 µg LPPC pre-adsorbed 100 µg BSA, and then adsorbed with 2.4 µg anti-CD3 mAb or with 2.4 µg anti-CD3 and 2.4 µg anti-CD28 mAb into 100 µl volume. After centrifuged, 2.5 µl LPPC complex treated PBMCs or splenocytes for
11
72hrs. By using MTT assay, and then the cell proliferation rate was calculated as O.D. value of sample divide into O.D. value of PBMC alone or splenocyte alone.
2.2.7 The stability of immunostimulatory monoclonal antibodies adsorbed on LPPC in RPMI
40 µg LPPC previously adsorbed 100 µg BSA, and then adsorbed with 2.4 µg anti-CD3 mAb or 2.4 µg anti-CD3 and 2.4 µg anti-CD28 mAb into 100 µl volume. After centrifuged, put the LPPC complex into RPMI solution in 37 ℃ for 30 minutes. After 30 minutes, the solution was centrifuged divide into LPPC pellet and the supernatant. The LPPC pellet was resuspend into 100 ul DDW. The 2.5 µl LPPC complex and the 2.5 µl supernatant respectively treated PBMCs (1×105 cells per well) or splenocytes (2.5×105 cells per well) in 96-well culture plate, and estimated the cell proliferation of immune cells for investigating the efficiency of monoclonal antibodies on LPPC. By using MTT assay, and then the stimulation index was calculated as (O.D. value of sample –O.D.
value of PBMC alone or splenocytes alone) / O.D. value of PBMC alone or splenocytes alone.
2.2.8 The dose-effect of monoclonal antibodies adsorbed on LPPC in immune cells
Cell proliferation
PBMC (1×105 cells per well) or splenocyte (2.5×105 cells per well) were respectively dispensed into 96-well culture plates and then except for control treated with different condition. Addition different amounts of
12
immunostimulatory monoclonal antibodies were combined with 1 µg LPPC to stimulate the proliferation of immune cells, and the cell numbers was counted by MTT assay at 72 hrs. The cell proliferation rate was calculated as O.D. value of sample divide into O.D. value of PBMC alone or splenocyte alone.
Cytokines secretion
PBMC (4×105 cells per well) or splenocyte (106 cells per well) were dispensed into 24-well culture plates and then except for control treated with different condition. 4 µg LPPC adsorbed different amounts of monoclonal antibodies respectively to treat PBMCs or splenocytes. And the supernatants were collected at 24h and 72 h and frozen at −80 ℃. Supernatants concentrations of TNF-α, IL-2, and IFN-γ were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).
Pro-inflammatory cytokine profiles secretion
PBMC (4×105 cells per well) was dispensed into 24-well culture plates and then except for control treated with different condition. 4 µg LPPC treated PBMCs and then the supernatants were collected at 24 h, 48 h and 72 h and frozen at −80℃. Supernatants concentrations of IL-1β, IL-6, and IL-8 were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).
2.2.9 The comparison of activities of monoclonal antibodies on LPPC PBMC (1×105 cells per well) or splenocyte (2.5×105 cells per well) were respectively dispensed into 96-well culture plates for monitoring cell proliferation. 40 µg LPPC previously adsorbed 100 µg BSA, and then adsorbed with 2.4 µg anti-CD3 mAb or 2.4 µg anti-CD3 and 2.4 µg
13
anti-CD28 mAb into 100 µl volume. After centrifuged, 2.5 µl LPPC complex respectively treat PBMCs or splenocytes, and comparing to the same amount mAb that free from added into solution. The cell numbers was counted by MTT assay at 72 hrs. The cell proliferation rate was calculated as O.D. value of sample divide into O.D. value of PBMC alone or splenocyte alone.
On the other hand, PBMC (4×105 cells per well) or splenocyte (106 cells per well) were dispensed into 24-well culture plates for monitoring cytokines secretion. 40 µg LPPC previously adsorbed 100 µg BSA, and then adsorbed with 6 µg anti-CD3 mAb or 6 µg anti-CD3 and 6 µg anti-CD28 mAb into 100 µl volume. After centrifuged, 10 µl LPPC complex respectively treat PBMCs or splenocytes, and comparing to the same amount mAb that free from added into solution. And the supernatants were collected at 24h and 72 h and frozen at −80 ℃. Supernatants concentrations of TNF-α, IL-2, and IFN-γ were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).
2.2.10 The uptake protein ability of P338D1
50 µg BSA-FITC as a green fluorescence protein was previously adsorbed by 150 µg LPPC or was not adsorbed, and then respectively co-cultured two hours with 5 ×105 mouse macrophage, P338D1. Added 100 µl trypan blue to quench the green fluorescence from BSA-FITC that was not uptaken or only adhered to cell surface, and FACS analysis was performed. In addition, 50 µg BSA-FITC as a green fluorescence protein was previously adsorbed by 150 µg LPPC or 10 µg LPPC, and then
14
respectively co-cultured two hours with 5 ×105 mouse macrophage, P338D1.
2.2.11 DC harvest
Balb/C mice were sacrificed by dislocation. Make a long transverse cut through the skin in the middle of the abdominal area. Reflect skin from the hindquarters and the hind legs. Removed the feet, and then removed all muscle from the femurs and tibiae. Separate the legs from the body at the hip joint (one leg each time). Transfer the bones to a 15 mL centrifuge tube containing cold RPMI. Place the bones in a 10 cm bacterial dish containing 70 % ethanol for less 2~5 min for disinfection, then washed with RPMI. Separate femurs and tibiae. Cut both ends of the bone with scissors and the marrow flushed with RPMI10 using a Syringe with a 25 G needle. Collect cell suspension in a 10 cm bacterial dish. Clusters within the cell suspension were disintegrated by vigorous pipetting.
Transfer the cell suspension to a 15-mL centrifuge tube. Centrifuges at RT, 300g for 5 min and then discard the supernatant. Add 2 mL of ACK lysis buffer to lyse red cells for 45 sec. The mixture is then added with 10 mL of RPMI10and centrifuges at RT, 300g for 5 min to wash out ACK.
Discard the supernatant, and then suspend the cell pellet and then add with 10 mL of RPMI. Transfer the suspension to another tube to remove the settled debris and clumps. Centrifuges at RT, 300g for 5 min and discard the supernatant. Count cell number and then BM leukocytes were seeded at 2.5×106 per 100 mm dish in 10 mL R10 medium containing 200 U/mL rmGM-CSF. At day 3, another 10 mL RPMI10 medium containing 200 U/mL rmGM-CSF were added to the plates. At days 6, half of the
15
culture supernatant was collected (10 mL/dish), centrifuged at RT, 300g for 5 min, and the cell pellet resuspended in 10 mL fresh RPMI10 containing 200 U/mL rmGM-CSF/dish, and given back into the original plate. At days 8, half of the culture supernatant was collected (10 mL/dish), centrifuged at RT, 300g for 5 min, and the cell pellet resuspended in 10 mL fresh R10 containing 200 U/mL rmGM-CSF/dish, and given back into the original plate. At day 9 or 10, non-adherent cells were collected by gentle pipetting. Cells were centrifuged at 300g for 5 min at RT, and resuspended in 10 mL fresh R10 (106 per mL) into a fresh 100 mm tissue culture plastic dish containing 100 U rmGM-CSF and 0.5
µg/mL LPS (–20
℃, A11, 100 µg/mL). Cells were then cultured for 1 or 2 days for further experiment (complete maturation). The mature dendritic cells were checked by staining with anti-mouse CD11 conjugated PE and analyzed by flow cytometry.Purification of DC membrane protein
Harvested DC cells (1×107 cells) were by centrifuging the cell suspension or culture at 900g for 10 min at 4℃. Resuspend the cell pellet in 10 ml PBS buffer and centrifuged at 900g for 10 min at 4℃. Resuspend the cells in 10 ml HEPES-KOH buffer. Homogenize the cells on ice to fine homogenate using an appropriate cell homogenizer. The cells were centrifuged at 9000g for 15 min at 4℃. Transfer the supernatant into fresh ultracentrifuge tubes and discard the pellet. The fresh ultracentrifuge tubes centrifuged at 50000g at 4℃. Discard the supernatant, briefly air dry, and save the membrane pellet. The membrane pellet was dissolve in the PBS buffer. The concentration of membrane proteins was analyzed by
16
using commassie plus test.
Dendritic cell surface marker staining
106 DCs were centrifuged the cells at 4000 rpm for 5min. Resuspend the cells with 500 µl staining buffer (0.5% skim milk in PBS). Stain the cells with antibody on ice in the dark for 30min. After washing the cells with 500 µl staining buffer, centrifuge the cells at 4000 rpm for 5min.
Repeat again. Analyze the cells on FACScan with dot plots with quadrant line. Figure 11 indicated that the surface marker expression of DCs, such as CD11c, MHC II, and CD86.
17
2.2.12 The experimental strategy of animal immunization Bovine serum albumin (BSA)
1st immunize (1mg/mouse) 2 weeks 1st boost (1mg/mouse) 1 week 2nd boost (1mg/mouse) 1 week 3rd boost (1mg/mouse)
Experiment
Six- to eight-weeks old female BALB/c mice were purchased from the National Laboratory Center and housed in a temperature- and light-controlled room (12L:12D) at the Animal Maintenance Facility of National Chiao Tung University. The mice were first immunized by subcutaneously (s.c.) injection of 1mg/100µl BSA emulsified in CFA.
The mice were boosted by subcutaneously (s.c.) injection of 1mg/100µl BSA emulsified in IFA, and all mice were sacrificed postchallenge. The experiment strategy of mice immunization followed above the protocol.
The negative group was injected with 100 µl PBS alone.
18
Heat shock protein 60 of Helicobacter pylori
1st immunize (100µg/mouse) 2 weeks 1st boost (100µg/mouse) 1 week 2nd boost (100µg/mouse) 1 week 3rd boost (100µg/mouse)
Experiment
Six- to eight-weeks old female BALB/c mice were purchased from the National Laboratory Center and housed in a temperature- and light-controlled room (12L:12D) at the Animal Maintenance Facility of National Chiao Tung University. The mice were first immunized by subcutaneously (s.c.) injection of 100µg/300µl HpHsp60 emulsified in CFA. The mice were boosted by subcutaneously (s.c.) injection of 100µg/300µl HpHsp60 emulsified in IFA, and all mice were sacrificed postchallenge. The experiment strategy of mice immunization followed above the protocol. The negative group was injected with 300 µl PBS alone.
19
HPV E7 epitope
1st immunize (30µg /mouse) 2 weeks 1st boost (30µg /mouse) 1 week Experiment
Six-eight weeks female C57BL/6-Tg(HLA-A2.1) mice were kindly provided from Dr. Shih-Jen Liu (National Health Research Institutes) and housed in a temperature- and light-controlled room (12L:12D) at the Animal Maintenance Facility of National Chiao Tung University. The mice were first immunized by subcutaneously (s.c.) injection of 30µg/100µl YML peptides emulsified in CFA. The mice were boosted by subcutaneously (s.c.) injection of 30µg/100µl YML peptides emulsified in IFA, and all mice were sacrificed postchallenge. The experiment strategy of mice immunization followed above the protocol. The negative group was injected with 100 µl PBS alone.
20
2.2.13 The enhancement of antigen presentation of APCs by LPPC Cell proliferation
First, 100 µg BSA proteins were adsorbed by 40 µg LPPC into 100 µl volume. Splenocytes that isolated from were prior immunized by BSA (2.5×105 cells per well) were respectively dispensed into 96-well culture plates for monitoring cell proliferation. The 2.5 µl LPPC-complex co-cultured with splenocytes, and MTT assay was used to estimate at 72 hrs. The cell proliferation rate was calculated as O.D. value of sample divide into O.D. value of splenocyte alone. Negative control was the splenocytes from naive mice.
Cytokines secretion
First, 100 µg BSA proteins were adsorbed by 40 µg LPPC into 100
µl volume. Splenocytes that isolated from were prior immunized by
HpHSP60 (106 cells per well) were respectively dispensed into 24-well culture plates for monitoring cytokines secretion. The 10 µl LPPC-complex co-cultured with splenocytes, and the supernatants were collected at 24h and 72 h and frozen at −80 ℃. Supernatants concentrations of TNF-α, IL-2, IL-10, IL-4, and IFN-γ were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).2.2.14 Membrane proteins with specific antigen coated LPPC Cell proliferation
50 µg membrane proteins contained BSA or HpHSP60 antigens isolated from DCs and 100 µg BSA proteins were for 40 µg LPPC adsorption into 100 µl volume. Splenocytes that isolated from were prior
21
immunized by HpHSP60 (2.5×105 cells per well) were respectively dispensed into 96-well culture plates for monitoring cell proliferation.
The 2.5 µl LPPC-complex co-cultured with splenocytes, and MTT assay was used to estimate at 72 hrs. The cell proliferation rate was calculated as O.D. value of sample divide into O.D. value of splenocyte alone.
Negative control was the splenocytes from naive mice.
Cytokines secretion
50 µg membrane proteins contained BSA or HpHSP60 antigens isolated from DCs and 100 µg BSA proteins were for 40 µg LPPC adsorption into 100 µl volume. Splenocytes that isolated from were prior immunized by HpHSP60 (106 cells per well) were respectively dispensed into 24-well culture plates for monitoring cytokines secretion. The 10 µl LPPC-complex co-cultured with splenocytes, and the supernatants were collected at 24h and 72 h and frozen at −80 ℃. Supernatants concentrations of TNF-α, IL-2, IL-10, IL-4, and IFN-γ
were measured by
Enzyme-Linked ImmunoSorbent Assay (ELISA).2.2.15 The specific peptide-loaded HLA-A2 adsorbed on LPPC Cell proliferation
50 µg YML peptide-loaded HLA-A2 molecules and 100 µg BSA proteins were for 40 µg LPPC adsorption into 100 µl volume. Splenocyte that immunized by YML antigen (2.5×105 cells per well) were respectively dispensed into 96-well culture plates for monitoring cell proliferation. The 2.5 µl LPPC-complex co-cultured with splenocytes, and MTT assay was used to estimate at 72 hrs. The cell proliferation rate
22
was calculated as O.D. value of sample divide into O.D. value of splenocyte alone. Negative control was the splenocytes from naive mice.
Cytokine secretion
50 µg YML peptide-loaded HLA-A2 molecules and 100 µg BSA proteins were for 40 µg LPPC adsorption into 100 µl volume. Splenocyte that prior immunized by YML antigen (106 cells per well) were respectively dispensed into 24-well culture plates for monitoring cytokines secretion. The 10 µl LPPC-complex co-cultured with splenocytes, and the supernatants were collected at 24h and 72 h and frozen at −80 ℃. Supernatants concentrations of TNF-α, IL-2, and IFN-γ were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).
2.2.16 The animal immunization of the immuno-LPPC in vivo
Six- to eight-weeks old female Balb/c mice were purchased from the National Laboratory Center and housed in a temperature- and light-controlled room (12L:12D) at the Animal Maintenance Facility of National Chiao Tung University. The 200µg LPPC previously adsorbed 250 µg peptide-loaded membrane proteins. And then the mice were immunized by intravenous (i.v.) injection of membrane proteins /LPPC complex and all mice were sacrificed after two weeks. The negative group was injected with 300 µl PBS alone.
Six-eight week female C57BL/6-Tg(HLA-A2.1) mice were kindly provided from Dr. Shih-Jen Liu (National Health Research Institutes) and housed in a temperature- and light-controlled room (12L:12D) at the Animal Maintenance Facility of National Chiao Tung University. The
23
200µg LPPC previously adsorbed 25 µg peptide-loaded HLA-A2 molecules and 25 µg anti-CD28 mAb. And then the mice were immunized by intravenous (i.v.) injection of LPPC complex and all mice were sacrificed after two weeks. The negative group was injected with 300 µl PBS alone.
2.2.17 The animal immunization efficiency of immuno-LPPC in vivo Cell proliferation
Splenocytes that isolated from were prior immunized by immuno-LPPC or PBS (2.5×105 cells per well) were respectively dispensed into 96-well culture plates for monitoring cell proliferation.
HpHsp60 or YML peptides (2µg/ml) were co-cultured with splenocytes, and MTT assay was used to estimate at 72 hrs. The cell proliferation rate was calculated as O.D. value of sample divide into O.D. value of splenocyte alone. Negative control was the splenocytes from naive mice.
Cytokine secretion
Splenocytes that isolated from were prior immunized by immuno-LPPC or PBS (106 cells per well) were respectively dispensed into 24-well culture plates for monitoring cytokines secretion. HpHsp60 or YML peptides (2µg/ml) were co-cultured with splenocytes, and the supernatants were collected at 24h, 48 h and 72 h and frozen at −80 ℃. Supernatants concentrations of TNF-α, IL-2, IL-10, IL-4, or IFN-γ were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).
2.2.18 Statistical analysis
24
All figures are expressed as mean ± SD. All data were computed by student-test. All statistical significant was set at p < 0.05.
25
Chapter 3 Result
3.1 The characters of LPPC
As figure 1a shown, the shape of LPPC was approximately round and the particle size was about 200 nm. In addition, the dark shadow of LPPC was hair-like, which might be PEI and PEG polymers. LPPC is a cationic liposome, and it was found that LPPC can adsorb proteins on its surface. Therefore, DLS was utilized to investigate the particle size of LPPC with or without protein adsorption. The results showed that the diameters of LPPCs with protein adsorption were about 358 ± 16 nm, which was larger than the LPPC without protein adsorption (Figure 1b).
Besides, the previous results have shown the empty LPPCs can be centrifuged and pelleted (Figure 1c) and the further experiments also indicated the protein adsorption did not affect this character. Because centrifugation is available for LPPC, unbound substances could be easily removed.
3.2 The characters of LPPC for protein adsorption
To understand the kinetic for protein adsorption to LPPC, the fluorescence of BSA-FITC was used to evaluate what time the LPPC need to adsorb proteins to their surface. The results showed that LPPC could adsorb 80% of proteins in ten minutes and reach the maximal adsorption in 20 minutes (Figure 2a). Moreover, the protein binding capacity of LPPC was estimated and the results revealed that the maximal adsorption of 40 µg LPPC was about 160 µg BSA (Figure 2b).
26
Surprisingly, the pre-adsorbed BSA-FITC on LPPC could not be replaced by the additions of different BSA dose (Figure 2c). The results showed that the pre-adsorbed proteins on LPPC were irreplaceable by the posterior added proteins.
3.3 The activities of immunostimulatory monoclonal antibodies adsorbed on LPPC
3.3.1 The cytotoxicity of LPPC to PBMCs or splenocytes
LPPC could adsorb proteins stably and remain their activities as previous experiments (Table 1). Therefore, to investigate whether LPPC could adsorb immunostimulatory monoclonal antibodies and stimulate immunity was further proceeded. First, the cytotoxicity of LPPC was determined for PBMCs or splenocytes at next experiment. The results indicated that 1 µg LPPC was an appropriate dosage for 105 PBMCs or 2.5×105 splenocytes, because the cells could survive without toxic damage in this concentration (Figure 3).
3.3.2 The effects of the bound antibodies in a dosage-dependent manner
In this study, anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) were utilized to activate T cell, which were used to determine whether the bound protein on LPPC could remain its biofunction. In order to understand the regulatory phenomenon of monoclonal antibodies on LPPC for activities, the different amounts of immunostimulatory mAbs were adsorbed on LPPC to stimulate the proliferation of PBMCs or
27
splenocytes. The results showed the cell proliferation rates of PBMCs and splenocytes were higher as anti-CD3 mAbs were increased. It would be
splenocytes. The results showed the cell proliferation rates of PBMCs and splenocytes were higher as anti-CD3 mAbs were increased. It would be