A major aim in immunotherapy is to generate specific cell-mediate immune responses to regulate host immunity. Efficiently resist pathogens and virus that relies on T cell immune responses (1). The optimal T-cell stimulation requires engagement of the T-cell receptor (TCR) through the major histocompatibility complex (MHC) bound to peptide, together with at least one interaction of a costimulatory molecule with an appropriate ligand on the T cell (2, 3).
The most potent and best-investigated costimulatory molecules are B7-1 and B7-2, which bind to CD28 (4, 5) on the T cell and induce cell proliferations. In contrast, B7 molecules might also bind cytotoxic T lymphocyte antigen 4 (CTLA-4) molecules on activated T cells and induce apoptosis in those T cells. Additional interactions might also regulate T-cell stimulation, including T-cell subtype differentiation, induction of maximal proliferation and prevention of apoptosis. Potential molecules of these interactions are other costimulatory molecules such as ICOS, 4-1BBL and OX40 (6-8).
Another major group of molecules are the adhesion molecules, which include leukocyte function-associated antigen (LFA) and intercellular adhesion molecule (ICAM) (9, 10). These molecules increase the interaction time between the T cell and APC, and enhance efficient activation (11). As discuss above, over the past decade many
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new costimulatory molecules have been identified, offering new insights into T-cell activation and regulation.
Naive T cells are activated to produce armed efficient T cells the first time they encounter their specific antigen in the form of a peptide:MHC complex on the surface of an activated antigen presenting cell (APC). Antigen presentation by APCs, most notably macrophages and dendritic cells (DCs), and infected B cells is critical for induction of specific T cells in the form of an adaptive immune response (12). Further, the induction of T cell-mediated immunity is controlled by antigen-presenting DCs, potent stimulators of specific T cell immunity (13). DCs in essence act as nature’s adjuvants and play an important role to generate adaptive immunity. They present immunogenic epitopes of antigens in the context of MHC class I and class II molecules in association with costimulatory molecules, and efficiently activate both cytotoxic T cells and T helper cells (14).
DCs are both efficient and specialized in antigen presentation, and they control the magnitude, quality, and memory of the ensuing immune response. Because of the exceptional ability of DCs to activate T-cell immunity in response to microbial pathogens and tumor cells, these cells have been exploited as ex vivo and in vivo tools for immunotherapy. For example, Dr. Lu demonstrate that a therapeutic vaccine made of inactivated SIV-pulsed DCs can elicit effective cellular and humoral immune responses against SIV, allowing the control of SIV replication in the secondary lymphoid tissues and the reduction of cell-associated viral
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DNA and cell-free viral RNA in blood of SIV-infected macaques (15). In addition, HSP105-pulsed BM-DC vaccine could induce specific T cells to inhibit the growth of intestinal tumors overexpressing HSP105 (16).
Moreover, Dr. Aldrich utilized rAAV with human tumor antigen, carcinoembryonic antigen (CEA), of gene to infect DC for induction of specific immunity (17). In another study, the coadministration of DNA vaccines encoding HPV16 E7 with siRNA targeting key proapoptotic proteins successfully prolongs the lives of DCs, enhances antigen specific CD8+ T-cell responses, and elicits potent antitumor effects against an E7-expressing tumor model (18).
As development of DC-based application, the challenges of these therapies need to be improved. Several virus- and nonvirus-based transduction methods have been used for DC-based therapy. However, all strategies result in different levels of gene expression depending on the transduction efficiency. Therefore, purification of expressing APCs is needed to avoid non-expect interaction (11). In addition, some articles indicated that pathogens or pathogens-derived factors, such as Candida albicans, Mycobacterium tuberculosis, mycobacterial LAM, and secretions of Candida respectively impact on efficiency of DCs and immune cells, affect cytokine expression and impair surface marker of DC (19-22). Moreover, mature DCs express high levels of costimulatory molecules such as B7.1 and B7.2, which could be contact to CTLA-4, an immuno-inhibitory ligand that suppresses T cell activation (23, 24). In addition, many tumors secrete immunosuppressive factors such as TGF-β, IL-10, or VEGF, which affect the function of DCs to influence
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the efficiency of DC vaccine (11, 25-27).
As an alternative strategy, artificial antigen-presenting cell systems (aAPC) have been recently developed and are rapidly expanding. They encompass both cellular-based and acellular-based technologies (28). In cellular-based, they utilized genetic methods that transduced into murine fibroblast cells to express immunoregulatory molecules, such as antigen-loaded MHC molecules and B7 molecules (29). In addition, a nonspecific cell-based aAPC (K32 cells) has also been developed, that were transfected with the costimulatory molecule 4-1BBL and the low-affinity Fc gamma receptor CD32 (30).
In acellular-based, they employed chemical method to conjugate immunoregulatory molecules on nanoparticles, bead or liposome. For example, a acellular aAPC was developed that can be used to induce and expand clinically relevant amounts of highly enriched peptide-specific T cells based on HLA-A2–Ig molecules and anti-CD28 monoclonal antibody (mAb) coupled to a magnetic bead (31, 32). In this strategy, peptide resident in the HLA–Ig molecule with any HLA-A2-restricted antigenic peptide can be modified. Thus, a single batch of HLA-A2–Ig-based aAPCs can be loaded with various different antigenic peptides for expansion of cells with different antigenic specificities.
These artificial APC-based strategies certainly diminished the effect of immunosuppression.
Our laboratory has developed a novel liposome, Lipo-PEI-PEG
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complex (LPPC), that was a biodegradable liposome with the characters that could strongly and rapidly adsorb proteins on its surface, and these proteins could maintain their activities. The purpose of this study, that manufacture artificial antigen presenting cells, or APC-liked liposome, which were combined the liposome with immunostimulatory molecules to develop as an immunoregulatory platform. Therefore, we exploited LPPC combined with immunostimulatory molecules as artificial antigen presenting cells to activate immunity. Here, we added anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), or addition of DCs’ membrane proteins, or addition of specific peptide-HLA-A2 complex for the LPPC adsorption. The results showed that LPPC indeed exhibited ability of enhancement of the cell proliferations and cytokine secretions of human peripheral blood mononuclear cells (PBMCs) and murine splenocytes in vitro. We also showed that the LPPC with immuno-molecules induced specific immune responses in vivo. Moreover, LPPC might have the potential of an adjuvant that enhance immune responses of APCs. In this study, we indeed demonstrated that LPPC showed quickness and good flexibility to construct an immunoregulatory platform as an artificial antigen presenting cell.
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Chapter 2 Material and method
2.1 Material
2.1.1 Reagent
The following reagents and chemicals were obtained as indicated:
RPMI 1640, Fetal Bovine Serum (FBS), and BSA from Invitrogen.
Penicillin/ streptomycin/ amphotericin (PSA) from Biological industries.
NaCl, Tris-HCl, Triton X-100, from Amresco. Ficoll-PaqueTM Plus from GE healthcare. Propidium iodide (PI) from CE. EDTA and chloroform from TEDIA. NaOH, H3PO4, KH2PO4, Na2HPO4, tween 20, KHCO3, NaN3, and KAc from SHOWA. KCl from Scharlau. Na2HPO4 from J. T.
Baker. Urea from USP.
2.1.2 Cell lines
P338D1 (Mouse macrophage-like cell line; ATCC number:
CCL-46.) 2.1.3 Antibody
The following antibodies were obtained as indicated: Mouse anti-human CD3 (OKT3) and Mouse anti-mouse CD3 (2C11) were kindly provided from Dr. Steve R. Roffler (ACADEMIA SINICA, Institute of BioMedical Sciences). Anti-human CD28 monoclonal antibody, and anti-mouse CD28 monoclonal antibody were purchased from Biolegent.
2.1.4 Kits
The following kits were obtained as indicated: Human IL-1β, IL-6,
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IL-8, IL-2, IFN- γ
and TNF-
α ELISA kit, and mouse IL-4, IL-10, IL-2, IFN- γ, and TNF-
α ELISA kit from R&D. MTT assay kit from Promega.2.1.5 Animals
Six-eight weeks old female BALB/C mice were purchased from the National Laboratory Animal Center, Taipei, Taiwan, R.O.C. Six-eight weeks old female C57BL/6-Tg (HLA-A2.1) mice were kindly provided from Dr. Shih-Jen Liu (National Health Research Institutes).
2.1.6 Others
Peptide-HLA-A2 monomer and one identify epitope of HPV type 16 E7 protein (YMLDLQPETT) were kindly from Dr. Shih-Jen Liu (National Health Research Institutes).
2.2 Method
2.2.1 LPPC preparation
Briefly, added each DOPC and DLPC 500µl (50 mg/ml) into the round bottom flask, and then added 1000µl methanol into the same container and mix well. The mixture was placed the container of lipid mixture to the rotary evaporator (37℃, without vacuum treatment, minimum rotary speed) until dry (about 2 days). Hydrated the lipid film by steam (about 37℃) for 2~3 hours. Added 5ml aqueous medium (containing 0.675g PEI and 0.22g PEG in 5 ml filtered DDW) gently to the container of dry lipid and agitating gently. The container was vortexed violently for 10 minutes. After vortexed, the LPPC was placed at RT overnight. The turbid medium of LPPC extruded through 200nm mesh
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nine times. The product stored into the container to 4℃ refrigerator.
2.2.2 Adsorption characters of LPPC Timing
Added 50 µg BSA-FITC into 40 µg/ml LPPC solution, and then centrifuged at 10,000 rpm for 5 min at different time. The fluorescence of LPPC pellet was estimated by Spectrofluorometer.
Capacity
Added different amounts of BSA into 40 µg/ml LPPC solution, and then centrifuged at 10,000 rpm for 5 min at 20 minutes. The amounts of BSA onto LPPC were measured by using coomassie plus reagent.
Competition
40 µg/ml LPPC prior to adsorb 50 µg BSA-FITC completely, and added different folds of BSA for competition in 20 minutes. And then centrifuged at 10,000 rpm for 5 min, the fluorescence of LPPC pellet was estimated by Spectrofluorometer. Positive control was the fluorescence of the LPPC solution without adding BSA. Negative control was the fluorescence of the LPPC alone solution.
2.2.3 PBMC isolation
Peripheral blood mononuclear cells (PBMCs) were separated from human white blood cell solution by using Ficoll-PaqueTM Plus. Dilute human white blood cells with equal volume of PBS. Add Ficoll-Paque PLUS (6 ml) to the 15 ml centrifuge tube and carefully layer the diluted blood sample (8 ml) on Ficoll-Paque PLUS. Centrifuge the tubes at 400g
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for 40min at 18℃. Remove the plasma layer and collect the PBMC layer.
Wash the cells with 2 volume of PBS for centrifuging at 1500 rpm for 15 min. Discard the supernatant and lyse the red blood cells by ACK buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA in DDW) at room temperature for 10min and followed by centrifuging at 1500 rpm for 15 min. Discard the supernatant and wash the cell with 10 ml PBS.
Centrifuge for another 15 min. Discard the supernatant and count the cell number. For the cell proliferation of PBMC, cells were plated in a 96-well at 1 x 105 per well. For the cytokine profiles of PBMC, cells were plated in a 24-well plate at 4 x 105 per well.
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
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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
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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
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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
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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
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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
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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
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