To summarize, we have established a novel platform for immunoregulation which utilized the LPPC to combine with certain immunostimulatory molecules, such as mAbs and MHC-loaded peptides molecules, and in this study the LPPC display its potential to be an artificial APC. First, the LPPC is easily adsorbing a variety of immunostimulatory proteins and the LPPC-bound proteins can remain their activities. Second, the LPPC have an adjuvant effects to enhance proinflammatory cytokine expression, antigen uptake and presentation of APC. Furthermore, our study provided the evidence that the regulatory molecules/LPPC complexes dramatically increase the specific anti-antigen activities of immune responses in vivo. As understanding the mechanism about immune responses, it is proposed that certain immunostimulatory molecules could be applied in this platform to regulate immunity for disease therapy. For example, the combination of microbeads with HLA-A2 monomers, anti-4-1BB antibodies and anti-CD28 antibodies can expand large numbers of antigen specific CTLs (33). Moreover, the addition of anti-LFA-1 mAbs with anti-CD3, anti-CD28 could enhance the T-cell expansion for adoptive cell therapy (34). Therefore, certain ligands or costimulatory molecules might be utilized for adsorption of LPPC to regulate immunity as artificial APCs.
The major defect for DC-based therapy was that the maturation of DCs was weaken by certain immunosuppressive substances such as TGF-β, IL-10, or VEGF which derived from tumor cells (11, 25-27).
32
Moreover, Candida albicans, Mycobacterium tuberculosis, the secretions of mycobacterial, and the secretions of Candida respectively impact on efficiency of DCs and immune cells, affect cytokine expression and impair surface marker of DC (19-22). Many Comparing to our strategy, previous DC-based therapy strategies were DCs incubated with antigens such as tumor antigen HSP105, HPV E7 protein, breast tumor cell lysate, HIV, or SIV to successfully induce host immunity against tumor or pathogens (15, 16, 35-37). We modified these previous strategies in this study, the membrane proteins of DCs which were prior treated and up-take antigens (HpHsp60) were isolated and were adsorbed on LPPC, later the membrane antigen/LPPC complexes would be used as antigen to immunize animals. The results revealed the LPPC adsorbed membrane proteins could induce specific immune responses efficiently (Figure 13).
The modified strategy excludes the usage of alive DC to prevent the above problem.
Moreover, immuno-molecules such MHC molecules are covalently coupled to beads or liposomes and used to expand CTL ex vivo (38, 39).
However, such chemical modification might impair the original functions or activities of immunoregulatory or targeting molecules (40). In contrast, mAb non-covalently adsorbed on the nanoparticle surface could provide a better activity than covalent coupling (41). In this study, the LPPC provided an ability to strongly adsorb proteins on its surface, which was proposed that the activities of the bound immunoregulatory molecules could maintain higher activities without chemical damage. Moreover, figure 2c and figure 8 indicated that the pre-adsorbed proteins on the
33
surface of LPPC may be irreplaceable in serum without the dissociated doubt.
On the other hand, the results in this study indicated that the LPPC had the interesting characters, the effects of adjuvant. Figure 9 showed LPPC promoted antigen uptake ability of APC in a dose-dependent fashion. In addition, BSA adsorbed on the LPPC provided a better presentation of APC than free BSA to induce T-cell proliferations and cytokine secretions (Figure 10). Moreover, LPPC showed it could directly induced PBMCs or splenocytes to secret proinflammatory cytokines, IL-1β, IL-6, IL-8 or TNF-α. It is well-known that the function of the proinflammatory cytokines could recruit or activate macrophage or other immune cells (42). Similarly, the adjuvant MPL reagent, a liposome-like reagent could also improve the capacity of APCs by enhancements of antigen uptake and cytokine release (43). Furthermore, their primary adjuvant mechanism of cationic liposomes is to target to the membrane of APCs and to induce the uptake and increased presentation of antigen (44).
Together these results indicated that the LPPC can be a good adjuvant.
In vivo experiments, the figure 13 and figure 14 showed that the proteins/ LPPC complexes induced a better specific immune response than free proteins of immunization. We proposed that the LPPC might provide a little positive charge to attract immune cells, and the higher density of the immunoregulatory molecules on the LPPC than free form of molecules might also provide good affinity. Moreover, the LPPC reagent had an adjuvant effect. Therefore, the LPPC should be a good
34
immune platform of enhancement of immune response.
Many investigators have started to develop artificial APCs and Dr.
Mathias Oelke classified artificial APCs according to cell-based or non-cell-based in current approaches for immunotherapy (1). Early, murine MHC class I molecules with the costimulatory molecules B7-1 and ICAM-1 were co-expressed on drosophila cell to activate T cells, which resulted in specific killing for tumor cells in vitro and in vivo (45).
However, the transfected drosophila cells were unstable in mouse body.
Another cell-based approach was also used. The NIH/3T3 murine fibroblast cells were transduced with the costimulatory molecule B7-1, ICAM-1 and LFA-3, and a single HLA–peptide complex (HLA-A2) as APCs. These aAPCs have proved that they can induce specific CTLs to against tumor cells when the aAPC were pulsed with tumor antigens MART-1 or hTERT (29, 46). In acellular systems, magnetic beads were usually used to assemble as aAPCs, (31, 47, 48). In addition, latex microspheres (49) or liposomes (32) were also developed as aAPC.
Generally, the immunoregulatory molecules on these aAPC are MHC molecules or immunoregulatory antibodies. For instance, HLA-A2–Ig molecules, biotinylated murine MHC class I or MHC class II-peptide molecules have been combined with costimulatory molecules such as anti-CD28 antibodies or B7 molecules, which were coupled on particle surface to induce and expand antigen-specific T cells (31, 32, 49).
In this study, the LPPC adsorbing with anti-CD3 and anti-CD28 mAbs or peptide–loaded HLA-A2 provided immunoregulatory function
35
to stimulate T cells, similar to other acellular-based APCs. But the difference between LPPC and other aAPC is that LPPCs do not need chemical modification and complicated purification. In addition, LPPC can also adsorb membrane proteins to trigger the specific T cells responses (Figure 11 and 13). As our knowledge, no other aAPC coupled with DC’s membrane proteins to induce specific immunity. Moreover, figure 11 and 12 showed that neither DC’s membrane proteins nor peptide-loaded HLA-A2 could induce stronger immune responses than these antigens alone except these molecules combined with LPPC. Figure 13 also revealed that LPPC/ DC’s membrane proteins complex could enhance antigen-induced Th-1 cytokine releases but its abilities to induce Th-2 cytokine releases were as same as antigen alone immunization.
Therefore, LPPC should be developed as a convenient and efficient immunoregulatory platform to mediate host immunity.
Interestingly, the results showed that LPPC alone could stimulate immune cells to release pro-inflammatory cytokines (Figure 5 and 6). Its mechanism may result from PEI, one component of LPPC, has been proved it was an adjuvant effects to enhance the expressions of both Th1 and Th2 cytokines (50). However, LPPC alone could only induce the proinflammatory cytokine releases including TNF-α (Figure 5), IL-1β, IL-6 and IL-8 (Figure 6) but it had no effects on the releases of Th-1 and Th-2 cytokines including IL-2 (Figure 5a and 5d) , IFN-γ (Figure 5b and 5e), IL-4 (Figure 10) and IL-10 (Figure 10). Because the proportion of PEI in LPPC is 3%, only 1.62 µg PEI would interact with immune cells in our study. In contrast, more than 33.75 µg PEI was used in reaction
36
(50), the difference between the dosages may result in different immunoregulation (51, 52). Except for the adjuvant effect, LPPC preferentially enhance Th-1 responses. Th-1 responses such as Th1-CD4+T cell or CTL have been demonstrated that they are important and necessary against malignant tumors or intracellular pathogens. Thus, the scientists attempted to design the strategies of vaccination by enhancement of Th-1 immunity (53-55). According to our results, LPPC seem to be a good adjuvant which can be developed as the vaccines against tumor or intracellular pathogens.
In conclusion, we demonstrated that LPPC could show its flexible and convenient characters as an artificial APC that stimulate immune responses and induce specific immunity in vitro and in vivo. The immuno-LPPC has immuno-function by adsorbing certain immunoregulatory molecules. Except activation of immunity, the LPPC may also combine with immunosuppressive molecules or apoptosis ligand, such as CTLA-4 and FasL, to provide different function to suppress abnormal immunity (11, 56). In the future, the immuno-LPPC may have broad functional immuno-regulatory abilities to treat human diseases by mediating host immunity.
37
Figure 1
(a)
(b)
(c)
Before adsorbing After adsorbing
0 20 40 60 80 100
0 100 200 300 400 500
particle size (nm)
relative percentage (%)
LPPC alone LPPC-BSA complex
38
Figure 1. The characters of LPPC. (a) The TEM photo of LPPC; (b) The particle size distribution of LPPC with proteins or without proteins were estimated by utilizing DLS. The average particle size of LPPC
without BSA proteins was about 216 ± 4 nm, and the average particle size of LPPC with BSA proteins was about 358 ± 16 nm; (c) The centrifuged property of LPPC with or without proteins.
39
Figure 2
(a)
Timing Course
NC 5 10 15 20 25 30
0 50 100 150
Time (min)
Adsorbing Persentage(%)
(b)
Capacity
0 40 80 120
160 200
240 0
50 100 150 200
addition of BSA (µµµµg)
detection of BSA (µµµµg)
40
Competition
0.0 0.5 1.0 1.5
Fluorescence Index
(c)
BSA-FITC - + + + + + BSA (µg) - - 100 200 300 400
Figure 2. The properties of LPPC adsorption. (a) The time consumption that LPPC adsorbed BSA-FITC by utilizing
spectrofluorometer, and twenty minutes was determined for completely adsorption. (b) To understand the quantity of BSA could be adsorbed by LPPC, added different amount of proteins into LPPC solution. The result show that 160 µg BSA was probably the maximal capacity of 40 µg LPPC. (c) Added a great quantity of BSA for competition 50µg
BSA-FITC previously adsorbed on 40 µg LPPC, and the result showed that fluorescence index was no significant difference even added 400µg BSA. Results are representative at least two independent experiment.
41
Table 1
Proteins Assay method
Bovine serum albumin (BSA) Coomassie Plus Reagent FITC-conjugated BSA Spectrofluorometer Beta-galactosidase Enzyme activity (PNPG) HRP-conjugated Antibody Enzyme activity (TMB
solution)
Hp Hsp60 OD 280
Urease B OD 280
Table 1. The various proteins were adsorbed by LPPC. Added different kinds of proteins into LPPC solution, and centrifuged to
estimate various proteins whether adsorbed on LPPC or not. The results were cited from Yen-Ku Liu (NCTU).
42 0 0.1
0.5 1 5 10 30 50
0 50 100 150
LPPC (µµµµg)
Survival rate (%)
0 0.5 1 3 5 10 30 50
0 50 100 150
LPPC (µµµµg)
Survival rate (%)
Figure 3
(a) PBMC
(b) Splenocyte
Figure 3. The cytotoxicity of LPPC for PBMC and Splenocyte .
Different amounts of LPPC were respectively added into growth medium containing 105 PBMC (a) or 2.5 ×105 splenocytes (b), and incubated in 96-well plate at 72 h. Utilized MTT assay to determine what dosage of LPPC treated immune cells without influenced cells survival. The results showed that 1 µg LPPC was an appropriate amount for PBMC and
splenocytes next experiment. Results are representative at least three independent experiment.
43 0
200 400 600 800
anti-CD3 anti-CD3/28
cell proliferation rate (%)
0 100 200 300 400 500
anti-CD3 anti-CD3/28
cell proliferation rate (%)
Figure 4
(a) PBMC
**
Anti-CD3 0 60 90 120 150 (ng) Anti-CD3/28 0/0 60/60 90/90 120/120 150/150 (ng)
(b) Splenocyte
*
Anti-CD3 0 60 90 120 150 (ng) Anti-CD3/28 0/0 60/60 90/90 120/120 150/150 (ng)
44
Figure 4. The dose-dependent effect of cell proliferations was
stimulated by immunostimulatory monoclonal antibodies on LPPC.
For comprehending whether LPPC adsorbed monoclonal antibodies could stimulate immune cells proliferation. Addition different amounts of
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. The cell proliferation rate was calculated as O.D. value of sample divide into O.D. value of PBMC alone or
splenocyte alone. As the result showed, more amounts of anti-CD3 monoclonal antibody were added, the cell numbers of PBMC (a) and splenocytes (b) were larger, especially combined with anti-CD28
monoclonal antibody. Results are representative at least three independent experiment. (*︰P<0.05; **︰P<0.01)
45
46
47
Figure 5. The dose-dependent fashion of cytokine profiles secretion was activated by monoclonal antibodies on LPPC. Combined different amounts of immunostimulatory monoclonal antibodies with 4 µg LPPC to treat PBMC or splenocytes, and the cytokines in culture supernatants were measured by ELISA. The results indicated that LPPC adsorbed immunostimulatory monoclonal antibodies could stimulate immune cells’
secretion of cytokines, such as IL-2, IFN-γ and TNF-α. And the more dosage of antibodies was added, the higher the concentration of cytokines was monitored. Besides, LPPC alone could activate PBMCs and
splenocytes TNF-α cytokine secretion. Results are representative at least three independent experiment.
48
Figure 6. The induction of pro-inflammatory cytokine profiles was by the LPPC. Addition of 4 µg LPPC treated 4 ×105 PBMC/well in 24-well plate, and the cytokines in culture supernatants were collected to measure by ELISA. The results indicated that the LPPC could stimulate immune cells’ secretion of cytokines, such as IL-1β, IL-6 and IL-8. Results are representative at least three independent experiment. (**︰P<0.01; ***︰
P<0.001)
49
Table 2. The induction of cytokine profiles was by the LPPC. The LPPC could induce the pro-inflammatory cytokine secretions (TNF-α, IL-1β, IL-6, or IL-8), but not Th1 and Th2 related cytokine expressions.
(**︰P<0.01; ***︰P<0.001)
50
51
52
Figure 7. The activities of immunostimulatory monoclonal antibodies on LPPC. LPPC might adsorb the immunostimulatory monoclonal
antibodies by electric force and non-covalent force. To compare the activities of monoclonal antibodies with LPPC complexes or without LPPC, the cell proliferations and cytokine secretions were estimated. The results indicated that the activities of monoclonal antibodies were no significant difference between them, such as cell proliferation, IL-2 and IFN-γ secretion. But, LPPC with monoclonal antibodies could enhance TNF-α secretion more than mAbs alone. Results are representative at least three independent experiment. (anti-hCD3 60ng/96well ;
anti-hCD28 60ng/96well) (anti-mCD3 60ng/96well ; anti-mCD28 60ng/96well) (anti-hCD3 0.6µg/24well ; anti-hCD28 0.6µg/24well) (anti-mCD3 0.6µg/24well ; anti-mCD28 0.6µg/24well)
53
Figure 8 (a) PBMC
LPPC + + + + + + + + + Anti-CD3 - + + - + + - + + Anti-CD28 - - + - - + - - +
(b) Splenocyte
LPPC + + + + + + + + + Anti-CD3 - + + - + + - + + Anti-CD28 - - + - - + - - +
Original Pellet Supernatant
Original Pellet Supernatant
0 100 200 300
cell proliferation rate (%)
0 100 200 300 400
cell proliferation rate (%)
54
Figure 8. The stability of immunostimulatory monoclonal antibodies on LPPC in RPMI. LPPC previously adsorbed monoclonal antibodies, and then put it 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 and the supernatant respectively treated PBMCs or splenocytes, and estimated the cell proliferation for
investigating the stability of monoclonal antibodies on LPPC. 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. The results showed that approximately 90% activities of
monoclonal antibodies in LPPC pellet, in contrast, the cell proliferation of the supernatant groups were no significant different comparing to the control group. Results are representative at least three independent experiment. (anti-hCD3 60ng ; anti-hCD28 60ng) (anti-mCD3 60ng ; anti-mCD28 60ng)
55
Figure 9
(a)
(b)
(c)
BSA-FITC - + + + + + LPPC (
µg
) - - 20 100 200 300100 101 102 103 104
FL1-H
100 101 102 103 104
FL1-H
Control BSA-FITC
LPPC+BSA-FITC
Control 1X LPPC 15X LPPC
0 20 40 60 80 100
Fluorescence cell rate(%)
56
Figure 9. The increase of cellular uptake of P338D1 was by the LPPC.
To investigate that LPPC might have ability to enhance cellular uptake of antigen presenting cells. LPPC previously coated BSA-FITC
co-incubated 5 ×105 murine macrophages, P338D1, and then added trypan blue to quench the fluorescence of LPPC complexes without up-taken. (a) The result showed that added the same amounts of
BSA-FITC, LPPC could enhance the uptake ability of P338D1. (b) The result indicated that the fluorescence expression of P338D1 from addition fifteen fold amounts of LPPC was higher than that addition one fold amount of LPPC. (c) The fluorescence cell rate of P338D1 was estimated by flow cytometer, and the data showed that LPPC could enhance uptake ability of P338D1 in a dose-dependent manner. Results are representative at least two independent experiment.
57
58
Figure 10. The enhancement of antigen presentation of APC was by the LPPC. To further understand whether the other adjuvant effect of the LPPC has, such as the enhancement of antigen presentation of APC, using BSA proteins were mimic as one disease antigen, and then Balb/c mice were prior immunized by s.c. injection with BSA proteins to get anti-BSA T cells. The results showed that as compared with addition of the same amount of BSA, LPPC/ BSA complexes co-cultured with splenocyte that had been immunized by BSA could efficiently activate cell proliferation and induce cytokine secretions. Results are
representative at least three independent experiment. (*︰P<0.05; **︰
P<0.01)
59
Figure 11
(a) CD11c+ (b) MHC II
(c) CD86
Figure 11. The surface marker expressions of dendritic cells. (a) The average CD11c+ expression of cells were about 80%, and it indicated that the population of the cells were 80% DCs; (b) The MHC II expression of the cells(c) The costimulatory molecules CD86expression. Figure 11b and 11c indicated that high level expression of DCs’ surface markers which treated with HpHsp60 and LPS were actually mature. Black line was that DCs were harvested with HpHsp60 and LPS, and green line was that DCs were harvested without treatment, and Red line was negative control.
60
61
Figure 12. The specificties of DCs’ membrane proteins coated LPPC.
HpHsp60 was as one disease antigen, and Balb/c mice were immunized by s.c. injection with HpHsp60 proteins. And DCs’ membrane proteins contained HpHsp60 antigens were isolated for LPPC adsorption. The results showed that LPPC adsorbed membrane proteins with HSP60 antigens could stimulate splenocytes which prior to HSP60 immunized cell proliferation and cytokines secretion compared to LPPC with membrane proteins without HSP60 antigens group. Results are representative at least three independent experiment. (*︰P<0.05; **︰
P<0.01)
62
Figure 13. The specificities of antigen-loaded HLA-A2combined with anti-CD28 monoclonal antibody on LPPC. YML was one identify epitope of HPV E7 protein, and immunized transgenic mice previously.
Utilized specific antigen-loaded HLA-A2on LPPC, and co-cultured with splenocytes from previously immunized transgenic mice. The results showed that LPPC could remain the specific activities of antigen-loaded HLA-A2to stimulate cell proliferation and cytokines secretion. Moreover, added anti-CD28 monoclonal antibody as the second signal of the optimal T cell activation, the immune responses could be better than that only provided for one signal, antigen-loaded HLA-A2 molecules. Results are representative at least three independent experiment. (*︰P<0.05)
Splenocyte
63
64
Figure 14. The animal immunization of the bound membrane
proteins derived from DCs on LPPC. HpHsp60 was as one antigen and incubated with DCs for uptake and presentation. In addition, DCs’
membrane proteins contained HpHsp60 antigens were isolated for LPPC adsorption. Balb/c mice were immunized by i.v. injection with the
membrane proteins /LPPC complex. After two weeks, the mice were sacrificed and the splenocytes were isolated to incubate with 2 µg/ml HpHSP60. The results showed that splenocytes from mice that i.v.
injection with the membrane proteins /LPPC complex could activate immune responses efficiently comparing to the other groups. (*︰P<0.05
when compared to HpHsp60 group, and **︰P<0.01; #︰P<0.01 when compared to HpHsp60 mp group) Results are two independent
experiments.
65
66
Figure 15. The animal immunization of the LPPC combined HLA-A2 molecules with anti-CD28 mAb. Utilized YML peptide-loaded HLA-A2 and anti-CD28 mAb bound to LPPC, and then transgenic mice were immunized by i.v. injection with the the immuno-LPPC complex. After two weeks, the mice were sacrificed and the splenocytes were isolated to incubate with 2 µg/ml YML peptides. The results showed that splenocytes from mice that i.v. injection with the immuno-LPPC complex could
activate immune responses efficiently comparing to the other groups, such as cell proliferations and Th-1 cytokine secretions, but not Th-2 cytokine. (*︰P<0.05 when compared to HLA and anti-CD28 mAb group, and **︰P<0.01; #︰P<0.05 when compared to LPPC with HLA group and
activate immune responses efficiently comparing to the other groups, such as cell proliferations and Th-1 cytokine secretions, but not Th-2 cytokine. (*︰P<0.05 when compared to HLA and anti-CD28 mAb group, and **︰P<0.01; #︰P<0.05 when compared to LPPC with HLA group and