| Immunotolerance, the key mechanism to keep self antigen non-response, is very important to build and sustain the immune homeostasis and prevent the development of auto-immune diseases. However, during the process of cell malignancy and chronic infections, tumor antigens (over-expressed self antigen) and pathogenic antigens could somehow induce immune tolerance to them leading to the generation of tumor and chronic infectious disease.Tumor antigens, most of them are self-antigens, are inappropriately expressed or over-expressed when tumor occurs. Specific anti-tumor immune response could not be induced for self-antigen tolerance, consequently failed to tumor regression. In infectious and immunity, one of the pivotal reasons for Hepatitis B Virus (HBV) to cause Chronic Hepatitis B is the adaptive HBsAg-specific immune tolerance induced by HBV infection. Up to now there remains no effective strategy to prevent or cure tumor or viral chronic infection. On the other hand, in case of Crohn' disease and colitis, an immunopathologic intestinal inflammation caused by over-secretion of IL-12, is another evidence of self-antigen over expression leading to disease. The over-expression of self-IL-12 plays an important role in the pathogenesis. Administration of anti self-antigen Abs could relieve the inflammation of the intestine, however, problems such as antigenicity of the exogeneous Abs, side-effects; short half-life of the Abs and the high costs prevents its general application.Therefore, to eliminate those over-expressed selfantigens, tumor antigens and exogenous pathogenic antigens becomes more important to maintain homeostasis. It has been generally believed that break the immune tolerance so as to induce specific immune responses against the selfantigens, tumor antigens and the pathogenic antigens is the essential way. So the problem is raised to: how to induce or recall the immune stimulation to the naturally tolerated selfantigens, tumor antigens or adaptively tolerated pathogenic antigens? It has been well known that the antigen processing and presentation by the Antigen Presenting Cells (APCs) plays a key role in the immune priming. The numbers and maturation state of DCs infiltrated in the antigen-driven immune site, the expression of MHC molecules, co-stimulatory molecules to a great extent determines the deviation of immune activity or immune tolerance.Hugues and his colleagues observed that when the peptide-pulsed immature DCs were vaccination to mice, short-term contacts betweenT cells and DC occurred in the draining lymph nodes (DLN), resulted in immune tolerance. However, when the peptide-pulsed mature DCs were given to mice, the T-DC cell contacts persisted for a longer time, ultimately evoked immunity priming. In addition, Shortman also proposed that the process of DC migration was accompanied by its maturation. Antigen presentation by immature DCs induced immune tolerance, while that by mature DC could elicit specific immunity. The above studies indicated that proper modulation of the antigen processing and antigen presentation ability by DCs, might be a promising way to break the specific immune tolerance, recall the specific immune responses against antigens so as to clear the toleragens and lessen the relevant inflammatory diseases caused by the antigens.Therefore, we hypothesized that if DCs could be enriched by artificial means in the antigen-driven immunized sites and if meanwhile their maturation process could be accelarated, then the up-regulated DCs function (antigen processing and antigen presentation) could be speculated to convert tolerance to immunity leading to the immune response towards to the target antigens. This might be a prospective strategy to prevent or cure self-antigen driven diseases, tumors and chronic infective diseases.To chemoattract DC to the antigen-driven immune sites, chemokines are good candidate. Chemokines are groups of characteristic molecules which possess the function of chemoattracting immune cells to the infection sites or immunization sites and activating those immune cells. Therefore we hope to enrich DCs to the immunization sites and augment their antigen presenting function through utilizing chemokines or cytokines.We selected chemokine CCL20 and cytokine flt3L to fix our hypothesis. CCL20, a CC subtype chemokine, mainly attracts immature DCs, activated B cells and T cells. It was established that CCL20 could recruit large quantity of immature DCs to exert an anti-tumor function. F(?)t3L could impetus the growth of hematopoietic stem cells in bone marrow and up-regulate the differentiation and maturation of these precursor cells. Maraskovsky and his colleagues also demonstrated that flt3L could markedly promote DCs proliferation and maturation.Therefore, to address our hypothesis, first, we used IL-12 p40 subunit antigen as our model selfantigens to study combination efficacy of the CCL20 and flt3L encoding genes to break the natural tolerance of murine IL-12 in a mouse inflammatory bowel disease model. The model self-antigen, IL-12p40 encoding plasmid DNA was delivered with the combination of CCL20 and flt3L plasmid DNA. Following a tri-molecule gene co-immunization, a large amount of recruited immature DCs was detected at the immunization site. With the help of flt3L and CCL20 molecules, although the same amounts of mature DCs resided in the spleen, more mature DCs were found in the DLN during DCs migration in which they interacted with T cells and helped T cells to break tolerance to self-antigen. And the increased anti-IL-12p40 specific immune responses were also detected to further confirm the effect of IL-12p40-CCL20-flt3L gene co-immunization.To confirm this tri-molecule gene co-immunization strategy works as a general way, we selected other two toleragens simultaneously, one self-antigen is mgp100 and the other is HBsAg. The similar tri-molecule gene immune strategies were performed in a B16F10 mice tumor model and in an HBV transgenic mice model to observe whether these two kinds of specific immune tolerance could be also broken. The tri-molecule gene co-immunization strategy created in this study would greatly benefit the theoretical basis of break the immune tolerance and meanwhile provide a promising approach to prevent and treat autoimmune diseases, tumor and chronic infective diseases.1. Construction, expression and functions of plasmids encoded with indicated target genes.Three eukaryotic expression plasmids, pcDNA3-CCL20 (pCCL20), pcDNA3-flt3L (pf3L), pcDNA3-IL-12p40 (p40), were constructed. And the combination plasmids pcDNA3-CCL20-flt3L (pCCL20-f3L) plasmid and pcDNA3-CCL20- IL-12p40-flt3L (pCCL20-40-f3L) fusion plasmid were also constructed. These plasmid DNAs were then electroporation to C2C12 muscular cells and the expression of CCL20, flt3L and IL-12p40 proteins in C2C12 cell culture supernatant were measured after 72 hours. Western blot was applied to analyze protein levels.Three visable bands representing the three proteins derived from the condensed culture supernatants were identified, indicating the successful expression of the three proteins in vitro. Biological function analysis showed that CCL20 attracted much more splenocytes with the chemotactic index as high as 9.18±0.78 compared to 0.84±0.40 of the control (p=0.001). In vivo function of CCL20 was reflected by the amounts of attracted DCs at the injection sites. The infiltrating index was as high as 1.67±0.33 for the tri-molecule gene co-immunization group which was significantly higher than that of control group (0.33±0.21) (p=0.007). And at the same time, CCL20 enhanced both the production of IL-12p40-specific antibody and the cellular immune response.The function analysis of flt3L showed that it could markedly upregulate the expression of co-stimulatory molecules of cultured DCs in vitro. Meanwhile, the lL-12p70 level in the culture supernatants of DCs was elevated as 660.9±25.44pg/ml compared to the control (110.4±11.03pg/ml) (p<0.01), suggesting that flt3L could promote the maturation and function of DCs.All results above suggested that CCL20, flt3L and IL-12p40 proteins could be effectively expressed in vitro and in vivo by the recombinant plasmid DNA we constructed. The expressed proteins maintained their biological functions in vitro.2. Co-immunization with the plasmids coding for CCL20/flt3L/p40 could abrogate the immunotolerance versus IL-12.To investigate if immune tolerance to autoantigen IL-12p40 could be broken by a tri-molecule gene co-immunization strategy, 6～8 weeks old female BALB/C mice were intramuscularly vaccinated with mixed DNAs consisting of pCCL20, pflt3L and pIL-12p40 or tri-molecule fusion DNA (pCCL20-40-f3L) and then boosted for one time with the same dose. The IL-12p40 specific serum antibody could be detected one week after the boost immunization both for the mixed or the fusion gene immunization groups, the IgG titer achieved 1: 600, and could not be improved even after one more boosting. In order to increase the antibody induction, the w/w/w combination ratio was optimized as 4:4:100 (pCCL20: pflt3L: pIL-12p40). This opitimized scheme resulted in the peak of the anti-p40 IgG titer as high as 1:8000 and the increased infiltrating immune cell numbers in the local immunization sites. If the concentrations of pCCL20 and pflt3L were further elevated, the immune cells infiltration was no more (p>0.05) than control group while the IgG level decreased yet (p<0.05). Many other w/w/w ratios were chosen, but the efficiency could not be promoted. Therefore, the w/w/w combination ratio for pCCL20:pflt3L:p40 DNA was fixed as 4:4:100 during all the following expeiments.The above results implied that the genetic co-immunization of pCCL20, pflt3L and pIL-12p40 may break the immune tolerance to the autoantigen IL-12p40. To further confirm this phenomenon, the titer, specificity and affinity of anti-p40 antibody were carefully studied. A 0wk-3wk-6wk immunization strategy was performed based on the previous data. The serum antibody was determined by ELISA at 2, 4, 6, 8, 10 and 12 week after the primary immunization. It was shown that the anti-p40 IgG could not be detected in many doses when pIL-12p40 was used alone. If the pIL-12p40 DNA was delivered together with pCCL20 or pflt3L DNA separately, yet no specific IgG was induced. Only in the tri-molecule gene co-immunization group using pIL-12p40, pCCL20 and pflt3L together, the specific IgG was detected and the titer at wk8 reached 1:3600. Moreover, the affinity detected by serial diluted NaSCN competitive inhibition at wk4 and wk8 was 1.3 and 1.5, respectively. And the anti-p40 IgG could be blocked by the addition of IL-12p40 protein since the OD value decreased from 0.97±0.14 to 0.149±0.03 (p=0.005) indicating that the induced serum IgG was IL-12p40 specific.Apart from the specific humoral response against self-antigen IL-12p40, the cellular immune response was also analyzed after tri-molecule (pCCL20, pflt3L and p40) gene co-immunization. It was shown that the lymphoproliferation stimulated with IL-12p40 (5μg/ml) could be inhibited by the addition of the serial diluted anti-sera derived from the tri-molecule co-immunized mice indicating the proliferation was p40-specific. FACS analysis showed that the percentage and amounts of CD8+ IFN-γ+ T cells for the tri-molecule co-immunization group were increased compared to other groups (p=0.000). And there are no significant difference among the other four groups (p>0.05). The percentage in pcDNA3, pIL-12p40, pIL-12p40/pCCL20, pIL-12p40/pflt3L and pIL-12p40/pCCL20/pflt3L group was 0.25±0.04%, 0.23±0.01%, 0.27±0.05%, 0.27±0.06% and 1.64±0.09%, respectively.The data demonstrated that tri-molecule (pCCL20-pflt3L-autoantigen p40) gene co-immunization could break the immune tolerance to p40 via the induction of anti-IL-12p40-specific humoral and cellular immune responses. 3. Co-immunization with the plasmids coding for CCL20/fIt3L/p40 could prevent and treat TNBS-induced inflammatory bowel disease.The inflammatory bowel disease induced by TNBS is a Thl type inflammation reaction. Over-expressed IL-12 is the key factor among the pathogenesis. As all known, IL-12 is a heterodimer comprised of p35 and p40 subunits, its biological function depending on the integrated structure. It has been reported that the colitis induced by TNBS was obviously attenuated in a p40-/- knockout mice. Therefore, production of anti-p40 antibody would probably have the same effect on colitis.The inflammatory bowel disease model induced by TNBS administration (two doses on day 0 and day 7, respectively) was established. For the 40% ethanol treated control group, the body weight of mice increased from 100% to 110% (103.30±0.75) during 9 days after drug use. While in TNBS treated group, the body weight significantly reduced on day 2 and day 9 due to diarrhea and dehydration, being only 84.5% and 87.2% of the basic weight (day 0). The body weight could not be restored to the basal level for a long period. Other parameters were also used to evaluate the bowel Inflammation. In TNBS treated group, the incidence of bowel inflammation was 100%, the macro-gross score was 11.63±0.53, MPO was 1.26±0.05 OD/g tissue, the colon/dry (w/w) ratio was 6.45±0.16, and the average food consumption was l～2g/day(1.53±0.14g/day). While in 40% ethanol treated mice, the disease incidence was only 6.25%, the macro-gross score was 0.88±0.40, MPO was 0.61±0.02 OD/g tissue, the colon/dry (w/w) ratio was 4.18±0.12 and food consumption was 4～6g/day(5.30±0.12g/day). Significant different colitis syndrome (P<0.001) was seen between the two groups.The gross morphology showed that in TNBS treated mice, no or little feces was seen in the colon while lots of feces could be observed in colon segments for the 40% ethanol treated group, The spleen of TNBS treated mice was much more enlarged than that of ethanol treated group. The histological observation showed that in 40% ethanol treated mice, the colon appeared normal, some microvilli extended into the intestinal space and only a light inflammatory infiltration was found. However, in the TNBS treated mice, the colon mucosal layer was segmentally destructed and local mucosa even completely disappeared. And there were many inflammatory cells infiltrating. Moreover, 5 weeks later, many irregular nodules could be seen on the surface of colon, and visable feces were not found in the colon. A number of cells infiltrated in the mucosal layer and in between the mucosal and muscular layers, exhibiting a chronic inflammation.First of all, the therapeutic effect of the tri-molecule gene co-immunization was evaluated. One week after the establishment of the TNBS-induced inflammatory bowel disease, pIL-12p40/pCCL20/pflt3L recombinant plasmids boost co-immunization was given three weeks later, at the 5th week, mice were sacrificed and the immunohistochemistry of the colons was done. In our tri-molecule gene co-immunization group, no obvious infiltrating cells were observed; no obvious destruction of the colon was shown and no obvious IC deposition in kidney could be seen; regular feces existed in most segments of the colons. While in the no-treating colitis mice, there appeared irregular nodules and edema on the colon surface, heavy mononuclear cells infiltration was observed and the local tissues were severely destroyed. Most colon segments had no feces in it. Thus, the therapeutic effect of the tri-molecule gene co-immunization was confirmed.Then, the prophylactic effect of the tri-molecule gene co-immunization was also analyzed. One week following the boost immunization, inflammatory bowel disease was induced by TNBS. Compared to the control group, the colitis incidence of the tri-molecule gene co-immunization group was reduced to 29.2±11.0% and its severity was largely lessened. The body weight was no less than 90% of the basic weight, and began increase on the second day following TNBS treatment, during the following 6～7days, their weights were restored. The food consumption was 2～5g/d(3.54±0.51) or so, MPO was 0.81±0.04 OD/g tissue and the macro-gross score was 2.00±0.87, the colon wet/dry (w/w)ratio was 5.21±0.20, all the characteristic disease markers were significantly reduced in the gene co-immunization (P<0.001) group. Morphological index such as spleen size and the colon gross morphology was not changed compared to control mice. Histological observation demonstrated that only light inflammatory infiltration was observed in the colon mucosal layer for some of the treated mice, while the gross structure of colon remained intact. These results suggested that the prophylactic efficiency of pIL-12p40, pCCL20 and pflt3L DNA co-immunization in a TNBS induced Thl type inflammatory bowel disease.4. The mechanisms involved in abrogation of immunotolerance versus IL-12p40 in IBD by co-immunization with the plasmids coding for CCL20/flt3L/p40.To further explore the mechanism underlying the immune tolerance breaking by the pIL-12p40, pCCL20 and pflt3L DNA co-immunization; BALB/c mice were i.m. immunized with the combined gene vaccines. The immune cell infiltration at the injection sites was first detected. There were only few cells infiltrating in the plL-12p40 immunized mice (Infiltrating index: 0.67±0.21). While there were more infiltrating cells in the pIL-12p40/pCCL20-treated mice (Infiltrating index: 1.67±0.33), and the largest amounts of infiltrating cells were observed in the mice immunized with the tri-molecule combined vaccine (Infiltrating Index: 2.67±0.21).Since CCL20 could only chemoattract CCR6+ cells to the immunization cite including T, B and DCs that expressed membrane CCR6, we tried to identify the local infiltrating cells by immunohistochemistry and FACS. The immunohistochemistry results showed most of muscle infiltrating cells were S100+CD19-CD3- cells, and FACS also showed that 81.26% of these population were CD11c+ CD19-CD3- cells. All of these two kinds of cells were DCs. When the mouse anti-CCR6 antibody was injected into the muscle together with the combined DNA (pCCL20/pflt3L/p40), the infiltrating cell numbers in the local immunization sites was significantly reduced from previous 3.00±0.00 to 1.50±0.34 (P=0.000), and moreover, the anti-p40 serum antibody could not be detected after the boost immunization. These results indicated that pCCL20/pflt3L/pIL-12p40 co-immunization could functionally chemoattract more CCR6+ immune cells to the injection cites, and most of these attracted cells are DCs. If this chemoattraction was blocked artificially, the immune cell infiltration was reduced significantly and the IL-12p40-specific immune tolerance could not be broken.It is believed that flt3L could stimulate the proliferation and maturation of bone marrow-derived cells such as immature DC which would help DCs Ag presenting function. So, whether or not the immune tolerance breaking was due to the alteration of DCs maturation state? To further elucidate this hypothesis, the number and maturation state of DCs in spleen and DLN were detected. It was shown that in the DLN, the amounts of DCs from the combined gene immunized mice were significantly increased (21.10±2.10×l03) compared to those from pcDNA3 (7.41±0.42×l03), pIL-12p40 (7.85±0.19×103), pIL-12p40/pCCL20 (9.05±0.23×103) and pIL-12p40/pflt3L (7.83±0.20×l03) immunized mice (p<0.001). There were no difference of DC amounts among the latter four groups (P>0.05). The co-stimulatory CD80/CD86/CD40 molecules and MHC class II molecules expression were upregulated in the combined gene immunization group compared to that in pcDNA3, pTL-12p40, pIL-12p40/pCCL20 or pIL-12p40/pflt3L immunized groups(P<0.001). As concerned with the spleen, there were no obvious differences of the infiltrated DCs numbers and DC maturation state among all the groups (p>0.05). These results suggested that more DCs infiltration in the injection cite and elevated matured DCs presence in the DLN might contribute to the autoantigen p40-specific immune tolerance breaking.In conclusion, as a natural immune tolerated autoantigen, IL-12p40 DNA administration could not induce a p40-specific immune response. However, this immune tolerance to p40 was successfully broken by a CCL20/flt3L and p40 gene co-immunization, resulting in the induction of specific immune response against IL-12p40 which served as both a prophylactic and a therapeutic strategy to treat the TNBS-induced IL-12 mediated Th1 inflammatory bowel disease. One possible mechanism might lie in that a great amount of DCs were chemoattracted to infiltrate into the local immunization site by the co-immunization with tri-plasmids, blocking this chemoattraction resulted in the block of DCs infiltration and the specific antibody production. The other mechanism might be that significantly more mature DCs were aggregated in the DLN by the means of co-injecting pCCL20/pflt3L/pIL-12p40 DNA which could stimulate more T cells.Our tri-molecule (pCCL20/pflt3L plus target gene) gene co-immunization strategy was demonstrated that effective to abrogate antigen-specific immune tolerance not only in the TNBS induced Th1 type inflammatory bowel disease model, but also in the natural HBsAg-tolerated HBV transgenic mice model and in an autoantigen mgp100 mice tumor model (As shown by the affiliated files). Above all, this is the first time we demonstrated that the immune tolerance could be abrogated through modulation of the amounts and the maturation of local infiltrating DCs. More DCs enrichment into the antigen injection site and accelerated DC maturation process during the DC movement from local injection site to the DLN would contribute greatly to the immunity. Our studies would provide a useful promising approach to abrogate immune tolerance so as to prevent or treat tumor or chronic infection caused by over-expression and/or inappropriate expression of tolerance antigens.
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