I would like to talk about the immune balance which plays a pivotal role in the immunoregulation of our bodies. It has not only led to my talk, but I would like to mention that disruption of the immune balance is a very big problem in the world, as well as climate change and emergency infectious disease problems, because disruption of the immune balance causes an increase of allergy in children, and children suffer very much with infectious disease.
In Japan, measles was once overcome, but recently group infectious diseases such as measles have been increasing concomitantly with the disruption of immune balance. This is because children cannot acquire Th1-dependent immune power until the early childhood days, because of their too-clean life and junk food, and stress through their lifestyle. This is a big problem in the world, especially in the developed countries. So we have to think about some strategy to control immune balance for improving the second disruption in the next generation.
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Recently a new paradigm for the regulation of immune balance has been proposed since the discovery of the IL-17-producing T cells. I show here that the Th1 cells, which produce interferon-gamma and IL-2, play a pivotal role in the cellular immune responses, and the Th2 cells, which produce IL-4 and IL-13, play a key role in the antibody production in humoral immunity.
It has been considered that most of the immune regulation mechanisms can be explained by the immune balance controlled by these Th1 and Th2 cells. But, recently, it has been demonstrated that Th17 and Treg cells are also involved in the control of immune balance. Treg cells are activated in the presence of TGF-β and Th17 is induced under the control of TGF- b and IL-6.
As you know, if we add TGF-β to the culture system, just one drop, both Th1 and Th2 responses can be stopped. But the difference occurred only when Th17 and Treg cells were activated.
These findings suggest to us that we have to re-examine the critical role of the immune balance regulated by Th1/Th2/Th17/Treg cells in anti-tumour immunity and infectious diseases.
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Today I will show new findings in terms of the negative immunoregulators at tumour local sites, which are regulated by TGF-β, IL-6, or the tumour-derived factors.
I will mention Treg, which was discovered by Dr Sakaguchi, and also talk about immature myeloid cells, and IL-17-producing γδT cells, infiltrating into the tumour local site.
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First, a negative immunoregulator is a Treg cell.
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I show here that with the increase of the tumour, the Tregs were activated only in the draining lymph node along the tumour site, so the immune response in the draining lymph node is very important for challenging the tumour, or suppressing anti-tumour immunity. Only the draining lymph nodes have Treg cells. Here you see that only the Foxp3 + cells, shown in green, were activated in draining lymph nodes of tumour.
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Carcinoma and sarcoma cells show different sensitivity against Treg-mediated immunosuppression. When Tregs infiltrate the tumour, the role is different in the carcinoma and sarcoma cells.
In the mouse tumour therapeutic system we always use sarcoma cells, because methylcholanthrene induces 100 per cent of the sarcoma cells, and we cannot get a carcinoma. But recently we found a method of inducing mouse carcinoma cells, with just one single injection of methylcholanthrene.
As you can see here, between the carcinoma and the sarcoma, more numbers of the Tregs were infiltrating in the carcinoma cells, compared with the sarcoma cells. And depletion of the Treg cells by injection of the αCD25 monoclonal antibody (depletion of the negative regulators) stimulated the anti-tumour effector cells so that in the case of the sarcoma cells, depletion of the Tregs resulted in the complete rejection of the tumour.
But in the case of the carcinoma, depletion of the Tregs had no effects on the tumour growth. This is because the carcinoma produced high amounts of the TGF-β, so that once the Tregs were depleted, soon the Tregs recovered at the tumour site because of their own produced TGF-β.
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This slide shows results of the injection of anti-CD25 mAb. Once depleted, the Tregs soon recovered. But combined treatment with anti-CD25 mAb for the depletion of Tregs and anti-TGF-β monoclonal antibody administration decreased the recovery of the Tregs and resulted in a complete rejection of the tumour.
Thus, the carcinoma produced high amounts of TGF-β essential for inducing Tregs, and was very refractory to the tumour immunotherapy. Usually, most mouse models use sarcoma cells, so the mouse model cannot be applicable to humans. So we should use the carcinoma model in the mouse tumour immunotherapy model.
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The next negative immunoregulator is the immature myeloid cells and myeloid-derived suppressor cells.
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Perhaps most people would recognise that at the late stage of the tumour-bearing stage, or at the late stage of infectious diseases, most mice had splenomegaly. This is because of the abnormal increase of CD11b + Gr-1 high-positive immature myeloid cells coming from the bone marrow cells.
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It has been reported that these immature myeloid cells are suppressor cells, but this is not true, because we isolated the immature myeloid cells from the spleen of the tumour-bearing mice and added them into a mixed lymphocyte culture, and there were no effects on the generation of CTLs. However, once these immature myeloid cells were cultured with a tumour-derived culture supernatants or with recombinant TGF-β and IL-6, they differentiated into phagocytic F4/80 + macrophages. If we added these cells into a mixed lymphocyte culture, we had a great inhibition of CTL generation.
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So our conclusion is that immature myeloid cells abnormally increased at the late stage of the tumour-bearing host, or at the late stage of the infectious disease, are not suppressor cells. But once affected by immunosuppressive factors such as TGF-b and IL-6 at tumour-local site, they were converted into suppressive ImC and F4/80 + MФ-like suppressor cells. Actually, these immature myeloid cells differentiated into the stimulatory dendritic cells (DCs) if they were cultured with Th1 cytokines.
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The third negative immunoregulator is IL-17-producing gd T cells.
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IL-17-producing cells are very involved in the regulation of autoimmune diseases. We recently reported in J. Exp. Med. 205, 1019-27, 2008 that IL-6-dependent spontaneous proliferation of IL-17-producing flora-specific CD8 + T (Tc17) cells, is the key process for inducing colitogenic effector cells. So IL-17 plays a crucial role in autoimmune disease.
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Our interest is in the role of IL-17-producing T cells at the tumour local site. We were surprised during the investigation that the IL-17-producing cell is not CD4 + T cell and not CD8 + T cell; it is γδT cells. Tγδ infiltrated into the tumour only produced IL-17, and the injection of the tumour in IL-17 knockout mice reduced the tumour growth in parallel with the decrease of angiogenesis. This indicates that IL-17 at the tumour local site promotes tumour growth via promoting angiogenesis, and the major IL-17-producing cell is the γδT cell.
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The differentiation and growth of IL-17-producing T cells are enhanced if IL-23 is added into the culture in addition to TGF-β and/or IL-6. And all of these factors were produced by the cells of tumour microenvironments. Thus, TGF-β, IL-6 and IL-23 produced by tumour microenvironments (tumour cells and APC) may be involved in the differentiation and propagation of IL-17-producing γδT cells.
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Actually, the addition of IL-23 into the γδT cells enhanced the production of IL-17, and the inhibition of one of these factors partially inhibited generation of the IL-17–producing γδT cells at the tumour local site.
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At the late stage of tumour-bearing mice, there are many immunosuppressive effector cells. It is very easy to induce anti-tumour immunity in tumour-free normal mice. And it is also easy to induce the immune response against viruses and bacteria, using normal mice. But once they were infected with viruses, the same immunosuppression occurred at the late stage as well as that of tumour-bearing mice.
So the problem is: how can we overcome such a strong immunosuppression, and how can we manipulate a positive immune response against tumours and infectious diseases?
Actually, many trials have been carried out all over the world in the last decade using tumour-derived MHC Class I-binding tumour peptide antigens and peptide-binding DCs, but the conclusion is that there are no effects. Why? This is because there is a great immunosuppression or tumour escape mechanisms in the tumour-bearing host, so we have to overcome a strong immunosuppression to develop a novel strategy to cure the tumour.
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There are two ways – stimulation of the innate immunity or stimulation of the acquired immunity. From our finding that Th1-dependent immunity is critical for inducing tumour-specific immunity in vivo, we have proposed that developing a strategy to activate Th1-immunity in vivo is essential to conquer the tumour. It is perhaps the same strategy for infectious diseases.
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For the activation of innate immunity, the Toll-like receptor is useful, and the ligand of the Toll-like receptor 9, CpG – we know that Pfizer has a patent for CpG. CpG is a great adjuvant for stimulating type-1 immunity. Toll-like receptor 9 is not expressed on the surface but it is expressed in cytoplasmic ER. So the internalisation of CpG is very important to trigger Th1 immunity.
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We capsulated the CpG with a liposome, to form liposome-CpG encapsulated tumour antigen proteins.
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Compared with the free CpG, in vivo administration of liposome-CpG resulted in a great enhancement of activation of NK and NKT cells. These are the primary innate effector cells. Compared with free CpG, liposome CpG induced over 80 per cent natural killing activity.
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We encapsulated a tumour antigen and CpG. We used EG7 tumour cells expressing OVA as an ideal tumour antigen, so we encapsulated CpG and OVA. Vaccination with the tumour antigen OVA alone had no effect, and the CpG alone had no effect. But encapsulation of CpG and OVA resulted in a complete rejection, and over 60 per cent of mice were free from tumour and we could induce tumour-specific tetramer-positive CTLs.
This is also applicable to primary methylcholanthrene in the tumour system.
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Time is limited, so I cannot show the detail, but the mechanism is that CpG can activate through the Toll-like receptor 9 and induce type-1 interferon. And type-1 interferon directly activates memory-type CD8 + T cells and allows the maturation of tumour-specific CTLs. In this case the CD4 + helper T cells are not essential, but if we supply the CD4 + helper T cells, more efficient CTL induction is possible.
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This is an example that the activation of innate immunity is possible to overcome a strong immunosuppression in the tumour-bearing host. But some days I asked myself why we are doing the DC-based vaccination. It is because we need to induce these antigen-specific Th1 cells, which are essential for inducing tumour-specific CTL. Then if we can induce tumour-specific Th1 cells from tumour patients and expand ex vivo, it is a very useful strategy to directly activate tumour-specific acquired cellular immunity.
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So next we tried to activate the overcoming effect on immunosuppression by the tumour-specific Th1 cells derived from the tumour-bearing host. In 1999 I first demonstrated the critical role of Th1 and Th2 cells in tumour-bearing hosts, using MHC class II-positive cells. In the Class II-positive cells, the Th1 cells alone can reject the tumour cells.
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But normally Class II expresses on limited tumour cells. Therefore, we re-examined Th-1 cell therapy using Class I-positive and Class II-negative EG7 tumour cells, which express OVA as a model tumour antigen. As shown here, adoptive cell transfer of OVA-specific Th1 cells alone cannot reduce the growth of ClassII-negative tumour mass of EG7. But when we injected Th1 cells with tumour antigen around the tumour, or systemically at a place distant from the tumour injection site – complete rejection of the tumour was induced.
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This is the mechanism of Th1 cell therapy. Th1 is labelled in green, and the OVA antigen is labelled in red. When tumour antigen, OVA and Th1 cells are injected together around the tumour, the antigen-capsulated DCs will soon migrate into the draining lymph nodes of tumour, and encounter the systemically circulated Th1 cells like this – only in the draining lymph nodes, but not in the distant lymph nodes. By cell-cell interaction between OVA-specific Th1 cells and OVA processed DC, a marked proliferation of the tumour-specific Th1 cells will occur at the draining lymph nodes to produce Th1-cytokines, which facilitate the inducuction of tumour-specific CTLs and finally reject the tumour mass.
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It is very interesting that such a Th1 cell adjuvant is also useful for inhibiting the accumulation of the Foxp3 + Treg at the tumour site. This inhibitory effect of the Th1 cells on Treg activation is cancelled by replacing with Th1 cells derived from the IFN- g knockout mice, so IFN- g is a very key cytokine for the inhibition of Treg migration at tumour local sites during Th1 cell adjuvant therapy.
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I have shown the tumour-specific Th1 cell story, but tumour-specific Th1 cells are not always necessary for tumour destruction. Th1 cells specific to tumour-irrelevant antigen such as PPD and OK-432 are also useful for Th1 cell therapy. When these Th1 cells were inoculated with antigen around the tumour, Th1 cells produced cytokine storm at tumour local site in response to PPD or OK-432, irrelevant to tumour antigen. Th1-derived cytokines sequentially accelerated the induction of tumour-antigen-specific CTL from endogenous activated CD8 + T cells at tumour local site concomitantly with inflammatory responses. Thus, Th1 cells, which are recognising tumour-irrelevant antigen trigger tumour-specific CTL at tumour local site.
So both tumour-specific Th1 and non-specific Th1 are useful for the efficient induction of tumour-specific CTLs essential for complete rejection of tumour tissue.
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Our conclusion is that the induction of Th1-dependent inflammatory responses to stimulate tumour-antigen processing by DC at the tumour local site and induction of Th1-dominant immunity at the draining lymph nodes are essential for induction of the anti-tumour CTL to eradicate tumour mass.
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This CpG-induced tumour immunity and Th1-induced immunity are augmented by combination therapy with radiation therapy.
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Usually the carcinoma is very refractory against this Th1-activation therapy and radiation therapy, but if we combine those therapies, first the radiation therapy and then Th1 activation by the CpG or Th1 cells, we can induce complete regression of the tumour. Thus, our finding indicates that combined therapy with radiation and Th1-cell therapy or CpG-based vaccination therapy will be a rational immunotherapy in future.
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Based on these basic experiments we are now planning to apply these findings to clinical trials. We have prepared the GMP Cell Processing Center. I am a founder of the venture company, BioImmulance – ‘immulance’ is an abbreviation for ‘immune balance’, so it is called BioImmulance.
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CpG now has a contract with Pfizer and the NY-ESO-1 was supplied by Dr Old, and we are starting CpG and NY-ESO-1 clinical trials. Soon we are also planning to do Th1 cell therapy combined with radiation- or chemo-therapy in humans.
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We have already defined new T helper epitopes of human tumours. There are many epitopes with Class I binding peptides, but by vaccination with only Class I peptides it is impossible to induce enough numbers of antigen-specific CTLs. If it is true, we can easily induce HIV CTLs in AIDS patients. AIDS patients are defect in CD4 + T cell function, so it is very difficult to induce HIV-specific CTL in those patients. So now the development of Class II-binding tumour peptides is essential, and we have developed and defined many helper-peptides useful for human Th1 cell therapy and vaccine therapy.
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Within two weeks, we can induce the antigen-tumour specific Th1 cells from the tumour patient’s PBMC. The Th1 cells show tumour-specific IFN- g production in ELISPOT-assay. Recently in clinical trials we confirmed that tumour-specific Th1 was increased in tumour patients after treatment with DC-based vaccination with CHP-Her2 tumour antigenic protein.
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So in future if this is true, I hope such a cartoon as this one will be demonstrated.
This represents that the injection of Class II-expressing tumour antigen-specific Th1 cells with tumour-derived helper peptide induced Th1-Th1 cell-cell interaction to activate each other and produce cytokines soon after injection into the tumour site. This induces inflammatory responses mediated by DC, M F , NK, NKT etcetera at the tumour site, which causes apoptotic cell death of tumour cells and enhanced processing of tumour antigens by DC. Migration and interaction of Th1 cells and antigen-processed DC at draining lymph node resulted in the induction of cytokine storm, which facilitate the generation of tumour-specific CTL from endogenously migrated tumour-CD8 + T cells. Finally, tumour-specific CTL infiltrated into tumour mass and destroyed the tumour to cure the patient.
This is my great dream for the future.
This work was carried out by the younger people in my university.
Question: A great talk. In regard to the Th17γδ-producing T cells, have you managed to find an NKG2D ligand in the tumour?
Takashi Nishimura: I have never done that. But it may be that they recognise some tumour antigens, including NKG2D.
Question (cont.): It seems, at least from Adrian Hayday’s stuff just recently, that early expression of NKG2D in tumours has effects on NKTs, potentially γδ’s, and that is causing early immune regression.
Takashi Nishimura: That is possible.
Question: Thank you for a very clear exposition of the work that you are doing. It is very interesting.
Fundamentally you have proposed two problems. One is locally in the tumour, and then one is with the sort of immune response that we generate through vaccination. From the work that you have done, which do you think is the most important problem we have to overcome in getting tumour immunotherapy to work? Is it better vaccines, or is it sorting out the problem locally in the tumour?
Takashi Nishimura: Vaccines are a very important area. Especially, what kind of adjuvants we use – the adjuvants which can stimulate the type-1 immune response so that the adjuvants don’t produce IL-10 and only stimulate IL-12 pathways – is very important in overcoming immunosuppression. And once we can induce type-1 immunity, we do not care about the existence of Tregs.
Question: Do you mean that the local environment becomes less important? I thought the essence I was getting from your talk was that the local environment was in fact absolutely critical, that there is nowhere for these effector T cells to go if there is massive immunosuppression locally.
Takashi Nishimura: Ah, whether there is degradation of immunosuppression at the local site?
Question (cont.): Yes.
Takashi Nishimura: The answer is both. If you first treat with radiation therapy, the radiation therapy will decrease the immunosuppression by immunosuppressive T cells and tumour-burden. I didn’t mention this today, but surprisingly radiation can induce tumour-specific CTLs in vivo.
And so the radiation damages immunosuppressive T cells and tumour, which then increases the anti-tumour effector cells, and then we should do the vaccination, which can stimulate Th1 cells. That is a rational strategy, maybe.
Question: I have noticed that you used CpG as an adjuvant to induce Th1 and CD8 response. Have you done any work, or what do you think, about the role of NKT cells to balance the Th1 and Th2 response, and the CD8 response?
Takashi Nishimura: In the mouse system, activation of the NKT cells and α-galactosylceramide or IL-12 alone can induce the regression of the tumour, but the combination treatment with IL-12 and α-galactosylceramide rather suppresses anti-tumour immunity. Because the NKT cells produce large amounts of IL-4, that stops Th1 immunity. In human systems, in Japan, Drs Taniguchi and Fuji are doing that work. In the human system, IL-4 production is generally low, and the activation of the α-galactosylceramide and especially α-galactosylceramide pulsed DC injection induces only interferon-gamma producing NKT cells. So in human cases I think that NKT cells are also involved in anti-tumour immunity.
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