Several lines of evidence have indirectly demonstrated a role for the Th1/Th2 balance in antitumor immunity. First, the cytokine IL-12, which stimulates Th1-dominant immunity in vivo, was shown to have strong in vivo antitumor activity against a variety of tumors, including primary tumors 18193132
. Second, in vivo neutralization of IFN-γ caused the inhibition of the antitumor effect of IL-12, suggesting that IFN-γ–producing Th1 cells may play an important role in tumor rejection. However, this conclusion is weakened by the observation that other T cells, including NKT cells and CD8+
T cells, can produce IFN-γ in response to IL-12, and that these cell types can activate Th1-dominant immunity 3334
. Indeed, it was recently demonstrated that the antitumor activities of IL-12 are mediated, in large part, by NKT cells 34
. Moreover, the finding that Th2-derived cytokines (IL-4, IL-5, IL-6, IL-10) show antitumor activities in vivo that are as strong as the antitumor activities of Th1 cytokines 2021222335
has made it difficult to conclude which cell type is the most effective for eliciting complete tumor regression in vivo.
To address this issue, we designed a new adoptive tumor immunotherapy model using tumor antigen–specific Th1 or Th2 cells. As tumor cells, we used OVA gene–transfected tumor cells, and Th1 and Th2 cells were induced from mice transgenic for an OVA-specific TCR. Our results demonstrated that both Th1 and Th2 cells show potent antitumor activities in vivo (). Interestingly, Th1 cells induced a marked lymphocyte infiltration into the tumor mass and eradicated the tumor mass via cellular immunity. In sharp contrast, Th2 cells induced inflammatory responses at the tumor site and induced tumor necrosis (). The finding that Th2 cells, which can produce high levels of IL-4, induced the inflammation characterized by eosinophils and neutrophils is consistent with previous results with IL-4 gene–transfected tumors, which were rejected by inflammatory cells that included eosinophils and neutrophils 2236
. However, it remains unclear how Th2 cells induce tumor necrosis.
Another important difference between Th1 and Th2 cell therapy is that Th1 therapy was able to induce a strong immunological memory suitable for the generation of CTLs, whereas Th2 cells did not induce the immunological memory for CTL generation very well (). Some of the cured mice from A20-OVA by Th1 cell therapy but not Th2 cell therapy showed resistance against rechallenged parental A20 tumor cells (data not shown), indicating that Th1 cell therapy might also be beneficial for the generation of CTLs, which recognize unknown tumor-rejection antigen expressed on parental A20 tumor. Based on our observation that in vivo administration of anti-CD4 mAb, anti-CD8 mAb, or anti–IFN-γ mAb blocked the therapeutic effect of Th1 cells against tumors ( A), we suggest that transferred Th1 cells migrate into local tumor sites, produce IFN-γ, and facilitate the induction of antitumor CD8+
CTLs in vivo. The requirement for CD8+
T cells in Th1 cell therapy is also demonstrated by our finding that DO11.10 TCR-Tg mice that have OVA-reactive CD4+
T cells, but not CD8+
T cells, were unable to permanently clear the tumor by Th1 cell therapy ( B). Direct evidence for the requirement for CD8+
T cells was demonstrated using adoptive transfer of CD8+
T cells into RAG2−/−
mice (). This experiment also indicated that NKT cells are not involved in Th1- and Th2-induced antitumor activity in vivo because NKT cells are not differentiated in RAG2−/−
mice, which are deficient in NKT cells, mainstream T cells, and B cells. Th2 cells contained <2% of IFN-γ–producing cells ( D), which might exhibit negligible cytotoxicity against A20-OVA ( H). Indeed, in some cases, Th2 cells exhibited low but significant cytotoxicity against A20-OVA in parallel with the increased production of IFN-γ. However, even in such cases, the IFN-γ produced by contaminating cells (Th1 or Th2) appeared not to be involved in the triggering of antitumor activity of Th2 cells, because administration of anti–IFN-γ mAb caused no significant blocking of the Th2-mediated therapeutic effect (data not shown). In mice that were cured from A20-OVA tumors by Th2 therapy, A20-OVA–specific CTLs were not detected. These findings suggest that antigen-nonspecific CD8+
killer T cells are involved in Th2-mediated adoptive immunotherapy. Alternatively, CD8+
TC2 cells 33
induced by IL-4 may contribute to tumor eradication in Th2 cell therapy. Since the immunological memory in mice cured by Th2 cell transfer may be mediated by humoral immunity, we are currently investigating whether Th2 immunological memory can be transferred into normal mice by serum isolated from tumor-cured mice.
The distinct antitumor immunity mediated by Th1 and Th2 cells may be due to the distinct cell adhesion interactions involved in the migration of these cells into tumor tissues across endothelia. Consistent with previous results 37
, we found that Th1 cells express higher levels of P-selectin ligands and produced higher amounts of chemokines compared with Th2 cells (data not shown). In addition, we found that Th1 cells exhibit strong LFA-1/ICAM-1–dependent cell–cell interactions ( C), which are critical for lymphocyte activation, cell-mediated cytotoxicity, and transmigration of lymphocytes into inflammatory tissues 3839
. In contrast, Th2 cells were defective in LFA-1/ICAM-1–mediated cell–cell interactions ( D), but were able to interact with the extracellular matrix on endothelia through the integrin αVβ3 (data not shown). These results suggest that Th1 cells express adhesion molecules that facilitate transmigration into tumor tissues across the tumor vessels. Indeed, antitumor therapeutic activity of Th1 cells was completely blocked by administration of anti–LFA-1 mAb, whereas the activities of Th2 cells were not affected by anti–LFA-1 mAb injection ( and ). From these results, we speculate that, at the tumor local site, Th1 cells actively respond to tumor cells and produce cytokines, which recruit other effector cells such as CD8+
T cells, NKT, or NK cells into the tumor tissue. In contrast, Th2 cells, which are unable to enter tumor tissue because of a defect of adhesion mechanisms, may accumulate on the endothelial cells around the tumor mass and induce tumor necrosis via molecules such as TNF-α that damage tumor vessels 40
. However, we have recently demonstrated that in vivo administration of anti–IL-4, anti–IL-10, or anti–TNF-α was unable to block the tumor necrosis induced by Th2 cell therapy (data not shown). Therefore, unknown mechanisms appear to be involved in Th2-induced tumor necrosis. One possibility would be that Th2-derived cytokines activated other inflammatory cells and the products of these cells damage endothelial cells to induce tumor necrosis. This hypothesis is strongly supported by recent findings by Hung et al. 41
T cells play an important role in inducing antitumor activity in vivo through activation of eosinophils and macrophages that produce superoxide and nitric oxide.
The finding that transfer of >107
Th1 or Th2 cells with CD8+
T cells is required for the complete rejection of tumor (data not shown) means that, at an early phase of tumor rejection, the bursting of a strong cytokine storm derived from Th1 or Th2 cells may be essential for overcoming a strong suppression in the tumor-bearing host and for induction of CD8+
CTL–mediated antitumor protective immunity in tumor-bearing mice. The present data demonstrate that Th1 and Th2 cells use distinct tumor eradication mechanisms. However, based on the following considerations, Th1 cells may be more suitable for adoptive tumor immunotherapy in the future: (a) Th1 cell therapy, but not Th2 cell therapy, induces strong immunological memory beneficial for CTL generation (); (b) Th2 cells produce high levels of IL-6, which can contribute to cachexia in late stage tumor-bearing hosts 42
; and (c) in our experience, IFN-γ–producing Th1 cells are easily expanded from total spleen or peripheral blood cell populations, while it is hard to induce pure Th2 cells producing IL-4 but not IFN-γ from total spleen or peripheral blood cells in humans and mice (data not shown). In a previous report 43
, we demonstrated that culture of tumor-infiltrating lymphocytes (TILs) with IL-2 plus IL-12 results in a profound increase in the development of autologous tumor-reactive CTLs. Moreover, we showed that this protocol enhanced the generation of autologous tumor-reactive Th1-dominant cells (our unpublished data). Therefore, if we can develop a large scale culture system for the generation of autologous tumor-reactive Th1 cells from TILs or PBLs of tumor patients, the adoptive tumor immunotherapy using tumor-specific Th1-dominant cells may be possible. The cytokine IL-12 shows great promise for the development of tumor immunotherapy 18193132
. However, recent findings have demonstrated that IL-12 also has adverse effects owing to overstimulation of Th1-dominant immunity 44
. In terms of side effects, adoptive transfer of in vitro IL-12–activated Th1-dominant cells may minimize side effects, and could make the management of side effects easier. Thus far, IL-12 has been suggested for cytokine therapy and gene therapy of cancer. This paper further indicates that IL-12 may be a useful tool for application to a novel tumor immunotherapy protocol using the adoptive transfer of Th1-dominant T cells and/or CTLs (Th1 helper/killer therapy).