Therapy‐induced antitumor vaccination in neuroblastomas by the combined targeting of IL‐2 and TNFα

L19‐IL2 and L19TNFα are fusion proteins composed of L19(scFv), specific for the angiogenesis‐associated ED‐B containing fibronectin isoform and IL‐2 or TNFα. Because of the tumor targeting properties of L19, IL‐2 and TNFα concentrate at therapeutic doses at the tumor vascular level. To evaluate the therapeutic effects of L19‐IL2 and L19mTNFα in neuroblastoma (NB)‐bearing mice, A/J mice bearing Neuro2A or NIE115 NB were systemically treated with L19‐IL2 and L19mTNFα, alone or in combination protocols. Seventy percent of Neuro2A‐ and 30% of NIE115‐bearing mice were cured by the combined treatment with L19‐IL2 and L19mTNFα, and further rejected a homologous tumor challenge, indicating specific antitumor immune memory. The immunological bases of tumor cure and rejection were studied. A highly efficient priming of CD4⁺ T helper cells and CD8⁺ CTL effectors was generated, paralleled by massive infiltration in the tumor tissue of CD4⁺ and CD8⁺ T cells at day 16 after tumor cell implantation, when, after therapy, tumor volume was drastically reduced and tumor necrosis reached about 80%. The curative treatment resulted in a long‐lasting antitumor immune memory, accompanied by a mixed Th1/Th2 type of response. Concluding, L19‐IL2 and L19mTNFα efficiently cooperate in determining a high percentage of NB cure that, in our experimental models, is strongly associated to the generation of adaptive immunity involving CD4⁺ and CD8⁺ T cells.     

3‐Methylcholanthrene‐induced transforming growth factor‐β‐producing carcinomas,but not sarcomas,are refractory to regulatory T cell‐depletion therapy

Regulatory T cells (Tregs) are major immunosuppressors in tumor‐bearing hosts. Although Treg‐depletion therapy has been shown to induce a complete cure in tumor‐bearing mice, this treatment is not always successful. Using 3‐methylcholanthrene‐induced primary mouse tumors, we examined the distinct regulation of Treg‐mediated immunosuppression between carcinomas and sarcomas. We showed that the number of Tregs was greatly increased in squamous cell carcinoma (SCC)‐bearing mice compared with sarcoma‐bearing mice. This appeared to be because SCC produced higher levels of active transforming growth factor (TGF)‐β, which is essential for inducing Tregs, compared with sarcoma. Moreover, SCC, but not sarcomas, were refractory to Treg‐depletion therapy by treatment with anti‐CD25 mAb. The refractoriness of SCC against Treg‐depletion therapy was due to the rapid recovery of Tregs in SCC‐bearing mice compared with sarcoma‐bearing mice. However, combination treatment of anti‐TGF‐β mAb with anti‐CD25 mAb caused a significant reduction in Treg recovery and induced a complete cure in SCC‐bearing mice. Thus, we showed the refractoriness of mouse carcinoma against Treg‐depletion therapy using anti‐CD25 mAb treatment. We also proposed a novel Treg‐blocking combination therapy using anti‐CD25 mAb and anti‐TGF‐β mAb to induce a complete cure of tumor‐bearing hosts. (Cancer Sci 2010; 101: 855–861)     

Blockade of TGF‐β enhances tumor vaccine efficacy mediated by CD8+ T cells

Though TGF‐β inhibition enhances antitumor immunity mediated by CD8⁺ T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF‐β blockade, we tested the effect of anti‐TGF‐β combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti‐TGF‐β antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti‐TGF‐β antibodies compared to vaccinated mice without anti‐TGF‐β, suggesting that anti‐TGF‐β synergistically enhanced irradiated tumor vaccine efficacy. CD8⁺ T‐cell depletion completely abrogated the vaccine efficacy, and so protection required CD8⁺ T cells. Depletion of CD25⁺ T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti‐TGF‐β did not change the number of CD25⁺ T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d‐restricted NKT cells, which have been reported to induce TGF‐β production by MDSC through an IL‐13‐IL‐4R‐STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti‐TGF‐β enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF‐β levels found in patients with cancer and that the effect is not dependent on TGF‐β solely from CD4⁺CD25⁺ T regulatory cells or the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway.     

Suppression of Tregs by anti‐glucocorticoid induced TNF receptor antibody enhances the antitumor immunity of interferon‐α gene therapy for pancreatic cancer

We have reported that interferon (IFN)‐α can attack cancer cells by multiple antitumor mechanisms including the induction of direct cancer cell death and the enhancement of an immune response in several pancreatic cancer models. However, an immunotolerant microenvironment in the tumors is often responsible for the failure of the cancer immunotherapy. Here we examined whether the suppression of regulatory T cells (Tregs) within tumors can enhance an antitumor immunity induced by an intratumoral IFN‐α gene transfer. First we showed that an intraperitoneal administration of an agonistic anti‐glucocorticoid induced TNF receptor (GITR) monoclonal antibody (mAb), which is reported to suppress the function of Tregs, significantly inhibited subcutaneous tumor growth in a murine pancreatic cancer model. The anti‐GITR mAb was then combined with the intratumoral injection of the IFN‐α‐adenovirus vector. The treatment with the antibody synergistically augmented the antitumor effect of IFN‐α gene therapy not only in the vector‐injected tumors but also in the vector‐uninjected tumors. Immunostaining showed that the anti‐GITR mAb decreased Foxp3⁺ cells infiltrating in the tumors, while the intratumoral IFN‐α gene transfer increased CD4⁺ and CD8⁺ T cells in the tumors. Therefore, the combination therapy strongly inclined the immune balance of the tumor microenvironment in an antitumor direction, leading to a marked systemic antitumor effect. The CCR5 expression on Tregs was downregulated in the antibody‐treated mice, which may explain the decrease of tumor‐infiltrating Tregs. The combination of Treg‐suppression by GITR mAb and the tumor immunity induction by IFN‐α gene therapy could be a promising therapeutic strategy for pancreatic cancer.     

Distinct chemotherapy‐associated anti‐cancer immunity by myeloid cells inhibition in murine pancreatic cancer models

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy associated with an extremely poor prognosis. Chemotherapy, such as gemcitabine (GEM), is the only treatment for PDAC patients who are not suitable for radical surgical treatment; however, its anti‐tumor efficacy is limited. In this study, we investigated the host immune system response in murine PDAC models undergoing GEM treatment. We found that PDAC tumor tissues were infiltrated with a substantial number of Gr‐1+ myeloid cells and had relatively small numbers of CD4+ and CD8+ cells. In addition, there were increased numbers of myeloid cells expressing CD11b+ and Gr‐1+ in peripheral blood. When mice with PDAC tumors in the intraperitoneal cavity or liver were treated with GEM, numbers of myeloid cells in tumor tissues and in peripheral blood decreased. In contrast, numbers of CD4+ or CD8+ cells increased. In peripheral blood, the numbers of CD8+ cells expressing interferon‐gamma (IFN‐γ) were higher in GEM‐treated mice than in untreated mice. In addition, GEM treatment in combination with myeloid cell depletion further prolonged the survival of PDAC mice. The gene expression profile of peripheral blood in myeloid cell‐depleted PDAC mice treated with GEM showed biological processes related to anti‐cancer immunity, such as natural killer cell‐mediated cytotoxicity, type I IFN signaling, and co‐stimulatory signaling for T cell activation. Thus, in PDAC murine models, GEM treatment was associated with an immune response consistent with an anti‐cancer effect, and depletion of myeloid‐lineage cells played an important role in enhancing anti‐cancer immunity associated with GEM treatment.     

Intermittent chemotherapy can retain the therapeutic potential of anti‐CD137 antibody during the late tumor‐bearing state

Immunomodulating monoclonal antibodies (mAb) can evoke antitumor T‐cell responses, which are attenuated by regulatory T cells (Treg) and myeloid‐derived suppressor cells (MDSC). Treatment with cyclophosphamide (CP) and gemcitabine (GEM) can mitigate the immunosuppression by Treg and MDSC, respectively. In the current study, we examined the antitumor effects of a combination of local injection with anti‐CD137 mAb and intermittent low‐dose chemotherapy using CP and GEM in subcutaneously established CT26 colon carcinoma. Although a significant antitumor effect was observed when local anti‐CD137 mAb therapy (5 μg) was started early in the tumor‐bearing stage (day 10), no therapeutic efficacy was observed when the mAb therapy was started at a later tumor‐bearing stage (day 17). Analyses of the tumor‐infiltrating immune cells revealed that the number of Gr‐1^high/low CD11b⁺ MDSC started to increase 13 days after tumor inoculation, whereas injection with low‐dose (50 mg/kg) CP and GEM mitigated this increase. In addition, although intermittent injections with low‐dose CP and GEM on days 10 and 18 suppressed tumor growth significantly, additional local injections of anti‐CD137 mAb on days 19, 21, and 23 further augmented the therapeutic efficacy. Cytotoxic T lymphocytes reactive to CT26 and a tumor antigen peptide were induced successfully from the spleen cells of tumor‐cured or tumor‐stable mice. In a bilateral tumor inoculation model, this combination therapy achieved systemic therapeutic effects and suppressed the growth of mAb‐untreated tumors. These results suggest that intermittent immunochemotherapy using CP and GEM could retain the therapeutic potential of anti‐CD137 mAb that is normally impaired during the late tumor‐bearing stage.     

IgG1 anti‐epidermal growth factor receptor antibodies induce CD8‐dependent antitumor activity

Anti‐EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR⁺ solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibody‐dependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab‐mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA‐A2 and adoptively transferred human HLA‐A2⁺ PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti‐EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti‐EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8⁺ T effector cells. This novel mechanism of ADCC conducted by CD8⁺ T effector cells was restricted to IgG1 anti‐EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8⁺ T effector cell‐mediated ADCC was enhanced in the presence of IL‐15 and strongly improved after glycosylation of anti‐EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.     

Combination immunotherapy with interleukin‐2 surface‐modified tumor cell vaccine and programmed death receptor‐1 blockade against renal cell carcinoma

Immunotherapy may be an effective way to prevent postoperative recurrence of renal cell carcinoma. Streptavidin‐interleukin‐2 (SA‐IL‐2) surface‐modified tumor cell vaccine developed through our protein‐anchor technology could induce specific antitumor T‐cell responses, but this immunotherapy cannot completely eradicate the tumor. These effector T cells highly expressed programmed death receptor‐1 (PD‐1), and the expression of programmed death ligand‐1 (PD‐L1) in the tumor environment also was upregulated after SA‐IL‐2‐modified vaccine therapy. PD‐1/PD‐L1 interaction promotes tumor immune evasion. Adding PD‐1 blockade to SA‐IL‐2‐modified vaccine therapy increased the number of CD4⁺, CD8⁺ and CD8⁺interferon‐γ⁺ but not CD4⁺Foxp3⁺ T cells. PD‐1 blockade could rescue the activity of tumor‐specific T lymphocytes induced by the SA‐IL‐2‐modified vaccine. Combination therapy delayed tumor growth and protected mice against a second Renca cells but not melanoma cells challenge. Taken together, PD‐1 blockade could reverse immune evasion in the treatment with SA‐IL‐2‐modified vaccine, and eventually induce a stronger specific antitumor immune response against renal cell carcinoma.     

Agonistic anti‐CD137 antibody treatment leads to antitumor response in mice with liver cancer

Immunotherapy is a promising strategy against hepatocellular carcinoma (HCC). We assessed the therapeutic effects of stimulating CD137, a member of the TNF receptor family, with agonistic monoclonal antibodies (mAb). Agonistic anti‐CD137 mAb treatment was tested on two in situ models of HCC in immunocompetent mice. We also studied the mediators involved at different time points. In an orthotopic HCC the treatment consistently leads to complete tumor regression in 40–60% of animals. The protection is long lasting in the animals responding to the treatment, which can reject a second tumor challenge more than 3 months after treatment and eradication of the first malignancy. The main mediators of the effect are T lymphocytes and NK cells, demonstrated through depletion experiments. In addition, adoptive transfer of splenocytes prepared from anti‐CD137 mAb‐treated and ‐cured mice to naive mice allowed them to, in turn, reject the tumor. The efficacy of anti‐CD137 mAb treatment is associated with early, sustained recruitment of iNOS‐positive macrophages within tumor nodules. Moreover, in the absence of treatment, tumor development is accompanied by infiltration by myeloid derived suppressor cells (MDSC) and regulatory T lymphocytes. In mice responding to the anti‐CD137 mAb treatment, this infiltration is very limited, and a combination treatment with a depletion of MDSC leads to the recovery of 80% of the mice. These results demonstrate that agonistic anti‐CD137 mAb is a promising therapeutic strategy for anti‐tumor immunity stimulation against HCC.     

Reduction of Foxp3+ T cell subsets involved in incidence of chronic graft‐versus‐host disease after allogeneic hematopoietic stem cell transplantation

Foxp3+ T cells (CD4+ Tregs and CD8+ Treg) have been demonstrated to play roles in the maintenance of tolerance after allogeneic hematopoietic stem cell transplantation (Allo‐HSCT). We have found that Foxp3+ γδTCR+ Treg cells (γδTregs) exerted regulatory functions. In the current study, patients were recruited and divided as non‐cGVHD, limited cGVHD and extensive cGVHD groups. Healthy volunteers were recruited as healthy group. Treg cells were evaluated by flow cytometry. Serum cytokine levels of IL‐2, tumour necrosis factor‐α, interferon‐γ and transforming growth factor‐β1 (TGF‐β1) were evaluated by ELISA. The results showed that percentages of CD4+ Tregs, CD8+ Tregs and γδTregs were all significantly increased in non‐cGVHD group compared with those in healthy group, limited cGVHD group and extensive cGVHD group. Moreover, compared with extensive cGVHD group, percentages of these three types of Tregs were significantly increased in limited cGVHD group. The levels of TGF‐β1 increased dramatically in non‐cGVHD group compared with other groups. Spearman's correlation analysis revealed that the increased levels of TGF‐β1 and IL‐2 were positively associated with increased Treg subsets, indicating that TGF‐β1 and IL‐2 participated in the expansion process of Foxp3+ Tregs in vivo. Our findings support that increasing the number of Tregs following allo‐HSCT would be a preferential strategy for controlling cGVHD. Copyright © 2015 John Wiley & Sons, Ltd.     

PD‐1 blockade enhances the antitumor efficacy of GM‐CSF surface‐modified bladder cancer stem cells vaccine

Eliminating cancer stem cells (CSCs) is a key issue in eradicating tumor. The streptavidin–granulocyte‐macrophage‐colony stimulating factor (SA–GM‐CSF) surface‐modified bladder CSCs vaccine previously developed using our protein–anchor technology could effectively induce specific immune response for eliminating CSCs. However, program death receptor‐1 (PD‐1)/program death ligand 1 (PD‐L1) signaling in tumor microenvironment results in tumor‐adaptive immune resistance. Although the CSCs vaccine could increase the number of CD8⁺T cells, a part of these CD8⁺T cells expressed PD‐1. Moreover, the CSCs vaccine upregulated the PD‐L1 expression of tumor cells, resulting in immune resistance. Adding PD‐1 blockade to the CSCs vaccine therapy increased the population of CD4⁺, CD8⁺ and CD8⁺IFN‐γ⁺ but not CD4⁺ Foxp3⁺T cells and induced the highest production of IFN‐γ. PD‐1 blockade could effectively enhance the functions of tumor‐specific T lymphocytes generated by the CSCs vaccine. This combination therapy improved the cure rate among mice and effectively protected the mice against a second CSCs cell challenge, but not a RM‐1 cell challenge. These results indicate that PD‐1 blockade combined with the GM‐CSF‐modified CSCs vaccine effectively induced a strong and specific antitumor immune response against bladder cancer.     

Combination of anti‐CD4 antibody treatment and donor lymphocyte infusion ameliorates graft‐versus‐host disease while preserving graft‐versus‐tumor effects in murine allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is not only a well‐established immunotherapy for hematologic malignancies, but is potentially useful for treating solid tumors refractory to available therapies. However, application of allo‐HSCT to solid tumors is limited, despite the beneficial antitumor effects, by the risk of graft‐versus‐host disease (GVHD). CD4⁺ T cells have been implicated in several aspects of GVHD, and suppress antitumor CD8⁺ T‐cell responses. In the present study, we investigated clinically applicable allo‐HSCT protocols designed to maximize antitumor effects while reducing the risk of GVHD. We used a mouse model of allo‐HSCT with s.c. tumors. We found that myeloablative conditioning was associated with better inhibition of tumor growth but with severe acute GVHD. Early treatment with anti‐CD4 mAb substantially ameliorated GVHD while preserving antitumor effects, leading to improved survival in myeloablative allo‐HSCT. Late treatment with anti‐CD4 mAb also ameliorated GVHD to some extent. Donor lymphocyte infusion in GVHD mice treated with anti‐CD4 mAb further suppressed tumor growth without exacerbating GVHD. Collectively, our results suggest that myeloablative allo‐HSCT followed by anti‐CD4 mAb treatment and donor lymphocyte infusion could be a potent and safe immunotherapy for patients with cancers refractory to available therapies.     

MiR‐15a/16 deficiency enhances anti‐tumor immunity of glioma‐infiltrating CD8+ T cells through targeting mTOR

MiR‐15a/16, a miRNA cluster located at chromosome 13q14, has been reported to act as an immune regulator in inflammatory disorders besides its aberrant expression in cancers. However, little is known about its regulation in tumor‐infiltrating immune cells. In our study, using an orthotropic GL261 mouse glioma model, we found that miR‐15a/16 deficiency in host inhibited tumor growth and prolonged mice survival, which might be associated with the accumulation of tumor‐infiltrating CD8+ T cells. More importantly, tumor‐infiltrating CD8+ T cells without miR‐15a/16 showed lower expression of PD‐1, Tim‐3 and LAG‐3, and stronger secretion of IFN‐γ, IL‐2 and TNF‐α than WT tumor‐infiltrating CD8+ T cells. Also, our in vitro experiments further confirmed that miR‐15a/16^?/? CD8+ T displayed higher active phenotypes, more cytokines secretion and faster expansion, compared to WT CD8+ T cells. Mechanismly, mTOR was identified as a target gene of miR‐15a/16 to negatively regulate the activation of CD8+ T cells. Taken together, these data suggest that miR‐15a/16 deficiency resists the exhaustion and maintains the activation of glioma‐infiltrating CD8+ T cells to alleviate glioma progression via targeting mTOR. Our findings provide evidence for the potential immunotherapy through targeting miR‐15a/16 in tumor‐infiltrating immune cells.     

An ISCOM vaccine combined with a TLR9 agonist breaks immune evasion mediated by regulatory T cells in an orthotopic model of pancreatic carcinoma

Vaccines based on immune stimulatory complexes (ISCOM) induce T‐cell responses against tumor antigen (Ag). However, immune responses are impaired in pancreatic cancer patients. We investigated the efficacy of an ISCOM vaccine in a murine pancreatic carcinoma model. Panc02 cells expressing OVA as a model Ag were induced subcutaneously or orthotopically in the pancreas of C57BL/6 mice. Treatment consisted of an OVA containing ISCOM vaccine, either used alone or in combination with the TLR9 agonist CpG. The ISCOM vaccine effectively induced Ag‐specific CTL capable of killing tumor cells. However, in mice with established tumors CTL induction by the vaccine was inefficient and did not affect tumor growth. Lack of efficacy correlated with increased numbers of Treg. Depletion of Treg with anti‐CD25 mAb restored CTL induction and prolonged survival. Adding low‐dose CpG to the ISCOM vaccine reduced Treg numbers, enhanced CTL responses and induced regression of pancreatic tumors in a CD8⁺ T cell–dependent manner. Mice cured from the primary tumor mounted a memory T‐cell response against wild‐type Panc02 tumors, indicative of epitope spreading. Combining ISCOM vaccines with TLR agonists is a promising strategy for breaking tumor immune evasion and deserves further evaluation for the treatment of pancreatic carcinoma.     

Newcastle disease virus co‐expressing interleukin 7 and interleukin 15 modified tumor cells as a vaccine for cancer immunotherapy

Interleukin 15 (IL15) and IL7 are two cytokines essential for T cell development and homeostasis. In order to improve the antitumor activity by Newcastle disease virus (NDV)‐modified tumor vaccine, we generated a recombinant NDV co‐expressing IL15 and IL7 (LX/IL(15+7)) through incorporation of a 2A self‐processing peptide into IL15 and IL7 using reverse genetics. B16 cells infected with LX/IL(15+7) expressed both IL15 and IL7 stably. The cytotoxicity assay showed that murine melanoma cells modified with LX/IL(15+7) could significantly enhance the antitumor immune response in vitro. Then, the antitumor effects of tumor vaccine modified with recombinant virus were tested in the murine tumor models. We observed strong antitumor responses induced by LX/IL(15+7)‐modified tumor cells both in prophylaxis and therapeutic models. Although the tumor‐infiltrating CD4⁺ T cells and CD8⁺ T cells were both increased, the antitumor activity of the tumor vaccine modified with LX/IL(15+7) was dependent on CD8⁺ T cells. Taken together, our data strongly indicated that tumor vaccine modified with NDV strain LX/IL(15+7) is a promising agent for cancer immunotherapy.     

Toll‐like receptor‐4 deficiency inhibits ultraviolet radiation‐induced tumor development by modulation of immune and inflammatory responses

Ultraviolet (UV) B irradiation of the skin induces acute inflammation, as characterized by erythema, edema, and immunosuppression, and is subsequently linked to the progression of skin cancer. Toll‐like receptor 4 (TLR4), a component of innate immunity, has been shown to play an important role in cancer. To elucidate the role of TLR4 in UVB‐induced tumor development, TLR4‐proficient (C3H/HeN) and TLR4‐deficient (C3H/HeJ) mice were exposed to multiple doses of UVB radiation (200 mJ/cm²) for 40 weeks. Photocarcinogenesis was retarded in terms of tumor incidence, and tumor latency, in mice deficient in TLR4 compared with TLR4‐proficient mice, whereas significantly greater numbers of tumors occurred in TLR4‐proficient mice. There was significant upregulation of inflammatory markers like COX‐2, PGE₂, S100A8, and S100A9 in the skin of TLR4‐proficient mice than the skin of TLR4‐deficient mice. Furthermore, we found that TLR4‐proficient mice had a significantly higher number of Gr1+CD11b+ myeloid cells CD4+CD25+ regulatory T‐cells than TLR4‐deficient mice. Furthermore, the levels of interferon (IFN)‐γ cytokine was increased and the levels of interleukin (IL)‐4, IL‐10, and IL‐17 cytokines were decreased in serum, skin, and tumor lysates of TLR4‐deficient mice in comparison with samples from TLR4‐proficient mice. Together, our data indicate that TLR4‐mediated inflammation may cause suppression of antitumor responses and trigger the development of UVB‐induced skin cancers. Thus, strategies to inhibit TLR4‐mediated immune suppression may allow us to develop preventive and therapeutic approaches for the management of UVB‐induced cutaneous tumors.     

Intratumoral injection of interferon‐α and systemic delivery of agonist anti‐CD137 monoclonal antibodies synergize for immunotherapy

CD137 artificial costimulation results in complete tumor rejection in several mouse models. Type I interferons (IFN) exert antitumor effects through an array of molecular functions on malignant cells, tumor stroma and immune system cells. The fact that agonist anti‐CD137 mAb induce tumor regressions in mice deficient in the unique receptor for Type I IFNs (IFNAR^?/?) indicated potential for treatment combinations. Indeed, combination of intratumor injections of mouse IFN‐α and intraperitoneal injections of anti‐CD137 mAb synergized as seen on subcutaneous lesions derived from the MC38 colon carcinoma, which is resistant to each treatment if given separately. Therapeutic activity was achieved both against lesions directly injected with IFN‐α and against distant concomitant tumors. Experiments in bone marrow chimeras prepared with IFNAR^?/? and WT mice concluded that expression of the receptor for Type I interferons is mainly required on cells of the hematopoietic compartment. Synergistic effects correlated with a remarkable cellular hyperplasia of the tumor draining lymph nodes (TDLNs). Enlarged TDLNs contained more plasmacytoid and conventional dendritic cells (DC) that more readily cross‐presented. Importantly, numbers of both DC subtypes inversely correlated with the tumor size. Numbers of CD8 T cells specific for a dominant tumor antigen were increased at TDLNs by each separate treatment but only with slight augments due to the combination. Combined antitumor effects of the therapeutic strategy were also seen on subcutaneous TC‐1 tumors established for 24 days before treatment onset. The described strategy is realistic because (i) agents of each kind are clinically available and (ii) equivalent procedures in humans are feasible.     

A reduction of recipient regulatory T cells by cyclophosphamide contributes to an anti-tumor effect of nonmyeloablative allogeneic stem cell transplantation in mice

We have recently established a unique model system of nonmyeloablative allogeneic stem cell transplantation (SCT) for treatment of murine solid tumors, based on cyclophosphamide‐induced tolerance. An injection of allogeneic donor spleen cells and bone marrow cells (BMC) followed by cyclophosphamide treatment induced a stable mixed chimerism with long lasting tolerance to the allografts. A donor lymphocyte infusion (DLI) in the cyclophosphamide‐induced tolerant mice exerted strong anti‐tumor effects on an MBT‐2 murine bladder tumor, MBT‐2 via their graft versus tumor (GVT) activity. In the present study, we determined whether a cyclophosphamide‐induced reduction of naturally occurring regulatory T cells (Tregs) was associated with the anti‐tumor activity in our nonmyeloablative SCT system. The number of recipient CD4+ CD25+ Foxp3+ Tregs significantly decreased 3 days after an intraperitoneal injection of cyclophosphamide in C3H/HeN mice that had been injected with spleen cells and BMC of donor AKR/J mice, compared with the number of CD4+ CD25+ Foxp3‐ T cells. An adoptive transfer of CD4+ CD25+ T cells from naïve C3H/He x AKR/J F1 mice into recipient mice 1 day after DLI significantly suppressed the expansion and IFN‐γ production of host‐reactive donor CD4+T cells and hampered the MBT‐2 anti‐tumor activity when compared with the transfer of CD4+ CD25‐ T cells. These results indicated that cyclophosphamide‐induced reduction of recipient Tregs is associated with retardation of tumor progression via the expansion of host‐reactive donor T cells and IFN‐γ production after DLI in our nonmyeloablative SCT system.     

Lack of interleukin‐6 in the tumor microenvironment augments type‐1 immunity and increases the efficacy of cancer immunotherapy

Conquering immunosuppression in tumor microenvironments is crucial for effective cancer immunotherapy. It is well known that interleukin (IL)‐6, a pleiotropic cytokine, is produced in the tumor‐bearing state. In the present study, we investigated the precise effects of IL‐6 on antitumor immunity and the subsequent tumorigenesis in tumor‐bearing hosts. CT26 cells, a murine colon cancer cell line, were intradermally injected into wild‐type and IL‐6‐deficient mice. As a result, we found that tumor growth was decreased significantly in IL‐6‐deficient mice compared with wild‐type mice and the reduction was abrogated by depletion of CD8⁺ T cells. We further evaluated the immune status of tumor microenvironments and confirmed that mature dendritic cells, helper T cells and cytotoxic T cells were highly accumulated in tumor sites under the IL‐6‐deficient condition. In addition, higher numbers of interferon (IFN)‐γ‐producing T cells were present in the tumor tissues of IL‐6‐deficient mice compared with wild‐type mice. Surface expression levels of programmed death‐ligand 1 (PD‐L1) and MHC class I on CT26 cells were enhanced under the IL‐6‐deficient condition in vivo and by IFN‐γ stimulation in vitro. Finally, we confirmed that in vivo injection of an anti‐PD‐L1 antibody or a Toll‐like receptor 3 ligand, polyinosinic‐polycytidylic acid, effectively inhibited tumorigenesis under the IL‐6‐deficient condition. Based on these findings, we speculate that a lack of IL‐6 produced in tumor‐bearing host augments induction of antitumor effector T cells and inhibits tumorigenesis in vivo, suggesting that IL‐6 signaling may be a promising target for the development of effective cancer immunotherapies.