Induction of tumor-specific acquired immunity against already established tumors by selective stimulation of innate DEC-205+ dendritic cells

Two major distinct subsets of dendritic cells (DCs) are arranged to regulate our immune responses in vivo; 33D1+ and DEC-205+ DCs. Using anti-33D1-specific monoclonal antibody, 33D1+ DCs were successfully depleted from C57BL/6 mice. When 33D1+ DC-depleted mice were stimulated with LPS, serum IL-12, but not IL-10 secretion that may be mediated by the remaining DEC-205+ DCs was markedly enhanced, which may induce Th1 dominancy upon TLR signaling. The 33D1+ DC-depleted mice, implanted with syngeneic Hepa1-6 hepatoma or B16-F10 melanoma cells into the dermis, showed apparent inhibition of already established tumor growth in vivo when they were subcutaneously (sc) injected once or twice with LPS after tumor implantation. Moreover, the development of lung metastasis of B16-F10 melanoma cells injected intravenously was also suppressed when 33D1+ DC-deleted mice were stimulated twice with LPS in a similar manner, in which the actual cell number of NK1.1+CD3− NK cells in lung tissues was markedly increased. Furthermore, intraperitoneal (ip) administration of a very small amount of melphalan (l-phenylalanine mustard; l-PAM) (0.25 mg/kg) in LPS-stimulated 33D1+ DC-deleted mice helped to induce H-2Kb-restricted epitope-specific CD8+ cytotoxic T lymphocytes (CTLs) among tumor-infiltrating lymphocytes against already established syngeneic E.G7-OVA lymphoma. These findings indicate the importance and effectiveness of selective targeting of a specific subset of DCs, such as DEC-205+ DCs alone or with a very small amount of anticancer drugs to activate both CD8+ CTLs and NK effectors without externally added tumor antigen stimulation in vivo and provide a new direction for tumor immunotherapy

Natural Splice Variant of MHC Class I Cytoplasmic Tail Enhances Dendritic Cell-Induced CD8+ T-Cell Responses and Boosts Anti-Tumor Immunity

Dendritic cell (DC)-mediated presentation of MHC class I (MHC-I)/peptide complexes is a crucial first step in the priming of CTL responses, and the cytoplasmic tail of MHC-I plays an important role in modulating this process. Several species express a splice variant of the MHC-I tail that deletes exon 7-encoding amino acids (Δ7), including a conserved serine phosphorylation site. Previously, it has been shown that Δ7 MHC-I molecules demonstrate extended DC surface half-lives, and that mice expressing Δ7-Kb generate significantly augmented CTL responses to viral challenge. Herein, we show that Δ7-Db-expressing DCs stimulated significantly more proliferation and much higher cytokine secretion by melanoma antigen-specific (Pmel-1) T cells. Moreover, in combination with adoptive Pmel-1 T-cell transfer, Δ7-Db DCs were superior to WT-Db DCs at stimulating anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival. Human DCs engineered to express Δ7-HLA-A*0201 showed similarly enhanced CTL stimulatory capacity. Further studies demonstrated impaired lateral membrane movement and clustering of human Δ7-MHC-I/peptide complexes, resulting in significantly increased bioavailability of MHC-I/peptide complexes for specific CD8+ T cells. Collectively, these data suggest that targeting exon 7-encoded MHC-I cytoplasmic determinants in DC vaccines has the potential to increase CD8+ T-cell stimulatory capacity and substantially improve their clinical efficacy

Human Monocytes Undergo Excessive Apoptosis following Temozolomide Activating the ATM/ATR Pathway While Dendritic Cells and Macrophages Are Resistant

Immunodeficiency is a severe therapy-limiting side effect of anticancer chemotherapy resulting from sensitivity of immunocompetent cells to DNA damaging agents. A central role in the immune system is played by monocytes that differentiate into macrophages and dendritic cells (DCs). In this study we compared human monocytes isolated from peripheral blood and cytokine matured macrophages and DCs derived from them and assessed the mechanism of toxicity of the DNA methylating anticancer drug temozolomide (TMZ) in these cell populations. We observed that monocytes, but not DCs and macrophages, were highly sensitive to the killing effect of TMZ. Studies on DNA damage and repair revealed that the initial DNA incision was efficient in monocytes while the re-ligation step of base excision repair (BER) can not be accomplished, resulting in an accumulation of DNA single-strand breaks (SSBs). Furthermore, monocytes accumulated DNA double-strand breaks (DSBs) following TMZ treatment, while DCs and macrophages were able to repair DSBs. Monocytes lack the DNA repair proteins XRCC1, ligase IIIα and PARP-1 whose expression is restored during differentiation into macrophages and DCs following treatment with GM-CSF and GM-CSF plus IL-4, respectively. These proteins play a key role both in BER and DSB repair by B-NHEJ, which explains the accumulation of DNA breaks in monocytes following TMZ treatment. Although TMZ provoked an upregulation of XRCC1 and ligase IIIα, BER was not enhanced likely because PARP-1 was not upregulated. Accordingly, inhibition of PARP-1 did not sensitize monocytes, but monocyte-derived DCs in which strong PARP activation was observed. TMZ induced in monocytes the DNA damage response pathways ATM-Chk2 and ATR-Chk1 resulting in p53 activation. Finally, upon activation of the Fas-receptor and the mitochondrial pathway apoptosis was executed in a caspase-dependent manner. The downregulation of DNA repair in monocytes, resulting in their selective killing by TMZ, might impact on the immune response during cancer chemotherapy

HER2-based recombinant immunogen to target DCs through FcγRs for cancer immunotherapy

Dendritic cell (DC)-based immunotherapy is an attractive approach to induce long lasting antitumor effector cells aiming to control cancer progression. DC targeting is a critical step in the design of DC vaccines in order to optimize delivery and processing of the antigen, and several receptors have been characterized for this purpose. In this study, we employed the FcγRs to target DCs both in vitro and in vivo. We designed a recombinant molecule (HER2-Fc) composed of the immunogenic sequence of the human tumor-associated antigen HER2 (aa 364–391) and the Fc domain of a human IgG1. In a mouse model, HER2-Fc cDNA vaccination activated significant T cell-mediated immune responses towards HER2 peptide epitopes as detected by IFN-γ ELIspot and induced longer tumor latency as compared to Ctrl-Fc-vaccinated control mice. Human in vitro studies indicated that the recombinant HER2-Fc immunogen efficiently targeted human DCs through the FcγRs resulting in protein cross-processing and in the activation of autologous HER2-specific CD8+ T cells from breast cancer patients

Lipid bodies containing oxidatively truncated lipids block antigen cross-presentation by dendritic cells in cancer

Tumor-associated dendritic cells are defective in their ability to cross-present antigens, and they accumulate lipid bodies. Here the authors show that this defect is due to an impaired trafficking of peptide-MHC class I caused by the interaction of electrophilic lipids with chaperone heat shock protein 70

Phenotypic and functional analysis of lymphocytes infiltrating osteolytic tumors: use as a possible therapeutic approach of osteosarcoma

BACKGROUND: Osteosarcoma is the most common type of primary bone tumor. The use of aggressive chemotherapy has drastically improved the prognosis of the patients with non-metastatic osteosarcomas, however the prognosis of the patients with metastasis is still very poor. Then, new and more effective treatments for curing osteosarcoma, such as immunotherapy are needed. Tumor-infiltrating lymphocytes (TIL) have been involved in the control of tumor development and already assessed with success for the treatment of several cancers including melanoma. While TIL represent a fascinating therapeutic approach in numerous malignant pathologies, there is few report concerning adult bone-associated tumors including osteosarcoma. METHODS: Human TIL were isolated and characterized (phenotype, lytic activity) from twenty-seven patients with bone-associated tumors (osteosarcoma, Ewing's sarcoma, giant cell tumor, chondrosarcoma, plasmocytoma and bone metastases). Similar experiments were performed using rat osteosarcoma model. RESULTS: While TIL with a main CD4(+ )profile were easily isolated from most of the tumor samples, only TIL extracted from osteosarcoma were cytotoxic against allogeneic tumor cells. In all cases, TIL lytic activity was significantly higher compared to autologous peripheral blood leukocytes. Similar data were observed in rat osteosarcoma model where TIL were characterized by a main CD4(+ )profile and high lytic activity against allogeneic and autologous tumor cells. Moreover, rat TIL expansion was not accompanied by refractoriness to further activation stimulus mainly by tumor antigens. CONCLUSION: These results demonstrated that TIL therapy could be a very efficient strategy for the treatment of adult osteosarcoma

Effect of B7.1 Costimulation on T-Cell Based Immunity against TAP-Negative Cancer Can Be Facilitated by TAP1 Expression

Tumors deficient in expression of the transporter associated with antigen processing (TAP) usually fail to induce T-cell-mediated immunity and are resistant to T-cell lysis. However, we have found that introduction of the B7.1 gene into TAP-negative (TAP−) or TAP1-transfected (TAP1+) murine lung carcinoma CMT.64 cells can augment the capacity of the cells to induce a protective immune response against wild-type tumor cells. Differences in the strength of the protective immune responses were observed between TAP− and TAP1+ B7.1 expressing CMT.64 cells depending on the doses of γ-irradiated cell immunization. While mice immunized with either high or low dose of B7.1-expressing TAP1+ cells rejected TAP− tumors, only high dose immunization with B7.1-expressing TAP− cells resulted in tumor rejection. The induced protective immunity was T-cell dependent as demonstrated by dramatically reduced antitumor immunity in mice depleted of CD8 or CD4 cells. Augmentation of T-cell mediated immune response against TAP− tumor cells was also observed in a virally infected tumor cell system. When mice were immunized with a high dose of γ-irradiated CMT.64 cells infected with vaccinia viruses carrying B7.1 and/or TAP1 genes, we found that the cells co-expressing B7.1 and TAP1, but not those expressing B7.1 alone, induced protective immunity against CMT.64 cells. In addition, inoculation with live tumor cells transfected with several different gene(s) revealed that only B7.1- and TAP1-coexpressing tumor cells significantly decreased tumorigenicity. These results indicate that B7.1-provoked antitumor immunity against TAP− cancer is facilitated by TAP1-expression, and thus both genes should be considered for cancer therapy in the future

From ‘Hellstrom Paradox–to anti-adenosinergic cancer immunotherapy

Cancer therapy by endogenous or adoptively transferred anti-tumor T cells is considered complementary to conventional cancer treatment by surgery, radiotherapy or chemotherapy. However, the scope of promising immunotherapeutic protocols is currently limited because tumors can create a ‘hostile–immunosuppressive microenvironment that prevents their destruction by anti-tumor T cells. There is a possibility to develop better and more effective immunotherapies by inactivating mechanisms that inhibit anti-tumor T cells in the tumor microenvironment and thereby protect cancerous tissues from immune damage. This may be now possible because of the recent demonstration that genetic deletion of immunosuppressive A2A and A2B adenosine receptors (A2AR and A2BR) or their pharmacological inactivation can prevent the inhibition of anti-tumor T cells by the hypoxic tumor microenvironment and as a result facilitate full tumor rejection [Ohta A, Gorelik E, Prasad SJ et al (2006) Proc Natl Acad Sci USA 103(35):13132–3137]. This approach is based on in vivo genetic evidence that A2AR play a critical role in the protection of normal tissues from overactive immune cells in acutely inflamed and hypoxic areas. The observations of much improved T-cell-mediated rejection of tumors in mice with inactivated A2AR strongly suggest that A2AR also protects hypoxic cancerous tissues and that A2AR should be inactivated in order to improve tumor rejection by anti-tumor T cells

Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status

BACKGROUND: Macrophages play a dual role in multiple sclerosis (MS) pathology. They can exert neuroprotective and growth promoting effects but also contribute to tissue damage by production of inflammatory mediators. The effector function of macrophages is determined by the way they are activated. Stimulation of monocyte-derived macrophages in vitro with interferon-γ and lipopolysaccharide results in classically activated (CA/M1) macrophages, and activation with interleukin 4 induces alternatively activated (AA/M2) macrophages. METHODS: For this study, the expression of a panel of typical M1 and M2 markers on human monocyte derived M1 and M2 macrophages was analyzed using flow cytometry. This revealed that CD40 and mannose receptor (MR) were the most distinctive markers for human M1 and M2 macrophages, respectively. Using a panel of M1 and M2 markers we next examined the activation status of macrophages/microglia in MS lesions, normal appearing white matter and healthy control samples. RESULTS: Our data show that M1 markers, including CD40, CD86, CD64 and CD32 were abundantly expressed by microglia in normal appearing white matter and by activated microglia and macrophages throughout active demyelinating MS lesions. M2 markers, such as MR and CD163 were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. Double staining with anti-CD40 and anti-MR revealed that approximately 70% of the CD40-positive macrophages in MS lesions also expressed MR, indicating that the majority of infiltrating macrophages and activated microglial cells display an intermediate activation status. CONCLUSIONS: Our findings show that, although macrophages in active MS lesions predominantly display M1 characteristics, a major subset of macrophages have an intermediate activation status