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TG4010, a Modified Vaccinia virus Ankara (MVA) expressing human mucin1 (MUC1) has demonstrated clinical benefit for patients suffering from advanced non-small cell lung cancer (NSCLC) in combination with chemotherapy. To support its development, preclinical experiments were performed with either TG4010 or β-galactosidase-encoding MVA vector (MVA-βgal) in mice presenting tumors in the lung. Tumor growth was obtained after intravenous injection of CT26 murine colon cancer cells, engineered to express either MUC1 or βgal. Mice showed increased survival rates after repeated intravenous injections of TG4010 or MVA-βgal, compared to an empty MVA control vector. Treatment with MVA vectors led to the accumulation of CD3dimCD8dim T cells, with two subpopulations characterized as KLRG1+CD127− short-lived effector cells (SLECs), and KLRG1−CD127− early effector cells (EECs) comprising cells releasing IFNγ, Granzyme B and CD107a upon antigen-specific peptide stimulation. EECs were characterized by an up-regulation of PD-1. Tumor growth in the diseased lung correlated with the appearance of PD1+ Treg cells that partially disappeared after TG4010 treatment. At late stage of tumor development in the lung, PD-L1 was detected on CD45− tumor cells, on CD4+ cells, including Treg cells, on CD3+CD8+ and CD3dimCD8dim T lymphocytes, on NK cells, on MDSCs and on alveolar macrophages. We demonstrated that targeting the PD-1/PD-L1 pathway with blocking monoclonal antibodies several days after TG4010 treatment, at late stage of tumor development, enhanced the therapeutic protection induced by the vaccine, supporting the ongoing clinical evaluation of TG4010 immunotherapy in combination with Nivolumab.

Effector T-cell access to tumor tissue is a limiting step for clinical efficacy of antigen-specific T cell–based immunotherapies. Ectopic mouse tumor models, in which a subcutaneously (s.c.) implanted tumor is treated with s.c. or intramuscular therapeutic immunization, may not be optimal for targeting effector T cells to an organ-borne tumor. We used an orthotopic renal carcinoma model to evaluate the impact of injection routes on therapeutic efficacy of a Modified Vaccinia virus Ankara viral vector expressing the human mucin 1 tumor–associated xeno-antigen (MVA-MUC1). We show that intravenous (i.v.) administration of MVA-MUC1 displayed enhanced efficacy when compared with s.c. injection. Therapeutic efficacy of MVA-MUC1 was further enhanced by i.v. injection of a TLR9 agonist. In all cases, infiltration of tumor-bearing kidney by CD8+ lymphocytes was associated with control of tumor growth. Biodistribution experiments indicate that, following i.v. injection, MVA-encoded antigens are quickly expressed in visceral organs and, in particular, in splenic antigen-presenting cells, compared with those following s.c. injection. This appears to result in a faster generation of MUC1-specific CD8+ T cells. Lymphocytes infiltrating tumor-bearing kidneys are characterized by an effector memory phenotype and express PD-1 and Tim3 immune checkpoint molecules. Therapeutic efficacy was associated with a modification of the tumor microenvironment toward a Th1-type immune response and recruitment of activated lymphocytes. This study supports the clinical evaluation of MVA-based immunotherapies via the i.v. route. Cancer Immunol Res; 2(12); 1163–74. ©2014 AACR.

Women showing normal cytology but diagnosed with a persistent high-risk human papillomavirus (HR-HPV) infection have a higher risk of developing high-grade cervical intraepithelial neoplasia and cervical cancer than noninfected women. As no therapeutic management other than surveillance is offered to these women, there is a major challenge to develop novel targeted therapies dedicated to the treatment of these patients. As such, E1 and E2 antigens, expressed early in the HPV life cycle, represent very interesting candidates. Both proteins are necessary for maintaining coordinated viral replication and gene synthesis during the differentiation process of the epithelium and are essential for the virus to complete its normal and propagative replication cycle. In the present study, we evaluated a new active targeted immunotherapeutic, a modified vaccinia virus Ankara (MVA) vector containing the E1 sequence of HPV16, aimed at inducing cellular immune responses with the potential to help and clear persistent HPV16-related infection. We carried out an extensive comparative time course analysis of the cellular immune responses induced by different schedules of immunization in C57BL/6 mice. We showed that multiple injections of MVA-E1 allowed sustained HPV16 E1-specific cellular immune responses in vaccinated mice and had no impact on the exhaustion phenotype of the generated HPV16 E1-specific CD8+T cells, but they led to the differentiation of multifunctional effector T cells with high cytotoxic capacity. This study provides proof of concept that an MVA expressing HPV16 E1 can induce robust and long-lasting E1-specific responses and warrants further development of this candidate.

Cancer progression has been associated with the presence of tumor-associated M2-macrophages (M2-TAMs) able to inhibit anti-tumor immune responses. It is also often associated with metastasis-induced bone destruction mediated by osteoclasts. Both cell types are controlled by the CD115 (CSF-1R)/colony-stimulating factor-1 (CSF-1, M-CSF) pathway, making CD115 a promising target for cancer therapy. Anti-human CD115 monoclonal antibodies (mAbs) that inhibit the receptor function have been generated in a number of laboratories. These mAbs compete with CSF-1 binding to CD115, dramatically affecting monocyte survival and preventing osteoclast and macrophage differentiation, but they also block CD115/CSF-1 internalization and degradation, which could lead to potent rebound CSF-1 effects in patients after mAb treatment has ended. We thus generated and selected a non-ligand competitive anti-CD115 mAb that exerts only partial inhibitory effects on CD115 signaling without blocking the internalization or the degradation of the CD115/CSF-1 complex. This mAb, H27K15, affects monocyte survival only minimally, but downregulates osteoclast differentiation and activity. Importantly, it inhibits monocyte differentiation to CD163(+)CD64(+) M2-polarized suppressor macrophages, skewing their differentiation toward CD14(-)CD1a(+) dendritic cells (DCs). In line with this observation, H27K15 also drastically inhibits monocyte chemotactic protein-1 secretion and reduces interleukin-6 production; these two molecules are known to be involved in M2-macrophage recruitment. Thus, the non-depleting mAb H27K15 is a promising anti-tumor candidate, able to inhibit osteoclast differentiation, likely decreasing metastasis-induced osteolysis, and able to prevent M2 polarization of TAMs while inducing DCs, hence contributing to the creation of more efficient anti-tumor immune responses.

Athough the clinical efficacy of oncolytic viruses has been demonstrated for local treatment, the ability to induce immune-mediated regression of distant metastases is still poorly documented. We report here that the engineered oncolytic vaccinia virus VVWR-TK−RR−-Fcu1 can induce immunogenic cell death and generate a systemic immune response. Effects on tumor growth and survival was largely driven by CD8+ T cells, and immune cell infiltrate in the tumor could be reprogrammed toward a higher ratio of effector T cells to regulatory CD4+ T cells. The key role of type 1 IFN pathway in oncolytic virotherapy was also highlighted, as we observed a strong abscopal response in Ifnar−/− tumors. In this model, single administration of virus directly into the tumors on one flank led to regression in the contralateral flank. Moreover, these effects were further enhanced when oncolytic treatment was combined with immunogenic chemotherapy or with immune checkpoint blockade. Taken together, our results suggest how to safely improve the efficacy of local oncolytic virotherapy in patients whose tumors are characterized by dysregulated IFNα signaling. Cancer Res; 77(15); 4146–57. ©2017 AACR.

Vaccination with recombinant adenylate cyclase of Bordetella pertussis (CyaA) carrying antigen is a promising approach to target antigen-presenting cells. We have used Trogocytosis Analysis Protocol (TRAP) assays to monitor immune responses raised by different vaccination regimens with recombinant CyaA carrying the ovalbumin antigen. We find that the intradermal, intramuscular or subcutaneous routes are all superior to intravenous injections, and actually lead to a sufficiently high frequency of reactive CTL to be detected and characterized directly ex vivo by TRAP assay or other standard assays. Finally, for all routes, we find a clear boosting effect upon re-injection of the vaccine.

Targeting the human papillomavirus (HPV) E7 antigen to dendritic cells with the adenylate cyclase (CyaA) of Bordetella pertussis as a vaccine vector led to potent therapeutic immune responses against TC-1 tumors in a murine model of cervical carcinoma induced by HPV. However, as the time between tumor graft and vaccination increased, the antitumor efficacy of the CyaA-E7 vaccine gradually decreased. The vaccine had no effect if the tumor diameter was >8 mm. Analyses of regulatory cells recruited during TC-1 tumor growth revealed a high number of splenic MDSCs and a large percentage of regulatory T cells, particularly in the tumor. Administration of a tritherapy including CpG complexed with a cationic lipid, low-dose cyclophosphamide, and the CyaA-E7 vaccine completely overcame tumor-associated immunosuppression and eradicated large, established tumors in almost all treated animals. This strong antitumor response was followed by a large expansion of regulatory T cells in tumor, spleen, and tumor-draining lymph nodes and of splenic neutrophils. These findings indicate that immunotherapeutic strategies that simultaneously target innate, adaptive, and regulatory components of the immune system are effective in the eradication of large tumors. [Cancer Res 2007;67(18):8847–55]

Trogocytosis, the process whereby lymphocytes capture membrane components from the cells they interact with, is classically evidenced by the transfer of fluorescent lipophilic compounds or biotinylated proteins from target cells to T or B cells. A particular class of molecules, not studied explicitly so far in the context of trogocytosis is glycoconjugates. Here, we used a method to metabolically install chemical labels in target cell glycoconjugates. Working with those target cells, we describe the conditions allowing CTL to be detected based on glycoconjugate trogocytosis triggered by antigen or stimulatory antibodies. Accordingly, we used this method to monitor the CTL response triggered in mice after vaccination. In addition, we documented the applicability of this approach to the detection of CD4(+) T and B cells. Overall, glycoconjugates were transferred between target cells and lymphocytes during trogocytosis with efficiencies comparable or higher than measured for biotinylated proteins or lipophilic dyes incorporated into general membrane lipids. From a technological point of view, our approach can be employed to detect reactive lymphocytes via glycoconjugate trogocytosis. More generally, we believe that the ever-growing ability to employ chemistry in living systems to label particular compounds will be powerful in unraveling the contributions of glycosylation to various aspects of T and B cells biology.

Detection, quantification, separation and characterization of T and B cells reactive to specific antigens are important tasks in both basic and clinical immunology. Here, we describe an approach allowing the performance of all four tasks on a functional basis by flow cytometry. The assay is based on the property of lymphocytes to capture membrane components from the cells they interact with, in a process we call trogocytosis. Working with CD8+ CTL and target cells labeled with membrane markers, we describe the conditions allowing reactive lymphocytes to be detected rapidly and inexpensively within mixed populations. Accordingly, we used this method to monitor the CTL response triggered in mice after vaccination. In addition, we documented the applicability of this method to the detection of antigen-specific CD4+ T and B cells. While our method is, for the time being, not as sensitive as staining of CTL with MHC class I multimers, it allows the simultaneous quantitative identification of reactive CD8+, CD4+ and B cells. Altogether, our method offers a simple and general alternative to other methods previously described to detect and quantify lymphocyte reactivity, and it can also be used in combination with those.

International audience; The immune system is routinely confronted with cell death resulting from the physiological turnover of renewable tissues, as well as from pathological insults of several types. We hypothesize the existence of a mechanism that allows the immune system to discriminate between physiological and pathological instances of cell death, but the factors that determine whether cellular demise is perceived as a neutral, tolerogenic or immunogenic event remain unclear 1. Infectious insults are accompanied by so-called microbe-associated molecular patterns (MAMPs), i.e., viral or bacterial products that activate immune cells through a panel of pattern-recognition receptors (PRRs) 2. Moreover, intracellular pathogens generally trigger adaptive mechanisms aimed toward the re-establishment of homeosta-sis, including the unfolded protein response (UPR) and autophagy 3,4. In mammals, MAMPs coupled to the activation of stress responses Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell-mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN- and IFN- receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN-related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy. npg

To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293 cell lines double positive for human Toll-like receptors (TLRs) and an NF-κB-inducible reporter gene. Screening of a large variety of compounds and cellular extracts detected a TLR3-activating compound in a microsomal yeast extract. Fractionation of this extract identified an RNA molecule of 4.6 kb, named nucleic acid band 2 (NAB2), that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to that of the genome of the double-stranded RNA (dsRNA) L-BC virus of Saccharomyces cerevisiae . A large-scale process of production and purification of this RNA was established on the basis of chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin but neither NAB2 nor Lipofectin alone induced the secretion of interleukin-12(p70) [IL-12(p70)], alpha interferon, gamma interferon-induced protein 10, macrophage inflammatory protein 1β, or IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 also signaled via the cytoplasmic sensor for RNA recognition MDA-5. A significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding modified vaccinia virus Ankara (MVA) and NAB2-Lipofectin. This combination of immunotherapies strongly increased at the injection sites the percentage of infiltrating natural killer (NK) cells and plasmacytoid dendritic cells (pDCs), cell types which can modulate innate and adaptive immune responses. IMPORTANCE Virus-based cancer vaccines offer a good alternative to the treatment of cancer but could be improved. Starting from a screening approach, we have identified and characterized an unexplored biological molecule with immunomodulatory characteristics which augments the efficacy of an MVA-based immunotherapeutic agent. The immune modulator consists of the purified dsRNA genome isolated from a commercially used yeast strain, NAB2, mixed with a cationic lipid, Lipofectin. NAB2-Lipofectin stimulates the immune system via TLR3 and MDA-5. When it was injected at the MVA vaccination site, the immune modulator increased survival in a preclinical tumor model. We could demonstrate that NAB2-Lipofectin augments the MVA-induced infiltration of natural killer and plasmacytoid dendritic cells. We suggest indirect mechanisms of activation of these cell types by the influence of NAB2-Lipofectin on innate and adaptive immunity. Detailed analysis of cell migration at the vaccine injection site and the appropriate choice of an immune modulator should be considered to achieve the rational improvement of virus vector-based vaccination by immune modulators.

Expression of small stress proteins (shsp) enhances the survival of mammalian cells exposed to heat or oxidative injuries. Recently, we have shown that the expression of shsp from different species, such as human hsp27, Drosophila hsp27 or human alphaB-crystallin protected murine L929 cells against cell death induced by tumor necrosis factor (TNFalpha), hydrogen peroxide or menadione. Here, we report that, in growing L929 cell lines, the presence of these shsp decreased the intracellular level of reactive oxygen species (ROS). shsp expression also abolished the burst of intracellular ROS induced by TNFalpha. Several downstream effects resulting from the TNFalpha-mediated ROS increment, such as NF-kappaB activation, lipid peroxidation and protein oxidation, were inhibited by shsp expression. We also report that the expression of these different shsp raised the total glutathione level in both L929 cell lines and transiently transfected NIH 3T3-ras cells. This phenomenon was essential for the shsp-mediated decrease in ROS and resistance against TNFalpha. Our results therefore suggest that the protective activity shared by human hsp27, Drosophila hsp27 and human alphaB-crystallin against TNFalpha-mediated cell death and probably other types of oxidative stress results from their conserved ability to raise the intracellular concentration of glutathione.

High-risk human papillomaviruses (HPV) such as HPV16 are associated with the development of cervical cancer. The HPV16-E6 and HPV16-E7 oncoproteins are expressed throughout the replicative cycle of the virus and are necessary for the onset and maintenance of malignant transformation. Both these tumor-specific antigens are considered as potential targets for specific CTL-mediated immunotherapy. The adenylate cyclase (CyaA) of Bordetella pertussis is able to target dendritic cells through specific interaction with the alpha(M)beta(2) integrin. It has been previously shown that this bacterial protein could be used to deliver CD4(+) and CD8(+) T cell epitopes to the MHC class II and class I presentation pathways to trigger specific Th and CTL responses in vivo, providing protection against subsequent viral or tumoral challenge. Here, we constructed recombinant CyaA containing either the full sequence or various subfragments from the HPV16-E7 protein. We show that, when injected to C57BL/6 mice in absence of any adjuvant, these HPV16-recombinant CyaAs are able to induce specific Th1 and CTL responses. Furthermore, when injected into mice grafted with HPV16-E7-expressing tumor cells (TC-1), one of these recombinant proteins was able to trigger complete tumor regression in 100% of the animals tested. This therapeutic efficacy compared favorably to that of strongly adjuvanted peptide and was marginally affected by prior immunity to CyaA protein. This study represents the first in vivo demonstration of the antitumoral therapeutic activity of recombinant CyaA proteins carrying human tumor-associated antigens and paves the way for the testing of this vector in clinical trials.

The protective activity of small stress proteins (sHsp) against H2O2-mediated cell death in the highly sensitive murine L929 fibroblast has been analyzed. We report here that the human Hsp27- and murine Hsp25-mediated rise in glutathione (GSH) levels as well as the maintenance of this redox modulator in its reduced form was directly responsible for the protection observed at the level of cell morphology and mitochondrial membrane potential. sHsp expression also buffered the increase in protein oxidation following H2O2 treatment and protected several key enzymes against inactivation. In this case, however, the protection necessitated both an increase in GSH and the presence of sHsp per se since the pattern of protection against protein oxidation mediated by a simple GSH increase was different from that induced by sHsp expression. Among the enzymes analyzed, we noticed that sHsp significantly increased glucose-6-phosphate dehydrogenase (G6PD) activity and to a lesser extent glutathione reductase and glutathione transferase activities. Moreover, an increased GSH level was observed in G6PD-overexpressing L929 cell clones. Taken together our results suggest that sHsp protect against oxidative stress through a G6PD-dependent ability to increase and uphold GSH in its reduced form and by using this redox modulator as an essential parameter of their in vivo chaperone activity against oxidized proteins.

Tumor progression is promoted by Tumor-Associated Macrophages (TAMs) and metastasis-induced bone destruction by osteoclasts. Both myeloid cell types depend on the CD115-CSF-1 pathway for their differentiation and function. We used 3 different mouse cancer models to study the effects of targeting cancer host myeloid cells with a monoclonal antibody (mAb) capable of blocking CSF-1 binding to murine CD115. In mice bearing sub-cutaneous EL4 tumors, which are CD115-negative, the anti-CD115 mAb depleted F4/80(+) CD163(+) M2-type TAMs and reduced tumor growth, resulting in prolonged survival. In the MMTV-PyMT mouse model, the spontaneous appearance of palpable mammary tumors was delayed when the anti-CD115 mAb was administered before malignant transition and tumors became palpable only after termination of the immunotherapy. When administered to mice already bearing established PyMT tumors, anti-CD115 treatment prolonged their survival and potentiated the effect of chemotherapy with Paclitaxel. As shown by immunohistochemistry, this therapeutic effect correlated with the depletion of F4/80(+)CD163(+) M2-polarized TAMs. In a breast cancer model of bone metastasis, the anti-CD115 mAb potently blocked the differentiation of osteoclasts and their bone destruction activity. This resulted in the inhibition of cancer-induced weight loss. CD115 thus represents a promising target for cancer immunotherapy, since a specific blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasis-induced bone destruction through osteoclast inhibition.

Small stress proteins protect against the cytotoxicity mediated by oxidative stress. The relationship between Hsp25 expression and the integrity of the actin network was studied in H2O2-treated murine L929 fibrosarcoma cells overexpressing endogenous wild-type (wt-) or non-phosphorylatable mutant (mt-) Hsp25. We show here that both proteins prevented actin network disruption induced by a 1 h treatment with 400 microM H2O2. In contrast, SB203580, a p38 MAPkinase inhibitor which suppresses Hsp25 phosphorylation, abolished the protective activity conferred by both wt- and mt-Hsp25. Hence, phosphorylation does not appear essential for Hsp25 protective activity against H2O2-induced actin disruption, and SB203580-sensitive events other than Hsp25 phosphorylation may be important for actin network regulation. Since, in L929 cells, wt- or mt-Hsp25 expression modulates intracellular glutathione levels, analyses were performed which revealed a direct correlation between glutathione and the integrity of the actin network. Moreover, laser scanning confocal immunofluorescences revealed that only a small fraction of wt- or mt-Hsp25 colocalized with actin microfilaments. Taken together, our results suggest that, in L929 cells, the protection against actin network disruption is probably a consequence of the redox change mediated by Hsp25 rather than a direct effect of this stress protein towards actin.

The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.