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Soft agar colony formation assay of cells transfected with xCT

Analysis of the cellular response induced by anti-xCT vaccination

qPCR analysis of xCT expression in tumorspheres

Effect of immunotherapy with non CSC-specific targets

Evaluation of efficacy of anti-xCT vaccination on TUBO or 4T1 derived lung metastases

Cellular localization and correlation with breast cancer patient clinical outcome (CO) of genes differentially expressed in TUBO cells and tumorspheres

Resistance to therapy and lack of curative treatments for metastatic breast cancer suggest that current therapies may be missing the subpopulation of chemoresistant and radioresistant cancer stem cells (CSC). The ultimate success of any treatment may well rest on CSC eradication, but specific anti-CSC therapies are still limited. A comparison of the transcriptional profiles of murine Her2+ breast tumor TUBO cells and their derived CSC-enriched tumorspheres has identified xCT, the functional subunit of the cystine/glutamate antiporter system xc−, as a surface protein that is upregulated specifically in tumorspheres. We validated this finding by cytofluorimetric analysis and immunofluorescence in TUBO-derived tumorspheres and in a panel of mouse and human triple negative breast cancer cell-derived tumorspheres. We further show that downregulation of xCT impaired tumorsphere generation and altered CSC intracellular redox balance in vitro, suggesting that xCT plays a functional role in CSC biology. DNA vaccination based immunotargeting of xCT in mice challenged with syngeneic tumorsphere-derived cells delayed established subcutaneous tumor growth and strongly impaired pulmonary metastasis formation by generating anti-xCT antibodies able to alter CSC self-renewal and redox balance. Finally, anti-xCT vaccination increased CSC chemosensitivity to doxorubicin in vivo, indicating that xCT immunotargeting may be an effective adjuvant to chemotherapy. Cancer Res; 76(1); 62–72. ©2015 AACR.

Immunofluorescence analysis of xCT expression in human tissues

Background The success of immunotherapeutics in oncology and the search for further improvements has prompted revisiting the use of cancer vaccines. In this context, knowledge of the immunogenic epitopes and the monitoring of the immune response cancer vaccines generate are essential. MUC1 has been considered one of the most important tumor associated antigen for decades. Methods To identify HLA-restricted MUC1 peptides we used eight human MHC class I transgenic mouse lines, together covering more than 80% of the human population. MUC1 peptides were identified by vaccinating each line with full length MUC1 coding sequences and using an IFNγ ELIspot restimulation assay. Relevant peptides were tested in a flow cytometry-based tetramer assay and for their capacity to serve as target in an in vivo killing assay. Results Four previously identified MUC1 peptides were confirmed and five are described here for the first time. These nine peptide-MHC combinations were further characterized. Six gave above-background tetramer staining of splenocytes from immunized animals and three peptides were induced more than 5% specific in vivo killing. Conclusions These data describe for the first time five new HLA class I-restricted peptides and revisit some that were previously described. They also emphasize the importance of using in vivo/ex vivo models to screen for immunogenic peptides and define the functions for individual peptide-HLA combinations. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1254-0) contains supplementary material, which is available to authorized users.

Organ-specific cell types are maintained by tissue homeostasis and may vary in nature and/or frequency in pathological situations. Moreover, within a cell lineage, some sub-populations, defined by combinations of cell-surface markers, may have specific functions. Dendritic cells are the epitome of such a population as they may be subdivided into discrete sub-groups with defined functions in specific compartments of various organs. Technically, to study the distribution of DC sub-populations, it involves performing multiparametric immunofluorescence on well-conserved organ structures. However, immunodetection may be impacted by protein cross-linking and antigenic epitope masking by the use of 10% neutral-buffered formalin. To circumvent this and to preserve a good morphological tissue structure, we evaluated alternative fixatives such as Periodate Lysine Paraformaldehyde or Tris Zinc fixatives in combination with other embedding techniques. The cryosection protocols were adapted for optimal antigen detection but offered a poor morphological preservation. We therefore developed a new methodology based on Tris Zinc fixative, gelatin-sucrose embedding and freezing. Using multiple DC markers, we demonstrate that this treatment is an optimal protocol for cell-surface marker detection on high-quality tissue sections.

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.

Resistance to therapy and lack of curative treatments for metastatic breast cancer suggest that current therapies may be missing the subpopulation of chemoresistant and radioresistant cancer stem cells (CSC). The ultimate success of any treatment may well rest on CSC eradication, but specific anti-CSC therapies are still limited. A comparison of the transcriptional profiles of murine Her2+ breast tumor TUBO cells and their derived CSC-enriched tumorspheres has identified xCT, the functional subunit of the cystine/glutamate antiporter system xc−, as a surface protein that is upregulated specifically in tumorspheres. We validated this finding by cytofluorimetric analysis and immunofluorescence in TUBO-derived tumorspheres and in a panel of mouse and human triple negative breast cancer cell-derived tumorspheres. We further show that downregulation of xCT impaired tumorsphere generation and altered CSC intracellular redox balance in vitro, suggesting that xCT plays a functional role in CSC biology. DNA vaccination based immunotargeting of xCT in mice challenged with syngeneic tumorsphere-derived cells delayed established subcutaneous tumor growth and strongly impaired pulmonary metastasis formation by generating anti-xCT antibodies able to alter CSC self-renewal and redox balance. Finally, anti-xCT vaccination increased CSC chemosensitivity to doxorubicin in vivo, indicating that xCT immunotargeting may be an effective adjuvant to chemotherapy. Cancer Res; 76(1); 62–72. ©2015 AACR.

Cancer immunotherapy is hampered by the immunosuppression maintained by regulatory T cells (Tregs) in tumor-bearing hosts. Stimulation of the Toll-like receptor 2 (TLR2) by Pam3Cys is known to affect Treg-mediated suppression. We found that Pam3Cys increases the proliferation of both CD4(+) effector T cells (Teffs) and Tregs co-cultured in vitro, but did not induce the proliferation of Tregs alone upon CD3 and CD28 stimulation. In a mouse model of RMA-MUC1 tumors, Pam3Cys was administered either alone or in combination with a modified vaccinia ankara (MVA)-based mucin 1 (MUC1) therapeutic vaccine. The combination of Pam3Cys with MVA-MUC1 (1) diminished splenic Treg/CD4(+) T-cell ratios to those found in tumor-free mice, (2) stimulated a specific anti-MUC1 interferon γ (IFNγ) response and (3) had a significant therapeutic effect on tumor growth and mouse survival. When CD4(+) Teffs and Tregs were isolated from Pam3Cys-treated mice, Teffs had become resistant to Treg-mediated suppression while upregulating the expression of BclL-x(L). Tregs from Pam3Cys-treated mice were fully suppressive for Teffs from naïve mice. Bcl-x(L) was induced by Pam3Cys with different kinetics in Tregs and Teffs. Teff from Pam3Cys-treated mice produced increased levels of Th1 and Th2-type cytokines and an interleukin (IL)-6-dependent secretion of IL-17 was observed in Teff:Treg co-cultures, suggesting that TLR2 stimulation had skewed the immune response toward a Th17 profile. Our results show for the first time that in a tumor-bearing host, TLR2 stimulation with Pam3Cys affects both Tregs and Teffs, protects Teff from Treg-mediated suppression and has strong therapeutic effects when combined with an MVA-based antitumor vaccine.

First-generation adenovirus vectors, deleted in the E1 and E3 regions of the genome, induce a strong inflammatory response that affects persistence of vector DNA in transduced organs and causes toxicity in the host. Wild-type adenovirus encodes a number of proteins that are nonessential for viral propagation in vitro but that dampen the inflammatory and immune responses mounted by the host during infection. The adenovirus E3 region-encoded 14.7K protein inhibits tumor necrosis factor alpha (TNF-alpha)-induced apoptosis and arachidonic acid synthesis. To evaluate the impact of constitutive expression of the 14.7K protein on vector-induced pathology, toxicity, and DNA persistence, we constructed vectors that contain a cytomegalovirus promoter-driven 14.7K expression cassette. Although these vectors inhibit TNF-alpha-induced apoptosis in vitro, they do not show better vector DNA persistence, or lower inflammation or pathology than E3-deleted first-generation vector in mouse models. However, the 14.7K protein is functional in mice because animals injected intravenously with a 14.7K-constitutive vector were fully protected against a lethal dose of lipopolysaccharide 5 days after vector administration. These results open new applications for the E3-encoded 14.7K protein, which can be used to protect organs against inflammatory reactions and TNF-alpha-mediated apoptosis.

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.

Experimental evidence shows the importance of the immune system in controlling tumor appearance and growth. Immunotherapy is defined as the treatment of a disease by inducing, enhancing or suppressing an immune response. In the context of cancer treatment, it involves breaking tolerance to a cancer-specific self-antigen and/or enhancing the existing anti-tumor immune response, be it specific or not. Part of the complexity in developing such treatment is that cancers are selected to escape adaptive or innate immune responses. These escape mechanisms are numerous and they may cumulate in one cancer. Moreover, different cancers of a same type may present different combinations of escape mechanisms. The limited success of immunotherapeutics in the clinic as stand-alone products may in part be explained by the fact that most of them only activate one facet of the immune response. It is important to identify novel methods to broaden the efficacy of immunotherapeutics. Calcium signaling is central to numerous cellular processes, leading to immune responses, cancer growth and apoptosis induced by cancer treatments. Calcium signaling in cancer therapy and control will be integrated to current cancer immunotherapy approaches. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

The relation between an antigenic peptide that can stimulate a mature T cell and the natural peptide that promoted selection of this cell in the thymus is still unknown. An experimental system was devised to address this issue in vivo—mice expressing neopeptides in thymic stromal cells after adenovirus-mediated delivery of invariant chain-peptide fusion proteins. In this system, selection of T cells capable of responding to a given antigenic peptide could be promoted by the peptide itself, by closely related analogs lacking agonist and antagonist activity, or by ostensibly unrelated peptides. However, the precise repertoire of T cells selected was dictated by the particular neopeptide expressed.

We have generated a panel of transgenic mice expressing HLA-A*01:03, -A*24:02, -B*08:01, -B*27:05, -B*35:01, -B*44:02, or -C*07:01 as chimeric monochain molecules (i.e., appropriate HLA α1α2 H chain domains fused with a mouse α3 domain and covalently linked to human β2-microglobulin). Whereas surface expression of several transgenes was markedly reduced in recipient mice that coexpressed endogenous H-2 class I molecules, substantial surface expression of all human transgenes was observed in mice lacking H-2 class I molecules. In these HLA monochain transgenic/H-2 class I null mice, we observed a quantitative and qualitative restoration of the peripheral CD8+ T cell repertoire, which exhibited a TCR diversity comparable with C57BL/6 WT mice. Potent epitope-specific, HLA-restricted, IFN-γ–producing CD8+ T cell responses were generated against known reference T cell epitopes after either peptide or DNA immunization. HLA-wise, these new transgenic strains encompass a large proportion of individuals from all major human races and ethnicities. In combination with the previously created HLA-A*02:01 and -B*07:02 transgenic mice, the novel HLA transgenic mice described in this report should be a versatile preclinical animal model that will speed up the identification and optimization of HLA-restricted CD8+ T cell epitopes of potential interest in various autoimmune human diseases and in preclinical evaluation of T cell–based vaccines.

Therapeutic vaccines are different from the well-known prophylactic vaccines in that they are designed to treat patients already suffering from a disease instead of preventing the disease in healthy individuals. Several therapeutic vaccines are today in late-stage clinical development for non-small-cell lung cancer. These vaccines use different approaches including peptides, cell lines and viral vectors, and explore different settings within the pathology. Some are given in monotherapy while others are combined with the classic therapies used with non-small-cell lung cancer. This review gives a summary of the therapeutic vaccines currently in late-stage clinical development for non-small-cell lung cancer.

Aim. To investigate the ability of recombinant modified vaccinia virus Ankara (rMVA) vector to induce an immune response against a well-tolerated self-antigen.Methods. rMVA vectors expressing different form ofα-fetoprotein (AFP) were produced and characterized. Naïve mice were vaccinated with MVA vectors expressing the AFP antigen in either a secreted, or a membrane-bound, or an intracellular form. The immune response was monitored by an IFNΓ ELISpot assay and antibody detection.Results. Vaccination with the membrane-associated form of AFP induced a stronger CD8+T-cell response compared to the ones obtained with the MVA encoding the secreted or the intracellular forms of AFP. Moreover, the vaccination with the membrane-bound AFP elicited the production of AFP-specific antibodies.Conclusions. The AFP transmembrane form is more immunogenic. Expressing a membrane-bound form in the context of an MVA vaccination could enhance the immunogenicity of a self-antigen.

The aim of this study was to monitor in vivo with low field MRI growth of a murine orthotopic glioma model following a suicide gene therapy.The gene therapy consisted in the stereotactic injection in the mice brain of a modified vaccinia virus Ankara (MVA) vector encoding for a suicide gene (FCU1) that transforms a non toxic prodrug 5-fluorocytosine (5-FC) to its highly cytotoxic derivatives 5-fluorouracil (5-FU) and 5'-fluorouridine-5'monophosphate (5'-FUMP). Using a warmed-up imaging cell, sequential 3D T1 and T2 0.1T MRI brain examinations were performed on 16 Swiss female nu/nu mice bearing orthotopic human glioblastoma (U87-MG cells). The 6-week in vivo MRI follow-up consisted in a weekly measurement of the intracerebral tumor volume leading to a total of 65 examinations. Mice were divided in four groups: sham group (n=4), sham group treated with 5-FC only (n=4), sham group with injection of MVA-FCU1 vector only (n=4), therapy group administered with MVA-FCU1 vector and 5-FC (n=4). Measurements of tumor volumes were obtained after manual segmentation of T1- and T2-weighted images.Intra-observer and inter-observer tumor volume measurements show no significant differences. No differences were found between T1 and T2 volume tumor doubling times between the three sham groups. A significant statistical difference (p0.05) in T1 and T2 volume tumor doubling times between the three sham groups and the animals treated with the intratumoral injection of MVA-FCU1 vector in combination with 2 weeks per os 5-FC administration was demonstrated.Preclinical low field MRI was able to monitor efficacy of suicide gene therapy in delaying the tumor growth in an in vivo mouse model of orthotopic glioblastoma.

Cancer progression has been associated with the presence of tumor-associated M2-type macrophages (M2-TAMs) able to inhibit anti-tumor immune responses, stimulate neo-angiogenesis and facilitate metastasis. Colony-stimulating factor-1 (CSF-1, M-CSF) is a cytokine required for the survival and differentiation of myeloid cell lineages, and CSF-1 signaling is known to polarize macrophages towards the M2-type. M2-TAMs can represent the most abundant immunosuppressive cell population in the tumor microenvironment, notably recruited by CSF-1 and MCP-1/CCL2. Transgene has developed a monoclonal antibody (mAb), TG3003, directed against the CSF-1 cell-surface receptor, CD115 (CSF-1R, M-CSFR). This mAb does not block the binding of CSF-1 to its receptor, but down-modulates CD115 signaling. In contrast to other anti-CD115 mAbs currently in development, whose modes of action rely on the blockade of ligand binding, TG3003 is not cytotoxic to normal myeloid cells that require CD115-mediated signaling for their survival. In vitro, TG3003 skews monocyte differentiation from M2-type macrophages towards dendritic cells, most potent antigen-presenting cells capable of stimulating efficacious T cell responses. It inhibits the secretion of MCP-1/CCL2 by differentiating macrophages and decreases their IL-6 production. Through this inhibition of M2-TAMs, TG3003 may potentiate immune responses in patients and impact on tumor progression. Moreover, due to its unique non-competitive mode of action, TG3003 does not block the physiological pathway for CSF-1 clearing from the circulation, thus avoiding the issue of toxic or rebound effects in treated patients. To investigate the properties of TG3003 in vivo, we have generated a transgenic mouse strain where the mAb epitope has been inserted into murine CD115 without affecting murine CSF-1 binding nor signaling. We will present the results of preclinical proof-of-concept experiments validating the mechanism of action and the immunomodulatory properties of mAb TG3003. Citation Format: Hélène Haegel, Christelle Ziller-Remy, Luc Barraud, Jean-Yves Bonnefoy, Sandrine Cochin, Vanessa Duong, Michel Geist, Benoit Grellier, Rémy Hallet, Jean-Baptiste Marchand, Thierry Menguy, Ronald Rooke, Christine Thioudellet, Carine Reymann, Xavier Préville. TG3003, an immunomodulatory anti-CD115 mAb targeting M2-macrophage polarization in the tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 288. doi:10.1158/1538-7445.AM2015-288

We report here that the anticancer drug hexadecylphosphocholine (HPC) can control plasmid DNA-mediated gene transfer to renal carcinoma following intratumoral administration. Significant improvement of gene expression levels could be achieved depending on HPC dose administered. Optimal concentration of HPC co-injected with plasmid DNA was found to be 0.2% (w/v) showing up to a 10-fold increase in reporter gene expression levels when compared to DNA administered alone. In vivo gene transfer activity of HPC was not affected by the nature of the diluent used, i.e. glucose-based or saline-based isotonic solutions. Although in vitro transfection activity of HPC formulations could not be evidenced, a liposome leakage assay revealed that HPC could significantly destabilize stable lipid membranes suggesting that a possible membrane permeation enhancer activity of HPC combined to the physical stress induced by the intratumor injection may facilitate plasmid DNA entry inside the cells resulting in increased gene expression. HPC/plasmid formulations represent new and attractive non-viral gene delivery systems with potential in cancer gene therapy and vaccination.

Immune responses to tumor-associated antigens are often dampened by a tumor-induced state of immune anergy. Previous work has attempted to overcome tumor-induced T-cell anergy by the direct injection of vectors carrying the genes encoding one of a variety of cytokines. We hypothesised that the polyclonal stimulation of T cells, preferably through the TCR complex, would result in a cascade of cytokines associated with T-cell activation and would be best able to overcome T-cell anergy. Here we use the highly attenuated MVA poxvirus to express on tumor cells, in vitro and in vivo, either of three membrane-bound monoclonal antibodies specific for murine TCR complex. Using this system, we have expressed antibodies specific for the CD3 epsilon chain (KT3), TCR alpha/beta complex (H57-597), and V beta 7 chain (TR310). Tumor cells bristling with these antibodies are capable of inducing murine T-cell proliferation and cytokine production. When injected into growing tumors (P815, RenCa, and B16F10), these constructs induce the activation of immune effector cells and result in the rejection of the tumor. Histological and FACS analysis of tumor-infiltrating leukocytes reveal that the injection of recombinant virus-expressing antibodies specific for the TCR complex attracts and activates (CD25(+), CD69(+)) CD4 and CD8 lymphocytes. This approach represents a novel strategy to overcome T-cell anergy in tumors and allow the stimulation of tumor-specific T cells.

The MUC1 glycoprotein is a major tumor associated antigen in human epithelial cancers in which it is overexpressed and hypoglycosylated, in contrast to the heavily glycosylated form of MUC1 found at lower expression level in normal cells. The TG4010 immunotherapeutic product consists of a suspension of attenuated vaccinia virus (Ankara strain), genetically modified to express MUC1 and the cytokine IL-2. The TG4010 immunotherapeutic agent is currently in advanced clinical development for non-small-cell lung cancer (NSCLC) in combination with chemotherapy. Antigen-specific models were generated by transfecting murine tumor cell lines with the human MUC1 and were used to address in vitro and in vivo issues regarding the combinatorial approach of chemotherapy and immunization with MVATG9931, an isogenic construct of TG4010. First, in vitro sensitivity to drugs used as standard of care to treat advanced NSCLC patients of the lymphoma RMA cell line and of the Lewis Lung Carcinoma cell line (LLC) expressing, or not, MUC1, was determined. The parental or transfected cell lines had similar sensitivity to gemcitabine, pemetrexed or the combination of these drugs with cisplatin. The in vivo anti-tumoral activity of chemotherapy was assessed in syngeneic C57BL/6 mice bearing tumors. In contrast to the in vitro situation, in vivo anti-tumoral activity of chemotherapy was clearly superior in the RMA or LLC MUC1-transfectants as compared to their respective un-transfected counterpart. Further investigations of the LLC model in vivo showed that MUC1 expression was linked to increased tumor aggressiveness when compared to the un-transfected LLC tumor with faster tumor growth and increased splenomegaly. Analysis of inflammatory cells and notably myeloid-derived suppressor cells (MDSC) indicated that their proportion was increased in the blood, spleen and tumor in mice bearing LLCMUC1 tumors as compared to that of mice with LLC tumors. Finally, using a gemcitabine-based regimen known to affect MDSC viability, combination therapy of LLCMUC1 tumors with MVATG9931 immunizations and gemcitabine/cisplatin increased significantly mice survival as compared to that of mice treated with chemotherapy alone. These preclinical data argues for a strong rationale to combine TG4010 with chemotherapy in advanced NSCLC patients.

Recombinant adenovirus vectors are powerful tools for inducing de novo gene expression in vivo. Here we have exploited them to study the specificity of CD4/CD8 lineage commitment during thymocyte positive selection, transferring MHC class II genes directly into thymi of mice deficient in both class I and II molecules. Expression of class II molecules was induced on cortical stroma, provoking the selection of a large population of mature CD4(+)CD8(-) cells, as expected, but also of a significant number of CD4(-)CD8(+) cells. The latter constituted a diverse population, containing both immature precursors and, though less frequent, cells that were mature according to several criteria. CD4(-)CD8(+) cells appeared with the same kinetics as their CD4(+)CD8(-) counterparts, but tended to be more prevalent at early times or when thymocyte reconstitution was only modest. These observations, derived from a dynamic selection system, indicate that CD4/CD8 lineage commitment is not irredeemably linked to the class of MHC molecule driving positive selection, a conclusion most compatible with selective models of commitment.

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.

Objective To compare biological properties of zidovudine-resistant variants of HIV-1 isolated from subjects on long-term drug therapy with drug-sensitive parental isolates obtained from the same patients before initiation of treatment. Methods Clinical HIV-1 strains were isolated following co-incubation of patient peripheral blood mononuclear cells with mitogen-stimulated umbilical cord blood lymphocytes. Drug resistance was evaluated by infecting MT-4 cells pretreated with zidovudine and maintained under drug pressure. Results The drug-resistant phenotype remained stable, following many viral replication cycles in the absence of zidovudine. Drug-resistant variants contained fewer replication-competent viruses but were more cytopathogenic than their corresponding zidovudine-sensitive parental strains. Conclusions These results suggest that drug-resistant strains possess biological properties that may differ from those of drug-sensitive variants of HIV-1.

The reverse transcriptase enzyme of HIV-1 is known to be error-prone. We were interested in the possibility of isolating a variant HIV-1 strain that might be capable of replication in the presence of AZT, thought to act by antagonizing reverse transcriptase activity. Toward this end, chronically infected H-9 cells were exposed to various concentrations of AZT for at least 500 days. No mutant has yet arisen from such cultures, which continued to produce high levels of each of the viral proteins p24, p17, gp41, and gp51/66 in the presence of the drug. Notwithstanding such expression of viral antigens, culture fluids from these various AZT-treated cultures were not capable of infecting otherwise susceptible target cells. Electron microscopic observations of AZT-treated chronically infected H-9 cells indicated a lower production of viral structures, in comparison with control cultures. Furthermore, those particles seen at the plasma membrane of AZT-treated cells often appeared to be envelope-deficient. These data suggest that AZT may be able to interfere in some way with proper assembly and/or packaging of infectious progeny HIV-1 at the cell membrane, although other modes of action for a postintegrational effect of AZT cannot be excluded.

We determined whether drug-resistant variants of HIV-1 could be isolated from the peripheral blood mononuclear cells of 20 individuals with HIV infection (Centers for Disease Control groups II and III) on long-term zidovudine (AZT) therapy. Toward this end, zidovudine (10 microM) has been included in the tissue culture medium used to isolate HIV-1. Under these circumstances, virus with a zidovudine-resistant phenotype was successfully obtained in five out of 20 cases. This property of drug resistance appeared to be stable, and did not disappear upon extended replication of such virus in the absence of drug pressure. Drug-resistant virus could also be isolated from these subjects on subsequent occasions, but was not present in samples obtained prior to therapy. Replication of these zidovudine-resistant isolates in tissue culture was inhibited by each of four other nucleoside analogues. Thus, other drugs may be useful in controlling selective zidovudine-resistant variants of HIV-1.

De novo differentiation of CD4+ T cells was provoked in mice lacking major histocompatibility complex (MHC) class II molecules by intrathymic injection of adenovirus vectors carrying class II genes. This permits a new approach to questions concerning the dynamics of CD4+ T cell compartments in the thymus and peripheral lymphoid organs. Here two issues are explored. First, we show that mature CD4+CD8− cells reside in the thymus for a protracted period before emigrating to the periphery, highlighting the potential importance of, and our ignorance of, the postselection maturation period. Second, we demonstrate that the survival of CD4+ cells in peripheral lymphoid organs is markedly curtailed when class II molecules are absent and is not further reduced in the absence of both class II and class I molecules, raising the possibility that MHC-mediated selection may continue in the periphery.

Infection of a newly described human T lymphoid cell line, CEM-CL10, with three different variants of HIV-1 resulted in cytopathic effects followed by cell lysis. Following primary lytic infection, proviral DNA could not be detected by Southern blot analysis in the outgrowth of the surviving CEM-CL 10 cells 60 days after initial exposure to HIV-1. These surviving cells could be further grown as a separate line, derived from CEM-CL10, and were found to be resistant to subsequent infection by HIV-1. A marked decrease in CD4 antigen expression was observed in these latter cells but not of the CD3 and transferrin receptor antigens. This decline in cell surface CD4 expression was correlated with both an absence of specific CD4 mRNA and with changes in structure of the CD4 gene. Both the HIV-1-sensitive CEM-CL10 cell line and its CD4(-), HIV-1-resistant derivative line, will be made available to interested investigators.

Background Primary cutaneous lymphomas (CLs), characterized by an accumulation of clonal T or B lymphocytes preferentially localized in the skin, have been successfully treated with interferons (IFNs) which counterbalance the Th2-immunosuppressive state associated with this pathology. In a phase I/II clinical trial, we correlated the local immune infiltrate and the anti-tumor effects of repeated intralesional administrations of an adenovirus vector expressing human interferon-gamma (IFN-g) termed TG1042, in patients with advanced primary cutaneous T-cell lymphomas (CTCL) or multilesional cutaneous B-cell lymphomas (CBCL). Methods For each patient, variation in time of specific lymphocyte populations, defined by immunohistochemical stainings, was assessed in biopsies of injected lesions. For each patient, the change in local immune response was associated with the patient’s objective response at the end of the study. Results Immunohistochemical analyses of biopsies indicate that infiltration of CD8+ T lymphocytes and of TIA-1+ cytotoxic T-cells in lesions injected with TG1042 correlates with clinical benefit. Conclusions These data suggest for the first time that a CD8+ cytotoxic infiltrate, induced by local expression of IFN-g correlates with a clinical response. Trial registration The phase I step (TG1042.01) does not have a registration number. The phase II step (TG1042.06) registration number was NCT00394693.