ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma.

Identification of a novel population of highly cytotoxic c-Met-expressing CD8+ T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are critical mediators of anti-tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen-presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c-Met (c-Met+ CTLs). Phenotypic and functional analysis of c-Met+ CTLs reveals that they display enhanced cytolytic capacities compared to their c-Met- CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c-Met+ CTLs in cell-mediated cytotoxicity reactions in vitro and in vivo and abrogates T-cell responses against metastatic melanoma in vivo Finally, we establish in three murine tumor settings and in human melanoma tissues that c-Met+ CTLs are a naturally occurring CD8+ T-cell population. Together, our findings suggest that the HGF/c-Met pathway could be exploited to control CD8+ T-cell-mediated anti-tumor immunity.

Mechanistic insights into the efficacy of cell penetrating peptide-based cancer vaccines.

Immunotherapies are increasingly used to treat cancer, with some outstanding results. Immunotherapy modalities include therapeutic vaccination to eliminate cancer cells through the activation of patient's immune system against tumor-derived antigens. Nevertheless, the full potential of therapeutic vaccination has yet to be demonstrated clinically because many early generation vaccines elicited low-level immune responses targeting only few tumor antigens. Cell penetrating peptides (CPPs) are highly promising tools to advance the field towards clinical success. CPPs efficiently penetrate cell membranes, even when linked to antigenic cargos, which can induce both CD8 and CD4 T-cell responses. Pre-clinical studies demonstrated that targeting multiple tumor antigens, even those considered to be poorly immunogenic, led to tumor regression. Therefore, CPP-based cancer vaccines represent a flexible and powerful means to extend therapeutic vaccination to many cancer indications. Here, we review recent findings in CPP development and discuss their use in next generation immunotherapies.

Enhancing Antitumor Immune Responses by Optimized Combinations of Cell-penetrating Peptide-based Vaccines and Adjuvants.

Cell penetrating peptides (CPPs) from the protein ZEBRA are promising candidates to exploit in therapeutic cancer vaccines, since they can transport antigenic cargos into dendritic cells and induce tumor-specific T cells. Employing CPPs for a given cancer indication will require engineering to include relevant tumor-associated epitopes, administration with an appropriate adjuvant, and testing for antitumor immunity. We assessed the importance of structural characteristics, efficiency of in vitro transduction of target cells, and choice of adjuvant in inducing the two key elements in antitumor immunity, CD4 and CD8 T cells, as well as control of tumor growth in vivo. Structural characteristics associated with CPP function varied according to CPP truncations and cargo epitope composition, and correlated with in vitro transduction efficiency. However, subsequent in vivo capacity to induce CD4 and CD8 T cells was not always predicted by in vitro results. We determined that the critical parameter for in vivo efficacy using aggressive mouse tumor models was the choice of adjuvant. Optimal pairing of a particular ZEBRA-CPP sequence and antigenic cargo together with adjuvant induced potent antitumor immunity. Our results highlight the irreplaceable role of in vivo testing of novel vaccine constructs together with adjuvants to select combinations for further development.

Phenotypic switch of CD8(+) T cells reactivated under hypoxia toward IL-10 secreting, poorly proliferative effector cells.

CD8(+) T cells controlling pathogens or tumors must function at sites where oxygen tension is frequently low, and never as high as under atmospheric culture conditions. However, T-cell function in vivo is generally analyzed indirectly, or is extrapolated from in vitro studies under nonphysiologic oxygen tensions. In this study, we delineate the role of physiologic and pathologic oxygen tension in vitro during reactivation and differentiation of tumor-specific CD8(+) T cells. Using CD8(+) T cells from pmel-1 mice, we observed that the generation of CTLs under 5% O2, which corresponds to physioxia in lymph nodes, gave rise to a higher effector signature than those generated under atmospheric oxygen fractions (21% O2). Hypoxia (1% O2) did not modify cytotoxicity, but decreasing O2 tensions during CTL and CD8(+) tumor-infiltrating lymphocyte reactivation dose-dependently decreased proliferation, induced secretion of the immunosuppressive cytokine IL-10, and upregulated the expression of CD137 (4-1BB) and CD25. Overall, our data indicate that oxygen tension is a key regulator of CD8(+) T-cell function and fate and suggest that IL-10 release may be an unanticipated component of CD8(+) T cell-mediated immune responses in most in vivo microenvironments.

Novel Multi-Antigen Orf-Virus-Derived Vaccine Elicits Protective Anti-SARS-CoV-2 Response in Monovalent and Bivalent Formats.

Prime-2-CoV_Beta is a novel Orf virus (ORFV)-based COVID-19 vaccine candidate expressing both the nucleocapsid and spike proteins of SARS-CoV-2 with the receptor-binding domain (RBD) of the Beta strain. This candidate was shown to be safe and immunogenic in a first-in-human Phase I clinical trial. With the shift in the immune landscape toward the Omicron variant and the widespread vaccine- and/or infection-derived immunity, further pre-clinical research was needed to characterize Prime-2-CoV. Here, we quantified the humoral and cellular response to Prime-2-CoV_Beta in pre-immunized mice and compared the protective efficacy of mono- and bivalent variant-based Prime-2-CoV vaccine candidates in hamsters. Prime-2-CoV_Beta induced robust humoral and cellular immune responses in naïve animals but did not further boost antibody titers in the tested setting when given as repeat booster at short interval. We furthermore showed that Prime-2-CoV_Beta-based mono- and bivalent immunization strategies produced comparable immunogenicity and protection from infection. Our results highlight the potential of the Orf virus as a vaccine platform against SARS-CoV-2 and potentially other infectious viruses.

A novel orf virus vector-based COVID-19 booster vaccine shows cross-neutralizing activity in the absence of anti-vector neutralizing immunity.

Next-generation COVID-19 vaccines are being developed to expand the breadth of coverage against existing and future variants and to extend the duration of protection. Prime-2-CoV_Beta is an orf virus (ORFV) based multi-antigen COVID-19 vaccine that co-expresses Spike (S) and Nucleocapsid (N) antigens. The safety and immunogenicity of Prime-2-CoV_Beta is investigated in a phase 1 first-in-human (FIH) dose-finding trial (ORFEUS study, ClinicalTrials.gov: NCT05367843). Participants of two age groups (18-55 and 65-85 years) who previously completed at least two doses of mRNA vaccines were enrolled and sequentially assigned to different dose groups to receive one intramuscular dose of 3 × 105, 3 × 106, 1.5 × 107, or 3 × 107 plaque-forming units (PFU) of Prime-2-CoV_Beta on day 1 and a second dose on day 29. Here, we report safety and immunogenicity data collected up to 6 months after the first study vaccination. Prime-2-CoV_Beta is safe and well tolerated and elicits immune responses at higher dose levels in participants aged 18-55. A single dose of 3 × 107 PFU boosted binding and cross-neutralizing antibody responses that are maintained through 6 months after the first booster vaccination. Polyfunctional S-specific CD4+ and CD8+ T cell responses are observed after vaccination. No pre-existing or vaccine-induced neutralizing anti-vector antibodies are detected. Our findings highlight the potential of the ORFV vector as a safe platform for future vaccine design, which provides the ability to deliver multiple antigens and allows for repeat immunization.

A multiantigenic Orf virus-based vaccine efficiently protects hamsters and nonhuman primates against SARS-CoV-2.

Among the common strategies to design next-generation COVID-19 vaccines is broadening the antigenic repertoire thereby aiming to increase efficacy against emerging variants of concern (VoC). This study describes a new Orf virus-based vector (ORFV) platform to design a multiantigenic vaccine targeting SARS-CoV-2 spike and nucleocapsid antigens. Vaccine candidates were engineered, either expressing spike protein (ORFV-S) alone or co-expressing nucleocapsid protein (ORFV-S/N). Mono- and multiantigenic vaccines elicited comparable levels of spike-specific antibodies and virus neutralization in mice. Results from a SARS-CoV-2 challenge model in hamsters suggest cross-protective properties of the multiantigenic vaccine against VoC, indicating improved viral clearance with ORFV-S/N, as compared to equal doses of ORFV-S. In a nonhuman primate challenge model, vaccination with the ORFV-S/N vaccine resulted in long-term protection against SARS-CoV-2 infection. These results demonstrate the potential of the ORFV platform for prophylactic vaccination and represent a preclinical development program supporting first-in-man studies with the multiantigenic ORFV vaccine.

ATP128 Clinical Therapeutic Cancer Vaccine Activates NF-κB and IRF3 Pathways through TLR4 and TLR2 in Human Monocytes and Dendritic Cells.

The use of cancer vaccines is a promising therapeutic strategy able to stimulate anti-tumor immunity by inducing both humoral and cellular immunity. In this study, antigen presenting cells play a key role by inducing a strong activation of the T cell-mediated adaptive immune response, essential for the anti-tumor potential of cancer vaccines. The first human candidate vaccine created from the KISIMA platform, ATP128, bears three tumor-associated antigens highly expressed in colorectal cancer tissues. At the N-terminus, the cell-penetrating peptide allows the antigen delivery inside the cell and, together with the TLR agonist-derived peptide at the C-terminus, ensures the activation of the monocyte-derived dendritic cells. Here, we show that ATP128 leads to both NF-κB and IRF3 pathway activation, with subsequent pro-inflammatory cytokines and type I Interferon release, as well as an increase in the expression of costimulatory molecules, alongside an upregulation of MHC class I molecules. This cellular immune response involves TLR2 and TLR4, for both membrane and intracellular signaling. We demonstrated an endocytic component in ATP128's activity by combining the use of a variant of ATP128 lacking the cell-penetrating peptide with endocytosis inhibitors. Importantly, this internalization step is detemined essential for the activation of the IRF3 pathway. This study validates the design of the self-adjuvanting ATP128 vaccine for cancer immunotherapy.

STING Agonist Combined to a Protein-Based Cancer Vaccine Potentiates Peripheral and Intra-Tumoral T Cell Immunity.

Combining different immunotherapy approaches is currently building the future of immunotherapy, with the view to maximize anti-tumoral efficacy for larger patient population. The KISIMA™ platform allows the development of protein-based cancer vaccines able to induce tumor-specific T cell response resulting in anti-tumoral efficacy in various mouse models. Intra-tumoral administration of stimulator of interferon gene agonists (STINGa) was shown to induce a potent inflammatory response leading to the development of tumor-specific immunity. Here, we explored the efficacy and mechanisms of action of subcutaneous STINGa treatment combined with therapeutic vaccination in various mouse tumor models. This combinatory treatment highly enhanced frequency and effector function of both peripheral and intra-tumoral antigen-specific CD8 T cells, promoting potent IFNγ and TNFα production along with increased cytotoxicity. Moreover, combination therapy favorably modulated the tumor microenvironment by dampening immune-suppressive cells and increasing CD4 T cell infiltration together with their polarization toward Th1 phenotype. Combination with STINGa treatment improved the effect of therapeutic vaccination, resulting in a prolonged control and slower growth of B16-OVA and TC-1 tumors. Altogether, the results presented here highlight the potential of combining STINGa with a therapeutic protein vaccine for cancer treatment.

Heterologous Prime-Boost Vaccination with a Peptide-Based Vaccine and Viral Vector Reshapes Dendritic Cell, CD4+ and CD8+ T Cell Phenotypes to Improve the Antitumor Therapeutic Effect.

Heterologous prime-boost settings with a protein vaccine and the viral vector vesicular stomatitis virus, both expressing tumor-associated antigens (KISIMA-TAA and VSV-GP-TAA), have been previously shown to generate potent antitumor immunity. In the cold TC-1 model (HPV antigen) and the immune-infiltrate MC-38 model (Adpgk, Reps1 and Rpl18 neo-antigens), we further investigated pivotal immune cells that educate CD8+ T cells. Heterologous prime-boost vaccination induced a superior antitumor response characterized by the increase in number and functionality of antigen-specific CD8+ T cells, recruitment of cross-presenting dendritic cells, and polarization of CD4+ T cells towards an antitumor Th1 phenotype within the tumor and tumor-draining lymph nodes, turning the cold TC-1 tumor into a hot, inflamed tumor. In the inflamed MC-38 tumor model, treatment combination markedly prolonged the overall survival of mice. Treatment with multi-epitope vaccines also induced high frequencies of multiple antigen specificities in the periphery and in the tumor. Prime-boost treatment reduced tumor-infiltrating regulatory CD4+ T cells whilst increasing cross-presenting dendritic cells in tumor-draining lymph nodes. In conclusion, heterologous prime-boost vaccination possesses the ability to induce a potent anti-tumor response in both immune-excluded and immune-infiltrated mouse tumor models. Additionally, this study highlights the design of a multi-epitope vaccine for cancer immunotherapy.

Novel Protein-Based Vaccine against Self-Antigen Reduces the Formation of Sporadic Colon Adenomas in Mice.

Novel immunopreventive strategies are emerging that show great promise for conferring long-term protection to individuals at high risk of developing colorectal cancer. The KISIMA vaccine platform utilizes a chimeric protein comprising: (1) a selected tumor antigen; (2) a cell-penetrating peptide to improve antigen delivery and epitope presentation, and (3) a TLR2/4 agonist to serve as a self-adjuvant. This study examines the ability of a KISIMA vaccine against achaete-scute family bHLH transcription factor 2 (Ascl2), an early colon cancer antigen, to reduce colon tumor formation by stimulating an anti-tumor immune response. Vaccine administrations were well-tolerated and led to circulating antibodies and antigen-specific T cells in a mouse model of colorectal cancer. To assess preventive efficacy, the vaccine was administered to mice either alone or in combination with the immune checkpoint inhibitor anti-PD-1. When delivered to animals prior to colon tumor formation, the combination strategy significantly reduced the development of colon microadenomas and adenomas, as compared to vehicle-treated controls. This response was accompanied by an increase in the intraepithelial density of CD3+ T lymphocytes. Together, these data indicate that the KISIMA-Ascl2 vaccine shows great potential to be a safe and potent immunopreventive intervention for individuals at high risk of developing colorectal cancer.

A modular self-adjuvanting cancer vaccine combined with an oncolytic vaccine induces potent antitumor immunity.

Functional tumor-specific cytotoxic T cells elicited by therapeutic cancer vaccination in combination with oncolytic viruses offer opportunities to address resistance to checkpoint blockade therapy. Two cancer vaccines, the self-adjuvanting protein vaccine KISIMA, and the recombinant oncolytic vesicular stomatitis virus pseudotyped with LCMV-GP expressing tumor-associated antigens, termed VSV-GP-TAA, both show promise as a single agent. Here we find that, when given in a heterologous prime-boost regimen with an optimized schedule and route of administration, combining KISIMA and VSV-GP-TAA vaccinations induces better cancer immunity than individually. Using several mouse tumor models with varying degrees of susceptibility for viral replication, we find that priming with KISIMA-TAA followed by VSV-GP-TAA boost causes profound changes in the tumor microenvironment, and induces a large pool of poly-functional and persistent antigen-specific cytotoxic T cells in the periphery. Combining this heterologous vaccination with checkpoint blockade further improves therapeutic efficacy with long-term survival in the spectrum. Overall, heterologous vaccination with KISIMA and VSV-GP-TAA could sensitize non-inflamed tumors to checkpoint blockade therapy.

Detection of Molecular Residual Disease Using Personalized Circulating Tumor DNA Assay in Patients With Colorectal Cancer Undergoing Resection of Metastases.

PURPOSE: More than 50% of patients with stage IV colorectal cancer (metastatic colorectal cancer [mCRC]) relapse postresection. The efficacy of postoperative systemic treatment is limited in this setting. Thus, these patients would greatly benefit from the use of a reliable prognostic biomarker, such as circulating tumor DNA (ctDNA) to identify minimal or molecular residual disease (MRD). PATIENTS AND METHODS: We analyzed a cohort of 112 patients with mCRC who had undergone metastatic resection with curative intent as part of the PREDATOR clinical trial. The study evaluated the prognostic value of ctDNA, correlating MRD status postsurgery with clinical outcomes by using a personalized and tumor-informed ctDNA assay (bespoke multiple PCR, next-generation sequencing assay). Postresection, systemic therapy was given to 39.2% of the patients at the discretion of the treating physician. RESULTS: Postsurgical, MRD positivity was observed in 54.4% (61 of 112) of patients, of which 96.7% (59 of 61) progressed at the time of data cutoff (hazard ratio [HR]: 5.8; 95% CI, 3.5 to 9.7; P < .001). MRD-positive status was also associated with an inferior overall survival: HR: 16.0; 95% CI, 3.9 to 68.0; P < .001. At the time of analyses, 96% (49 of 51) of patients were alive in the MRD-negative arm compared with 52.4% (32 of 61) in the MRD-positive arm. Patients who did not receive systemic therapy and were MRD-negative in the combined ctDNA analysis at two time points had an overall survival of 100%. In the multivariate analysis, ctDNA-based MRD status was the most significant prognostic factor associated with disease-free survival (HR: 5.78; 95% CI, 3.34 to 10.0; P < .001). CONCLUSION: This study confirms that in mCRC undergoing resection of metastases, postoperative MRD analysis is a strong prognostic biomarker. It holds promises for being implemented in clinical decision making, informing clinical trial design, and further translational research.

A novel chalcone derivative which acts as a microtubule depolymerising agent and an inhibitor of P-gp and BCRP in in-vitro and in-vivo glioblastoma models.

BACKGROUND: Over the past decades, in spite of intensive search, no significant increase in the survival of patients with glioblastoma has been obtained. The role of the blood-brain barrier (BBB) and especially the activity of efflux pumps belonging to the ATP Binding Cassette (ABC) family may, in part, explain this defect. METHODS: The in-vitro activities of JAI-51 on cell proliferation were assessed by various experimental approaches in four human and a murine glioblastoma cell lines. Using drug exclusion assays and flow-cytometry, potential inhibitory effects of JAI-51 on P-gp and BCRP were evaluated in sensitive or resistant cell lines. JAI-51 activity on in-vitro microtubule polymerization was assessed by tubulin polymerization assay and direct binding measurements by analytical ultracentrifugation. Finally, a model of C57BL/6 mice bearing subcutaneous GL26 glioblastoma xenografts was used to assess the activity of the title compound in vivo. An HPLC method was designed to detect JAI-51 in the brain and other target organs of the treated animals, as well as in the tumours. RESULTS: In the four human and the murine glioblastoma cell lines tested, 10 muM JAI-51 inhibited proliferation and blocked cells in the M phase of the cell cycle, via its activity as a microtubule depolymerising agent. This ligand binds to tubulin with an association constant of 2 x 105 M-1, overlapping the colchicine binding site. JAI-51 also inhibited the activity of P-gp and BCRP, without being a substrate of these efflux pumps. These in vitro studies were reinforced by our in vivo investigations of C57BL/6 mice bearing GL26 glioblastoma xenografts, in which JAI-51 induced a delay in tumour onset and a tumour growth inhibition, following intraperitoneal administration of 96 mg/kg once a week. In accordance with these results, JAI-51 was detected by HPLC in the tumours of the treated animals. Moreover, JAI-51 was detected in the brain, showing that the molecule is also able to cross the BBB. CONCLUSION: These in vitro and in vivo data suggest that JAI-51 could be a good candidate for a new treatment of tumours of the CNS. Further investigations are in progress to associate the title compound chemotherapy to radiotherapy in a rat model.

DNA delivery by microinjection for the generation of recombinant mammalian cell lines.

Gene transfer methods for producing recombinant cell lines are often not very efficient. One reason is that the recombinant DNA is delivered into the cell cytoplasm and only a small fraction reaches the nucleus. This chapter describes a method for microinjecting DNA directly into the nucleus. Direct injection has several advantages including the ability to deliver a defined copy number into the nucleus, the avoidance of DNAses that are present in the cell cytoplasm, and the lack of a need for extensive subcloning to find the recombinant cells. The procedure is described for two cell lines, CHO DG44 and BHK-21, using green fluorescent protein as a reporter gene. However, this method could easily be adapted to other cells lines and using other recombinant genes.

Targeting self and neo-epitopes with a modular self-adjuvanting cancer vaccine.

Induction of a potent CD4 and CD8 T-cell response against tumor-specific and tumor-associated antigen is critical for eliminating tumor cells. Recent vaccination strategies have been hampered by an inefficacious and low amplitude immune response. Here we describe a self-adjuvanted chimeric protein vaccine platform to address these challenges, characterized by a multidomain construction incorporating (i) a cell penetrating peptide (CPP) allowing internalization of several multiantigenic Major Histocompatibility Complex (MHC)-restricted peptides within (ii) the multiantigenic domain (Mad) and (iii) a TLR2/4 agonist domain (TLRag). Functionality of the resulting chimeric protein is based on the combined effect of the above-mentioned three different domains for simultaneous activation of antigen presenting cells and antigen cross-presentation, leading to an efficacious multiantigenic and multiallelic cellular immune response. Helper and cytotoxic T-cell responses were observed against model-, neo- and self-antigens, and were highly potent in several murine tumor models. The safety and the immunogenicity of a human vaccine candidate designed for colorectal cancer treatment was demonstrated in a non-human primate model. This newly engineered therapeutic vaccine approach is promising for the treatment of poorly infiltrated tumors that do not respond to currently marketed immunotherapies.

Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection.

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.

Orf1/SpcS chaperones ExoS for type three secretion by Pseudomonas aeruginosa.

OBJECTIVE: Pseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type III secretion system (TTSS) to inject effector proteins directly into the cytosol of target cells to subvert the host cell's functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P. aeruginosa, we analyzed the role of a postulated chaperone termed Orf1. METHODS: By allelic exchange, we constructed the mutant with the deletion of gene Orf1. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orf1, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF. The role of Orf1 in the expression of exoS was evaluated by gene reporter analysis. RESULTS: Pull-down assay showed that Orf1 binds to ExoS and ExoT. Secretion profile analysis showed that Orf1 was necessary for the optimal secretion of ExoS and ExoT. However, Orf1 had no effect on the expression of exoS. CONCLUSION: Orf1 is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orf1 as SpcS for "specific Pseudomonas chaperone for ExoS".

In-vivo gp100-specific Cytotoxic CD8+ T Cell Killing Assay.

Cytotoxic CD8+ T lymphocytes (CTLs) represent a crucial component of the adaptive immune system and play a prominent role in the anti-tumor immune responses of both mice and humans. Cytotoxic CD8+ T cells are responsible for the lysis of cells expressing peptides associated with MHC class I molecules and derived from infection with a pathogen or from mutated antigens. In order to quantify in vivo this antigen-specific CD8+ T cell killing activity, we use the in vivo killing assay (IVKA). Here, we describe the protocol for the lysis of cells expressing a CD8+ T cell melanoma epitope of the hgp10025-33 protein (KVPRNQDWL). C57BL/6 recipient mice, receive first target cells, prepared from naive congenic (CD45.1) C57BL/6 spleen cells pulsed with the hgp10025-33 peptide and labeled with CFSE and of non-pulsed control cells labeled with Brilliant violet. One day later, the spleen cells of recipient mice are isolated and analyzed by FACS to measure the amount of CFSE cells and Brillant Violet (BV) cells. The percentage of lysis is calculated by the difference between CFSE versus BV. Measuring the ability of antigen-specific CD8+ T cells to lyse their antigen in vivo is very important to evaluate the adaptive cytotoxic response induced against a pathogen or a tumor antigen.