SOT101 induces NK cell cytotoxicity and potentiates antibody-dependent cell cytotoxicity and anti-tumor activity.

SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.

Myeloid - derived suppressor cells in Type 1 diabetes are an expanded population exhibiting diverse T-cell suppressor mechanisms.

Myeloid-derived suppressor cells (MDSC) represent a heterogeneous group of immature myeloid cells with immunoregulatory function in cancer and autoimmune diseases. In humans, two subsets of MDSC were determined based on the characteristic surface markers, monocytic MDSC (M-MDSC) and granulocytic MDSC (G-MDSC). Expansion of MDSC has been reported in some murine models and patients with autoimmune diseases and their immune-suppressive properties were characterized. However, the exact role of MDSC in the pathogenesis of autoimmune diseases is more complex and/or controversial. In type 1 diabetes mellitus (T1D), the increased frequency of MDSC was found in the blood of T1D patients but their suppressor capacity was diminished. In our study, we assessed the role of M-MDSC in the pathogenesis of T1D and showed for the first time the increased frequency of M-MDSC not only in the blood of T1D patients but also in their at-risk relatives compared to healthy donors. T1D patients with inadequate long term metabolic control showed an elevation of M-MDSC compared to patients with better disease control. Furthermore, we described the positive correlation between the percentage of M-MDSC and Th17 cells and IFN-γ producing T cells in T1D patients and their at-risk relatives. Finally, we found that the ability of M-MDSC to suppress autologous T cells is efficient only at the high MDSC: T cells ratio and dependent on cell-cell-contact and TGF-ß production. Our data show that the engagement of MDSC in the pathogenesis of T1D is evident, yet not entirely explored and more experiments are required to clarify whether MDSC are beneficial or harmful in T1D.

Detection of tumor antigens and tumor-antigen specific T cells in NSCLC patients: Correlation of the quality of T cell responses with NSCLC subtype.

Allogeneic cancer cell lines serve as universal source of tumor-associated antigens in cancer vaccines. Immunogenic high hydrostatic pressure-killed cancer cells derived from cell lines can be used for the generation of dendritic cell (DC)-based active cellular immunotherapy of non-small cell lung cancer (NSCLC). We investigated the expression of 12 known NSCLC tumor-associated antigens (TAA) (CEA, MAGE-A1, MAGE-A3, MAGE-A4, PRAME, hTERT, HER2, MUC1, Survivin, STEAP1, SOX2 and NY-ESO-1) in 6 NSCLC cell lines as candidates for the generation of DC-based lung cancer vaccine. We showed that the selected antigenic profile of these cell lines overlaps to various degrees with that of primary NSCLC tumors (n = 52), indicating that 4 out of 6 NSCLC cell lines would be suitable for DC-based vaccine generation. We further investigated the presence of TAA-specific T cells in blood of NSCLC patients (n = 32) using commercially available peptide mixes in an in vitro stimulation assay. IFN-γ+CD8+ and IFN-γ+CD4+ T cell responses to all antigens were detected in NSCLC patients. Interestingly, despite higher TAA expression in squamous cell carcinoma (SCC) the responsiveness of patients' T cells to stimulation was significantly lower in SCC patients than in adenocarcinoma (AC) patients. This suggests qualitative differences in T cell functionality between NSCLC subtypes. Based on this study, and in order to maximize the amount of treatable patients, we selected a mix of H520 and H522 NSCLC cell lines for DC-based vaccine preparation. We also established a minimal panel of antigenic peptide mixes (CEA, hTERT, PRAME, HER2) for immunomonitoring of T cell responses during the DC-based lung cancer immunotherapy in Phase I lung cancer clinical trial (NCT02470468).

Radiotherapy in Combination With Cytokine Treatment.

Radiotherapy (RT) plays an important role in the management of cancer patients. RT is used in more than 50% of patients during the course of their disease in a curative or palliative setting. In the past decades it became apparent that the abscopal effect induced by RT might be dependent on the activation of immune system, and that the induction of immunogenic cancer cell death and production of danger-associated molecular patterns from dying cells play a major role in the radiotherapy-mediated anti-tumor efficacy. Therefore, the combination of RT and immunotherapy is of a particular interest that is reflected in designing clinical trials to treat patients with various malignancies. The use of cytokines as immunoadjuvants in combination with RT has been explored over the last decades as one of the immunotherapeutic combinations to enhance the clinical response to anti-cancer treatment. Here we review mainly the data on the efficacy of IFN-α, IL-2, IL-2-based immunocytokines, GM-CSF, and TNF-α used in combinations with various radiotherapeutic techniques in clinical trials. Moreover, we discuss the potential of IL-15 and its analogs and IL-12 cytokines in combination with RT based on the efficacy in preclinical mouse tumor models.

High hydrostatic pressure in cancer immunotherapy and biomedicine.

High hydrostatic pressure (HHP) has been known to affect biological systems for >100 years. In this review, we describe the technology of HHP and its effect macromolecules and physiology of eukaryotic cells. We discuss the use of HHP in cancer immunotherapy to kill tumor cells for generation of whole cell and dendritic cell-based vaccines. We further summarize the current use and perspectives of HHP application in biomedicine, specifically in orthopedic surgery and for the viral, microbial and protozoan inactivation to develop vaccines against infectious diseases.

Invasion of Dendritic Cells, Macrophages and Neutrophils by the Bordetella Adenylate Cyclase Toxin: A Subversive Move to Fool Host Immunity.

Adenylate cyclase toxin (CyaA) is released in the course of B. pertussis infection in the host's respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC), macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of B. pertussis CyaA to intoxicate DC with high concentrations of 3',5'-cyclic adenosine monophosphate (cAMP), which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review.

Severe, but not mild heat-shock treatment induces immunogenic cell death in cancer cells.

The mechanisms of immunogenicity underlying mild heat-shock (mHS) treatment < 42°C of tumor cells are largely attributed to the action of heat-shock proteins; however, little is known about the immunogenicity of tumor cells undergoing severe cytotoxic heat-shock treatment (sHS > 43°C). Here, we found that sHS, but not mHS (42°C), induces immunogenic cell death in human cancer cell lines as defined by the induction of ER stress response and ROS generation, cell surface exposure of calreticulin, HSP70 and HSP90, decrease of cell surface CD47, release of ATP and HMGB1. Only sHS-treated tumor cells were efficiently killed and phagocytosed by dendritic cells (DCs), which was partially dependent on cell surface calreticulin. DCs loaded with mHS or sHS-treated tumor cells displayed similar level of maturation and stimulated IFNγ-producing CD8+ T cells without any additional adjuvants in vitro. However, only DCs loaded with sHS-treated tumor cells stimulated antigen-specific CD4+ T cells and induced higher CD8+ T-cell activation and proliferation. sHS-treated murine cells also exposed calreticulin, HSP70 and HSP90 and activated higher DC maturation than mHS treated cells. Vaccination with sHS-treated tumor cells elicited protective immunity in mice. In this study, we defined specific conditions for the sHS treatment of human lung and ovarian tumor cells to arrive at optimal ratio between effective cell death, immunogenicity and content of tumor antigens for immunotherapeutic vaccine generation.

High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy.

High hydrostatic pressure (HHP) can be used to generate dendritic cell (DC)-based active immunotherapy for prostate, lung and ovarian cancer. We showed here that HHP treatment of selected human cancer cell lines leads to a degradation of tumor antigens which depends on the magnitude of HHP applied and on the cancer cell line origin. Whereas prostate or ovarian cell lines displayed little protein antigen degradation with HHP treatment up to 300MPa after 2h, tumor antigens are hardly detected in lung cancer cell line after treatment with HHP 250MPa at the same time. On the other hand, quick reduction of tumor antigen-coding mRNA was observed at HHP 200MPa immediately after treatment in all cell lines tested. To optimize the DC-based active cellular therapy protocol for HHP-sensitive cell lines the immunogenicity of HHP-treated lung cancer cells at 150, 200 and 250MPa was compared. Lung cancer cells treated with HHP 150MPa display characteristics of immunogenic cell death, however cells are not efficiently phagocytosed by DC. Despite induction of the highest number of antigen-specific CD8+ T cells, 150 MPa-treated lung cancer cells survive in high numbers. This excludes their use in DC vaccine manufacturing. HHP of 200MPa treatment of lung cancer cells ensures the optimal ratio of efficient immunogenic killing and delivery of protein antigens in DC. These results represent an important pre-clinical data for generation of immunogenic killed lung cancer cells in ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa).

Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis.

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMß2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b- cells. The nonhemolytic AC+ Hly- bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly- mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b- cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.

Generation of dendritic cell-based vaccine using high hydrostatic pressure for non-small cell lung cancer immunotherapy.

High hydrostatic pressure (HHP) induces immunogenic death of tumor cells which confer protective anti-tumor immunity in vivo. Moreover, DC pulsed with HHP-treated tumor cells induced therapeutic effect in mouse cancer model. In this study, we tested the immunogenicity, stability and T cell stimulatory activity of human monocyte-derived dendritic cell (DC)-based HHP lung cancer vaccine generated in GMP compliant serum free medium using HHP 250 MPa. DC pulsed with HHP-killed lung cancer cells and poly(I:C) enhanced DC maturation, chemotactic migration and production of pro-inflammatory cytokines after 24h. Moreover, DC-based HHP lung cancer vaccine showed functional plasticity after transfer into serum-containing media and stimulation with LPS or CD40L after additional 24h. LPS and CD40L stimulation further differentially enhanced the expression of costimulatory molecules and production of IL-12p70. DC-based HHP lung cancer vaccine decreased the number of CD4+CD25+Foxp3+ T regulatory cells and stimulated IFN-γ-producing tumor antigen-specific CD4+ and CD8+ T cells from non-small cell lung cancer (NSCLC) patients. Tumor antigen specific CD8+ and CD4+ T cell responses were detected in NSCLC patient's against a selected tumor antigens expressed by lung cancer cell lines used for the vaccine generation. We also showed for the first time that protein antigen from HHP-killed lung cancer cells is processed and presented by DC to CD8+ T cells. Our results represent important preclinical data for ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa) in combination with chemotherapy and immune enhancers.

Case-Control Study: Smoking History Affects the Production of Tumor Antigen-Specific Antibodies NY-ESO-1 in Patients with Lung Cancer in Comparison with Cancer Disease-Free Group.

INTRODUCTION: Lung cancer is the leading cause of cancer mortality worldwide; therefore, understanding the biological or clinical role of tumor-associated antigens and autoantibodies is of eminent interest for designing antitumor immunotherapeutic strategies. METHODS: Here we prospectively analyzed the serum frequencies of New York esophageal squamous cell carcinoma 1 (NY-ESO-1), human epidermal growth factor 2/neu, and melanoma-associated antigen A4 (MAGE-A4) antibodies and expression of the corresponding antigens in tumors of 121 patients with NSCLC undergoing an operation without prior neoadjuvant chemotherapy and compared them with those in 57 control age-matched patients with no history of a malignant disease. RESULTS: We found that only antibodies specific for NY-ESO-1 (19.8% [n = 24 of 121]) were significantly increased in the group of patients with NSCLC compared with in the controls. NY-ESO-1 seropositivity was significantly positively associated with an active smoking history in patients with NSCLC but not in smokers from the control group. In tumors, the frequency of NY-ESO-1 mRNA expression was 6.3% (in four of 64 patients), the frequency of human epidermal growth factor 2/neu (HER 2/neu) expression was 11.9% (five of 42), and the frequency of MAGE-A4 expression was 35.1% (20 of 57). MAGE-A4 expression in tumors correlated with smoking status and male sex in patients with NSCLC. Patients with squamous cell carcinoma displayed higher expression of NY-ESO-1 and MAGE-A4 in tumors than did patients with adenocarcinoma. On the other hand, 94.7% of nonsmoking patients in our study had adenocarcinoma (of whom 73.7% were women). CONCLUSION: These results confirm the reported high immunogenicity of NY-ESO-1 and suggest that a smoking-induced chronic inflammatory state might potentiate the development of NY-ESO-1-specific immune responses. Moreover, smoking might contribute to the expression of other cancer/testis antigens such as MAGE-A4 at early stages of NSCLC development.

Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

Bordetella adenylate cyclase toxin differentially modulates toll-like receptor-stimulated activation, migration and T cell stimulatory capacity of dendritic cells.

Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4(+) and CD8(+) T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4+ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4(+)CD25(+)Foxp3(+) T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8(+) T cell proliferation and limited the induction of IFN-γ producing CD8(+) T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.

Physical modalities inducing immunogenic tumor cell death for cancer immunotherapy.

The concept of immunogenic cancer cell death (ICD), as originally observed during the treatment with several chemotherapeutics or ionizing irradiation, has revolutionized the view on the development of new anticancer therapies. ICD is defined by endoplasmic reticulum (ER) stress response, reactive oxygen species (ROS) generation, emission of danger-associated molecular patterns and induction of antitumor immunity. Here we describe known and emerging cancer cell death-inducing physical modalities, such as ionizing irradiation, ultraviolet C light, Photodynamic Therapy (PDT) with Hypericin, high hydrostatic pressure (HHP) and hyperthermia (HT), which have been shown to elicit effective antitumor immunity. We discuss the evidence of ICD induced by these modalities in cancer patients together with their applicability in immunotherapeutic protocols and anticancer vaccine development.

Heterosubtypic protection against influenza A induced by adenylate cyclase toxoids delivering conserved HA2 subunit of hemagglutinin.

The protective efficacy of currently available influenza vaccines is restricted to vaccine strains and their close antigenic variants. A new strategy to obtain cross-protection against influenza is based on conserved antigens of influenza A viruses (IAV), which are able to elicit a protective immune response. Here we describe a vaccination approach involving the conserved stem part of hemagglutinin, the HA2 subunit, shared by different HA subtypes of IAV. To increase its immunogenicity, a novel strategy of antigen delivery to antigen presenting cells (APCs) has been used. The HA2 segment (residues 23-185) was inserted into a genetically detoxified adenylate cyclase toxoid (CyaA-E5) which specifically targets and penetrates CD11b-expressing dendritic cells. The CyaA-E5-HA2 toxoid induced HA2(93-102), HA2(96-104) and HA2(170-178)-specific and Th1 polarized T-cell responses, and also elicited strong broadly cross-reactive HA2-specific antibody response. BALB/c mice immunized with three doses of purified CyaA-E5-HA2 without any adjuvant recovered from influenza infection 2days earlier than the control mock-immunized mice. More importantly, immunized mice were protected against a lethal challenge with 2LD(50) dose of a homologous virus (H3 subtype), as well as against the infection with a heterologous (H7 subtype) influenza A virus. This is the first report on heterosubtypic protection against influenza A infection mediated by an HA2-based vaccine that can induce both humoral and cellular immune responses without the need of adjuvant.

Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores.

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca²âº into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²âº influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²âº influx promoted by molecules locked in a Ca²âº-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.

Bacteria and their toxins tamed for immunotherapy.

Bacterial toxins share the ability to enter host cells to target various intracellular proteins and to modulate host immune responses. Over the last 20 years, toxins and their mutated variants, as well as live attenuated bacteria, have been exploited for vaccination and immunotherapy of various infectious, malignant and autoimmune diseases. The ability of Bordetella pertussis adenylate cyclase toxin to translocate its adenylate cyclase domain across the host cell membrane, as well as the pathways of intracellular trafficking of Bacillus anthracis lethal and edema toxins, Shigella dysenteriae shiga toxin or Escherichia coli shiga-like toxin, have been repeatedly exploited for the delivery of antigenic epitopes into host cells and for stimulation of antigen-specific T cell responses. Similarly, E. coli α-hemolysin, or effector proteins of Yersinia and Salmonella secreted by the type III secretion systems, were used to facilitate the delivery of fused heterologous proteins or peptides for antigenic presentation. Vibrio cholerae cholera toxin, E. coli heat-labile enterotoxin, B. pertussis pertussis toxin or the Cry1A protein of Bacillus thuringiensis have shown a great potential to act as adjuvants and to stimulate mucosal as well as systemic immune responses. The immunotherapeutic potential of some toxins, like Clostridium perfringens perfringolysin O, Streptococcus intermedius intermedilysin, or Streptococcus pneumoniae pneumolysin needs to be evaluated further. The Bordetella adenylate cyclase toxoid used as a vaccine delivery tool, or Corynebacterium diphtheriae diphtheria toxin and Pseudomonas aeruginosa exotoxin A-based immunotoxins, are currently in various phases of clinical trials for cancer immunotherapy, as are some antigen-delivering Salmonella and Listeria monocytogenes strains.

RTX proteins: a highly diverse family secreted by a common mechanism.

Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca(2+) ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest.

Y. enterocolitica inhibits antigen degradation in dendritic cells.

Yersinia enterocolitica (Ye) disrupts the ability of dendritic cells (DC) to prime CD4+ T cells suggesting that Ye may subvert uptake and/or processing of soluble antigens (Ag). To investigate this Ye-infected DC were loaded with fluorescently labelled ovalbumins as markers for Ag uptake and processing, and analysed by flow cytometry, fluorometry and microscopy. Wild type pYV+ as well as plasmidless pYV(-) bacteria inhibited Ag degradation in DC by 40% compared to non-infected cells. Microscopic analyses of pYV(-)-infected DC revealed that 40% of DC contained intracellular bacteria, and that DC without intracellular bacteria had degraded more Ag. When internalization of pYV(-) was blocked by cytochalasin D, Ag degradation was no longer inhibited indicating the competition between degradation of bacteria and ovalbumin. In contrast, cytochalasin D pre-treated DC infected with pYV+ inhibited Ag degradation by a mechanism dependent on the presence of virulence plasmid pYV encoding YopE, YopH, YopM, YopP, YopT and YopO. As no single Yop inhibited Ag degradation, interaction of multiple Yops might account for this effect, possibly by inhibiting Rho GTPases, because of a significant decrease of Ag degradation observed in DC incubated with toxin B of C. difficile. However, the contribution of other pYV-encoded factors cannot be excluded.

Differential roles of Yersinia outer protein P-mediated inhibition of nuclear factor-kappa B in the induction of cell death in dendritic cells and macrophages.

Yersinia outer protein P (YopP) induces cell death in macrophages and dendritic cells (DC). In DC this YopP-dependent cell death coincides with the inhibition of nuclear factor-kappa B (NF-kappaB) activation. However, as shown by measurement of propidium iodide uptake via disrupted cellular membranes, the preincubation of DC with several NF-kappaB inhibitors prior to infection with Yersinia did not restore the death-inducing capacity of a YopP-deficient Yersinia mutant. These results suggest that in contrast to macrophages, in DC the YopP-dependent inhibition of NF-kappaB activation is not causative for the induction of cell death. Instead, in DC, the inhibition of mitogen-activated protein kinases (MAPKs), in particular, p38 and c-Jun N-terminal kinase, prior to infection with a YopP-deficient Yersinia mutant substituted the death-inducing capacity of the Yersinia wild-type strain, indicating that the YopP-dependent inhibition of MAPKs mediates Yersinia-induced DC death. The differences between DC and macrophages in the mechanisms of cell death induction by YopP presented herein might be crucial for the function of these antigen-presenting cells.