Heat shock fusion protein gp96-Ig mediates strong CD8 CTL expansion in vivo

PROBLEM: As shown previously, gp96-Ig peptide complexes secreted by an ovalbumin transfected tumor (EG7) mediate strong, specific tumor immunity through a CD4 T cell independent CD8+ CTL response. In this study, we set out to develop a system to quantitatively determine the CD8 CTL response to gp96-Ig and to evaluate the influence of an established wild type tumor. METHODS: Secreted heat shock protein gp96-Ig was constructed by replacement of the endoplasmic reticulum retention signal with the Fc portion of IgGI, transfected into EG7 (EG7-gp96-Ig) and used to induce CD8+ CTL expansion in vivo. Adoptively transferred, ovalbumin specific T-cell receptor (TCR) transgenic CD8+ cells (OT-1) responded with clonal expansion to the immunization with EG7-gp96-Ig. OT-1 expansion was quantitated with K(b-peptide)-tetramers by flow cytometry. RESULTS: In response to primary immunization with EG7-gp96-Ig, OT-1 expand from an initial frequency of 0.5 to 25% of all CD8 cells, and to 50% of all CD8 cells after a booster immunization. Endogenous ovalbumin specific CD8 cells also expand strongly. Antigen specific effector function was measured by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) for interferon-gamma (IFN-gamma). While effector function was strongly induced by secreted gp96-Ig, not all expanded OT-1 produce IFN-gamma. EG7 does not cause OT-1 expansion, but rather induces anergy. If OT-1 are transferred into wild type EG7 tumor bearing mice to induce anergy of OT-1, immunization with EG7-gp96-Ig can partly overcome unresponsiveness. CONCLUSIONS: We conclude that secreted gp96-Ig is a powerful mediator of specific CD8+ CTL responses in vivo. Secretory gp96 mimics release of gp96 by damaged or necrotic cells that is able to activate dendritic cells without CD4 help. Gp96-Ig associated peptides have not been selected by binding to major histocompatibility complex (MHC). Specific immunization by secreted gp96-Ig therefore is expected to occur also in allogeneic settings.     

bcl-2 protects against Fas-based but not perforin-based T cell-mediated cytolysis

Fas ligand and perforin are the two key effector mechanismsin T cell-mediated cytotoxicity. These molecules mediate cytolysisof target cells by membrane damage and apoptosis. bcl-2 is knownto protect cells against apoptosis induced by many stimuli includinggrowth factor removal. However bcl-2s effect on Fas ligand andperforin-induced lysis has not been studied extensively. Weinvestigated the effect of overexpression of bcl-2 alone, Fasalone or their combined overexpression on lysis of a commonlyused target, P815, by perforin-sufficient, Fas ligand-sufficientand perforin-deficient or Fas ligand-deficient, allospecificcytotoxic T lymphocytes (CTL). Wild-type P815 are susceptibleto lysis by perforin-sufficient CTL, regardless of the presenceor absence (gld) of Fas ligand, but are poorly lysed by perforin-deficientCTL. Fas transfection of P815 makes target cells highly susceptibleto lysis by both perforin-sufficient and -deficient CTL, indicatingthe presence of the Fas ligand-mediated cytotoxicity on bothtypes of CTL. Co-transfection of P815-fas with bcl-2 abolishestheir increased susceptibility to Fas-mediated lysis, even inthe face of Fas overexpression on the cell membrane. The protectiveeffect of bcl-2 against cell lysis is evident with perforin-deficientCTL as effector cells or when perforin activity is eliminatedby the absence of extracellular calcium in perforin-sufficientCTL. bcl-2 overexpression by P815, however, does not protectagainst CTL lysis by the perforin pathway, regardless of Fasoverexpression, as demonstrated by Fas ligand mutated gld andwild-type perforin-sufficient CTL. Therefore bcl-2 can protectP815 target cells against Fas-mediated lysis when triggeredby the Fas ligand on CTL, but not against perforin-mediatedlysis.     

Allogeneic tumor-cell-based vaccines secreting endoplasmic reticulum chaperone gp96

Heat-shock proteins are chaperones for proteins including tumor antigens. Heat-shock protein gp96, also known as glucose-regulated protein grp94, is the primary chaperone of the endoplasmic reticulum and a natural adjuvant for priming the innate and adaptive immune system. By transfecting tumor cells with a genetically modified secretory form of gp96, the tumor cells are transformed into vaccine cells. Gp96 vaccines in murine studies trigger robust innate and antigen-specific cellular immune responses and cause tumor rejection followed by long-lasting tumor immunity. The authors briefly review here the generation of cytotoxic T lymphocyte responses by gp96 and the most up to date clinical data in the use of gp96-based cancer vaccines.     

Induction of a TRAIL-mediated suicide program by interferon alpha in primary effusion lymphoma.

Gammaherpes viruses are often detected in lymphomas arising in immunocompromised patients. We have found that Azidothymidine (AZT) alone induces apoptosis in Epstein Barr Virus (EBV) positive Burkitt's lymphoma (BL) cells but requires interferon alpha (IFN-alpha) to induce apoptosis in Human Herpes Virus Type 8 (HHV-8) positive Primary Effusion Lymphomas (PEL). Our analysis of a series of AIDS lymphomas revealed that IFN-alpha selectively induced very high levels of the Death Receptor (DR) tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HHV-8 positive PEL lines and primary tumor cells whereas little or no induction was observed in primary EBV+ AIDS lymphomas and EBV-Burkitt's lines. AZT and IFN-alpha mediated apoptosis in PEL was blocked by stable overexpression of dominant negative Fas Associated Death Domain (FADD), decoy receptor 2 (DcR2), soluble TRAIL receptor fusion proteins (DR-4 and DR-5) and thymidine. Trimeric TRAIL (in place of IFN-alpha) similarly synergized with AZT to induce apoptosis in HHV-8 positive PEL cells. This is the first demonstration that IFN-alpha induces functional TRAIL in a malignancy that can be exploited to effect a suicide program. This novel antiviral approach to Primary Effusion lymphomas is targeted and may represent a highly effective and relatively non-toxic therapy.     

Generation of Tumor-Specific Cytotoxic T Lymphocytes by Stimulation with HPV Type 16 E7 Peptide-Pulsed Dendritic Cells: An Approach to Immunotherapy of Cervical Cancer

OBJECTIVE: The aim of this study was to generate HPV-16 E7 peptide-specific cytotoxic T lymphocytes (CTLs) in vitro for future adoptive immunotherapy of cervical cancer. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from HLA-A2+ healthy donors. The PBMCs were incubated with HPV-16 E7(11-20) peptide and varying cytokines in the primary culture. Restimulation was performed weekly with peptide-pulsed, irradiated autologous PBMCs. Alternatively, the PBMCs were depleted of abundant CD4+ cells and stimulated with HPV-16 E7(11-20) peptide-pulsed dendritic cells. Cytolytic activity was determined by a standard 4-h (51)Cr-release assay. RESULTS: After 6 weeks in culture, we were able to establish peptide-specific CTL lines in one of seven donors by incubating PBMCs with HPV-16 E7(11-20) peptide. When we employed autologous peptide-pulsed dendritic cells to stimulate CD8+ cell-enriched PBMCs, we obtained CTL lines in four of seven donors. The primed CTLs were able to lyse the HLA-A2+ and HPV-16+ cervical cancer cell line Caski. SiHa, an HLA-A2-, but HPV 16+, cervical cancer cell line could be lysed only after transfection with HLA-A2. In addition, a high cytotoxicity (>80%) was obtained against peptide-pulsed, but not unpulsed, targets such as autologous Ebstein-Barr virus-immortalized B cells or allogeneic lipopolysaccaride-stimulated PBMCs. DCs were clearly the most potent of all tested antigen presenting cells to stimulate a CTL response in a proliferation assay. CONCLUSION: HPV-16 E7 peptide-specific CTLs could be generated in vitro. A practical protocol to expand the CTLs to a sufficient number for an application in a clinical trial is in progress.     

A critical analysis of the tumour immunosurveillance controversy for 3-MCA-induced sarcomas

The cancer immunoediting hypothesis has gained significant footing over the past decade as a result of work performed using sarcomas induced by 3-methylcholanthrene (3-MCA) in mice. Despite the progress made by several groups in establishing evidence for the three phases of immunoediting (elimination, equilibrium and escape), there continues to be active controversy on the nature of interaction between spontaneously formed tumour cells and the immune system during the early phases of tumourigenesis. At the root of this controversy is conflicting and unresolved evidence spanning back to the 1970s regarding the incidence and frequency of 3-MCA-induced sarcomas in immunocompetent mice as compared to immunodeficient mice. In this mini review we provide a critical analysis of both sides of this controversy.     

Immunotherapy as a strategy for the treatment of non-small-cell lung cancer

Positive modulation of a patient's immune system to produce antitumor immunity is an attractive strategy that may improve the dismal outcomes typically associated with non-small-cell lung cancer (NSCLC). Using methods that either augment specific antitumor immunity or positively influence the patient's immune system to allow the de novo generation of immunity to encompass current strategies used in recent clinical trials of NSCLC. Encouraging results of Phase II trials in antigen-specific immunotherapy have led to three subsequent Phase III trials, which are currently enrolling. Results of these trials will improve our understanding of the role that immunotherapy plays in the treatment of NSCLC. Successful application of a humoral vaccine in Cuba led to its approval for the treatment of advanced NSCLC patients in that country. To date, trials involving nonspecific immunotherapeutic interventions have failed to improve outcomes in NSCLC and may indicate a need to combine them with antigen-specific vaccines. Although these trials will greatly advance our knowledge of NSCLC immunotherapy, we believe truly efficacious immunotherapy may only result from implementation of strategies to both augment antitumor immunity and counteract tumor-mediated immunosuppression.     

Decrease in CD3-negative-CD8dim(+) and Vdelta2/Vgamma9 TcR+ peripheral blood lymphocyte counts,low perforin expression and the impairment of natural killer cell activity is associated with chronic hepatitis C virus infection

BACKGROUND/AIMS: As chronic hepatitis C virus (HCV) infection is associated with impaired natural killer (NK) cell cytotoxicity, we examined the phenotypes and perforin expression of peripheral blood lymphocytes, as well as the effect of interferon-alpha2b (IFN-alpha2b) therapy. METHODS: Thirty-three patients had chronic hepatitis C, and of them 12 had been on IFN-alpha2b treatment. Eleven individuals had been treated earlier with IFN-alpha2b and completely cured, and eight were HCV carriers with persistently normal serum alanine aminotransferase. Three-colour flow cytometry was used to measure the percentage of CD3(+/-)CD8+, CD3+CD4+, gammadeltaTcR+, Vdelta2 TcR+, Vgamma9 TcR+, Vdelta1 TcR+, CD3-CD16+, CD3-CD56+, CD19+ and perforin-positive cells. NK cell activity was assessed by single cell cytotoxic and flow cytometric assay. RESULTS: Patients with chronic hepatitis C showed an impaired NK cytotoxicity, decreased percentage of CD3-negative-CD8dim-positive (NK subtype) and Vgamma9/Vdelta2 TcR+ as well as perforin-positive T lymphocytes, compared to controls and to those who were cured from HCV infection. IFN-alpha2b increased NK cell cytotoxicity and the percentage of perforin-positive lymphocytes. CONCLUSIONS: Our findings suggest that in chronic HCV infection a decreased percentage of CD3(-)CD8+, Vgamma9/Vdelta2 TcR+ and perforin-positive T cells and simultaneous decreased peripheral NK activity may contribute to the impaired cellular immune response and the chronicity of the disease.     

Perforin is required for innate and adaptive immunity induced by heat shock protein gp96

Tumor-secreted gp96-Ig is highly immunogenic and triggers CD8 T cell-mediated tumor rejection. In vivo secreted gp96-Ig and gp96-myc cause NK activation and clonal expansion of specific CD8(+) CTL in wild-type and in Fas-ligand-deficient (gld) mice but not in perforin- (PKO) or IFN-gamma-deficient (GKO) mice. Transfer of perforin-competent NK cells restores the ability of PKO mice to clonally expand CD8 CTL in response to gp96-Ig. The data demonstrate an essential role for perforin-mediated functions in the activation of innate and adaptive immunity by heat shock protein gp96-peptide complexes. Crosspresentation of antigens by heat shock proteins seems to require a perforin-dependent positive feedback loop between NK and DC for both sustained NK activation and clonal CTL expansion. The studies also explain how depressed NK activity in patients with tumors or after viral infections could diminish CTL responses.