Immunosurveillance and immunoediting--can the immune response be made more "immunodemocratic"?

Increasing evidence has demonstrated that the immune system is able to mount responses against tumors and that these responses can be enhanced using a number of strategies. Several of these strategies are currently being evaluated in clinical trials, where their efficacy and cost effectiveness will be ascertained. Sadly, we have to admit that despite great expectations and enormous achievements in basic immunology and molecular biology, immunotherapeutic interventions relying on the elicitation of cytotoxic cellular immunity so far have had limited success. We have found that gene-based vaccination is effective in breaking tolerance to tumor-associated antigens, but the response is directed towards few of the potential epitopes due to immunodominance. Tumor cells that have lost the immunodominant epitope due to mutations would no longer be recognized and would evade immune surveillance. Designing a protocol for immunotherapy, therefore, necessitates stimulation of an immune response directed against a multitude of epitopes. Increasing the number of epitopes available for presentation to T cells is the initial step. It mandates increased degradation of the antigen following DNA immunization. A logical continuation involves manipulation of the intimate mechanisms controlling the processes of stimulation and/or suppression of T cells recognizing the "sub-dominant" epitopes thus offering to the immunologist ways for overcoming tumor-immune evasion strategies. We now have the necessary instruments to ask all the fundamental questions of tumor immunology. A better understanding of the immune escape mechanisms and those underlying tumor-induced immune suppression will help in designing novel and more efficient protocols for immunotherapy in the clinical setting.

Immune responses against PSMA after gene-based vaccination for immunotherapy-A: results from immunizations in animals.

Two plasmid DNA vaccines, encoding either products that are retained in the cytosol and degraded in the proteasome (tVacs; hPSMAt), or secreted proteins (sVacs; hPSMAs) were evaluated for stimulation of cytotoxic cell or antibody responses. Immunization with both vectors led to generation of cell cytotoxicity providing granulocyte-macrophage colony-stimulating factor was administered with the vaccine. Spleen cells from animals immunized with hPSMAt demonstrated stronger cytotoxicity to the target cells. Priming with a vector that encoded a xenogeneic protein (hPSMAt; 'xenogeneic' construct) and boosting with a vector that encoded an autologous protein (rPSMAt; 'autologous' construct) gave the best protection against tumor challenge. Immunization with tVacs did not lead to formation of antibodies to the target protein as detected by Western blot or ELISA, while immunization with sVacs or with the protein did. Antibodies were of mixed Th1-Th2 isotype. Priming with tVacs and boosting with protein also resulted in antibody formation, but in this case the antibodies were from the cytotoxic, Th1 isotype. The best strategy to obtain a strong cellular cytotoxic response, therefore, seems to be gene-based vaccinations with tVacs, priming with the 'xenogeneic' and boosting with the 'autologous' constructs. When cytotoxic antibody production is the goal, priming should be performed with the tVacs while boosting with the protein.

Biochemical nature and mapping of PSMA epitopes recognized by human antibodies induced after immunization with gene-based vaccines.

BACKGROUND: A possible new target for immunotherapy is the prostate-specific membrane antigen (PSMA). The aim of the present study was to define potential PSMA epitopes for antibody binding using sera from patients immunized with gene-based anti-PSMA vaccines. MATERIALS AND METHODS: Sera from prostate cancer patients, immunized repeatedly with plasmid and adenoviral vectors, each encoding for the extracellular portion of human PSMA, were tested for anti-PSMA antibodies by Western blot. PSMA-producing LNCaP cells were used as a control. Recombinant PSMA protein cleaved with different proteinases was used for epitope mapping. Different enzymes were used to cleave the PSMA molecule. RESULTS: Specific anti-PSMA antibodies were detected in the studied patients' sera, mainly against the PSMA protein core. An alignment of the predicted enzyme-cleavage fragments was compared with Western blot results and several antibody epitopes were determined. CONCLUSION: These data demonstrate that multiple gene-based vaccinations induce an anti-PSMA humoral immune response. The antibodies are predominantly specific for the PSMA protein core.

In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer--changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-gamma lymphokine profile.

In order to provoke an immune response, a tumor vaccine should not only maximize antigen-specific signals, but should also provide the necessary "co-stimulatory" environment. One approach is to genetically manipulate tumor cells to either secrete lymphokines (GM-CSF, IL-12, IL-15) or express membrane bound molecules (CD80, CD86). Furthermore, patient dendritic cells can be loaded with tumor-associated antigens or peptides derived from them and used for immunotherapy. Genetic modification of dendritic cells can also lead to presentation of tumor-associated antigens. Transfection of dendritic cells with DNA encoding for such antigens can be done in vitro, but transfection efficiency has been uniformly low. Alternatively, dendritic cells can also be modulated directly in vivo either by "naked" DNA immunization or by injecting replication-deficient viral vectors that carry the tumor specific DNA. Naked DNA immunization offers several potential advantages over viral mediated transduction. Among these are the inexpensive production and the inherent safety of plasmid vectors, as well as the lack of immune responses against the carrier. The use of viral vectors enhances the immunogenicity of the vaccine due to the adjuvant properties of some of the viral products. Recent studies have suggested that the best strategy for achieving an intense immune response may be priming with naked DNA followed by boosting with a viral vector. We have successfully completed a phase I and phase II clinical trials on immunotherapy of prostate cancer using naked DNA and adenoviral immunizations against the prostate-specific membrane antigen (PSMA) and phase I clinical trial on colorectal cancer using naked DNA immunization against the carcinoembryonic antigen (CEA). The vaccination was tolerated well and no side effects have been observed so far. The therapy has proven to be effective in a number of patients treated solely by immunizations. The success of the treatment clearly depends on the stage of the disease proving to be most efficient in patients with minimal disease or no metastases. A panel of changes in the phenotype of peripheral blood lymphocytes and the expression of intra-T-cell lymphokines seems to correlate with clinical improvement.

Naked DNA and adenoviral immunizations for immunotherapy of prostate cancer: a phase I/II clinical trial.

INTRODUCTION AND OBJECTIVES: Animal studies have indicated that the use of syngeneic dendritic cells that have been transfected ex vivo with DNA for tumor-specific antigen results in tumor regression and decreased number of metastases. Additional studies have also suggested the possibility to modulate the dendritic cells in vivo either by 'naked' DNA immunization or by injecting replication-deficient viral vectors that carry the tumor-specific DNA. Using the prostate- specific membrane antigen (PSMA) as a target molecule, we have initiated a clinical trial for immunotherapy of prostate cancer. The primary objective of the study was to determine the safety of the PSMA vaccine after repeated intradermal injections. METHODS: We have included the extracellular human PSMA DNA as well as the human CD86 DNA into separate expression vectors (PSMA and CD86 plasmids), and into a combined PSMA/CD86 plasmid. In addition, the expression cassette from the PSMA plasmid was inserted into a replication deficient adenoviral expression vector. Twenty-six patients with prostate cancer were entered into a phase I/II toxicity-dose escalation study, which was initiated in spring 1998. Immunizations were performed intradermally at weekly intervals. Doses of DNA between 100 and 800 microg and of recombinant virus at 5x10(8) PFUs per application were used. RESULTS AND CONCLUSION: No immediate or long-term side effects following immunizations have been recorded. All patients who received initial inoculation with the viral vector followed by PSMA plasmid boosts showed signs of immunization as evidenced by the development of a delayed-type hypersensitivity reaction after the PSMA plasmid injection. In contrast, of the patients who received a PSMA plasmid and CD86 plasmid, only 50% showed signs of successful immunization. Of the patients who received PSMA plasmid and soluble GM-CSF, 67% were immunized. However, all patients who received the PSMA/CD86 plasmid and sGM-CSF became immunized. The patients who did not immunize during the first round were later successfully immunized after a boost with the viral vector. The heterogeneity of the medical status and the presence in many patients of concomitant hormone therapy does not permit unequivocal interpretation of the data with respect to the effectiveness of the therapy. However, several responders, as evidenced by a change in the local disease, distant metastases, and PSA levels, can be identified. A phase II clinical study to evaluate the effectiveness of the therapy is currently underway.

Changes in donor leukocytes during blood storage. Implications on post-transfusion immunomodulation and transfusion-associated GVHD.

Freshly drawn peripheral blood granulocytes, monocytes and NK cells express Fas-L following activation. Fully functional soluble Fas-L (sFas-L) is released and found in fresh plasma samples from healthy volunteers coupled to a Fas-L binding factor (PFBF). In the absence of plasma leukocytes undergo rapid apoptosis with 15% of the granulocytes and 25% of the monocytes showing signs of apoptosis immediately following separation. Apoptosis progresses during refrigerated storage even in the presence of plasma and all granulocytes disintegrate by day 15 of storage. The absence of plasma increases the rate of apoptosis. The resulting increased expression of PS on apoptosing leukocytes enhances blood procoagulant activity. Whereas transfusions of fresh blood lead to alloimmunization, the functional impairment and death of antigen-presenting cells during storage eliminates their ability to stimulate cytotoxic immune responses. Additionally, loss of function and death of donor T cells during storage preclude the possibility for development of post-transfusion GVHD. Continued storage leads to the secondary necrosis of apoptosed leukocytes and the release of soluble antigens. Depending on dose, transfusion of such products may lead to immunosuppression due to a T2 bias of the immune response.

Fas and Fas ligand expression on human peripheral blood leukocytes.

OBJECTIVES: Study of Fas and Fas ligand (Fas-L) expression, as well as sFas-L release, by fresh human peripheral blood leukocytes. METHODS: Flow cytometry, cytotoxicity, immunofluorescence staining of fresh smears. Western blotting. RESULTS: Granulocytes and monocytes express a low level of Fas receptor, but no Fas-L. These cells, as well as NK cells, contain presynthesized depots of Fas-L which they express following activation by brief storage (60 min) at room temperature or during separation from whole blood. Such activation also leads to Fas receptor upregulation. NK cells do not express Fas receptor. Once expressed on blood leukocytes, fully functional Fas-L can be released from the membrane and can be detected in plasma-free cell supernatants. CONCLUSION: Human peripheral blood granulocytes, monocytes and NK cells contain intracellular presynthesized Fas-L which they readily express following blood anticoagulation, blood storage or cell separation. Soluble Fas-L is released from those cells and can be detected in protein-free supernatants by immunoblotting.

Mechanisms of alloimmunization and immunosuppression by blood transfusions in an inbred rodent model.

During refrigerated storage leukocytes in donor blood progressively undergo apoptosis followed by secondary necrosis. Using an inbred rodent transfusion model, recipient animals received viable, necrotic, or apoptotic cells. While transfusion of viable blood MNCs stimulated production of IgM, IgG1 (Th2 type) and IgG2a (Th1-type) antidonor antibodies, leading to a suppression of subsequent DTH to donor antigens, transfusion of apoptotic donor cells led to neither alloimmunization nor immunosuppression. On the other hand transfusion of lysed donor cells resulted in production of IgM and IgG1 (Th2-type) antidonor antibodies and to a strong suppression of subsequent DTH to donor antigens. Intravenously administered spleen cells that had been depleted of professional APCs and enriched for B cells stimulated IgM antidonor antibodies but not IgG antibodies. Transfusion of such cells also led to suppression of subsequent DTH to donor antigens, probably through induction of anergy or apoptosis in alloantigen-reactive recipient cells. Depending on the duration of blood storage any or all of these 4 classes of cells may be present and Th2 and/or Th1 effector mechanisms can be generated following blood transfusion.

Blood transfusion, blood storage and immunomodulation.

Allogeneic blood transfusion is the most frequent allotransplantation procedure performed on a routine basis with no prior HLA-typing. Roughly 50% of the recipients of unprocessed red cells and platelets become alloimmunized. Evidence also exists for some degree of transfusion-induced immunosuppression. Prior transfusion has been shown to enhance kidney transplant survival and evidence of an increase in tumor recurrence and of infectious complications has also been presented. The presence of donor antigen-presenting cells appears to be a prerequisite for alloimmunization and they must be both viable and capable of presenting a costimulatory signal in order to induce IL-2 secretion and proliferation of responding CD4 T cells. APCs presenting antigen but no costimulatory signal can induce non-responsiveness in CD4 T cells, a possible mechanism of transfusion-induced immunosuppression. APCs in refrigerated blood continue to present antigen but progressively lose their ability to provide costimulation. By day 14 costimulatory capacity is absent and transfusion of such blood should not alloimmunize but could induce some degree of immunosuppression. Further refrigerated storage in excess of 2 to 3 weeks leads to induction of apoptosis in contaminating leukocytes. We have found that alloantigens-expressed on such cells do not appear to be recognized by responder T cells and transfusion of blood stored in excess of 3 weeks should neither alloimmunize nor immunosuppress.

Blood transfusion and immunomodulation: a possible mechanism.

To induce an immunogenic response in vivo, an antigen-presenting (stimulator) cell must present both antigen-specific (class II MHC) and an accessory signal to the CD4 T cell. Failure to express the accessory signal has been shown in vitro to induce a state of specific unresponsiveness (anergy) in the T cell. We have shown that although stimulator cells in blood continue to express class II MHC molecules during refrigerated storage, their ability to present the accessory signal diminishes, reaching zero in all units tested by about 13 days. This implies that blood in excess of 2 weeks old should not alloimmunize but could induce some degree of immunosuppression. UV-B irradiation and, to a lesser extent, gamma-irradiation, were also shown to inhibit expression of the accessory signal by stimulator cells in blood.

Costimulatory signals necessary for induction of T cell proliferation.

Two separate signals are required for induction of T cell proliferation. In an attempt to identify them we used polyclonal T cell activation with Con A, which requires costimulation with autologous accessory cells. The costimulatory activity is not constitutively expressed on accessory cells since such cells fixed immediately after separation from whole blood are unable to provide the necessary signal(s), although such activity is readily expressed after activation by incubation and such cells subsequently fixed will support Con A-induced T cell proliferation. Addition of recombinant IL-1 plus IL-6 to T cell cultures in the absence of accessory cells does not result in T cell proliferation but addition of these factors to cultures containing fixed activated accessory cells results in further increase in proliferation. The expression of the costimulatory activity during incubation is inhibited in the presence of cycloheximide or tunicamycin. The costimulatory activity of fixed activated cells is partially inhibited by antibody against ICAM-1. This inhibition is not reversed by the addition of recombinant IL-1 and IL-6. When accessory cells are preactivated in the presence of chloroquine, they are unable to provide costimulation to T cells but addition of recombinant IL-1 and IL-6 restores their ability to support T cell proliferation. Accessory cells preactivated in the presence of colchicine show an increased ability to provide costimulation to T cells in culture.

Induction of primary mixed leukocyte reactions with ultraviolet B or chemically modified stimulator cells.

Treatment of stimulator cells with 0.1% paraformaldehyde for 60 sec or ultraviolet-B (UV-B) irradiation (1000 J/m2) eliminates their ability to elicit T cell proliferation in a primary mixed leukocyte reaction. However, a T cell response equal to 20-40% of control value could be elicited by paraformaldehyde fixed or UV-B irradiated cells providing the latter are incubated at 37 degrees C for 18 hr prior to treatment. The incubation also induces a one-log increase in the density of fluorescence when the cells are stained with monoclonal antibodies against class II molecules DR and DP as well as the intercellular adhesion molecule -1 (ICAM-1). We interpret this as an increase in the membrane expression of these structures following incubation. Chloroquine and cerulenin, known to inhibit protein degradation and antigen processing and presentation do not influence the upregulation in membrane expression of these class II and adhesion molecules, but do prevent incubation from overriding the effect of paraformaldehyde treatment. Colchicine, which reduces the traffic through tubular lysosomes, also has no effect on the upregulation but enhances allopresentation. We propose that incubation of stimulator cells in the presence of chloroquine and cerulenin results in the membrane expression of class II molecules without associated peptides. The inability of stimulator cells expressing such "nude" MHC molecules to elicit T cell proliferation after chemical modification could be due to easier crosslinking of the allodeterminants by paraformaldehyde when the binding site is empty but could also mean that nude MHC molecules are not per se immunogenic and become so only after acquisition of a peptide. It is also possible that chloroquine, NH4Cl, and cerulenin block the expression of signals other than the class II and cell adhesion molecules that are essential for induction of T cell proliferation.

Isolation and cryopreservation of human peripheral blood monocytes.

A modification of the Freundlich and Avdalovic method (J. Immunol. Methods 62, 31 (1983] is reported. Buffy coats, separated and pooled together, are used for isolation of monocytes (70% yield, 100% purity). Cell density of working suspension is increased up to 0.65 X 10(9) cells/75 cm2 surface by multiplication of the active fibronectin sites. For the purpose, cryoprecipitate is used instead of plasma for coating the glass-gelatin surface. Monocytes, isolated by that procedure, could be successfully cryopreserved with dimethyl sulfoxide cryoprotective solution.

Aggregate formation in cryopreserved leukocyte suspensions.

During cryopreservation of leukocytes, a great part of the granulocytes is injured. The latter is responsible for aggregate formation in the suspension and a loss in the number of preserved cells. The formation of aggregates in the suspension is potentiated in the presence of serum in the cryoprotective medium. On the other hand, the presence of sugar (sucrose, lactose, dextrose) in the washing solution diminishes the aggregate formation, the latter effect being directly proportional to the final concentration of the sugar in the suspension. The aggregates contain mainly cells and very rarely amorphous or fibrous zones. They appear only in the presence of serum. Their formation is not connected with presence of traces of fibrinogen in the serum.

A method for preparation of dry thrombin for topical application.

Thrombin for topical hemostasis can be prepared from bovine or human blood plasma. The prothrombin is isolated by means of adsorption on DEAE-Sephadex A-50 and consecutively activated by CaCl2 and thromboplastin. Thrombin is precipitated and purified by acetone. The specific activity of the thrombin preparation is 122 + 23 IU/mg protein while the yield is 36,360 +/- 6623 IU/liter plasma.