ovalbumin and Thymus-Neoplasms

Activation of RORγ with synthetic small-molecule agonists has been shown to enhance type 17 effector (CD4

Topics: Animals; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Granzymes; Immunotherapy, Adoptive; Interleukin-17; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nuclear Receptor Subfamily 1, Group F, Member 3; Ovalbumin; T-Lymphocyte Subsets; Thymoma; Thymus Neoplasms

Tumor metastasis and immune evasion present major challenges of cancer treatment. Radiotherapy can overcome immunosuppressive tumor microenvironments. Anecdotal reports suggest abscopal anti-tumor immune responses. This study assesses abscopal effects of radiotherapy in combination with mRNA-based cancer vaccination (RNActive. C57BL/6 mice were injected with ovalbumin-expressing thymoma cells into the right hind leg (primary tumor) and left flank (secondary tumor) with a delay of 4 days. Primary tumors were irradiated with 3 × 2 Gy, while secondary tumors were shielded. RNA and combined treatment groups received mRNA-based RNActive. Radiotherapy and combined radioimmunotherapy significantly delayed primary tumor growth with a tumor control in 15 and 53% of mice, respectively. In small secondary tumors, radioimmunotherapy significantly slowed growth rate compared to vaccination (p = 0.002) and control groups (p = 0.01). Cytokine microarray analysis of secondary tumors showed changes in the cytokine microenvironment, even in the non-irradiated contralateral tumors after combination treatment.. Combined irradiation and immunotherapy is able to induce abscopal responses, even with low, normofractionated radiation doses. Thus, the combination of mRNA-based vaccination with irradiation might be an effective regimen to induce systemic anti-tumor immunity.

Topics: Animals; Antibodies, Monoclonal; Combined Modality Therapy; Disease Models, Animal; Mice; Mice, Inbred C57BL; Ovalbumin; Radioimmunotherapy; RNA, Messenger; Thymoma; Thymus Neoplasms

The aim of the research presented here was to determine the characteristics and immunostimulatory capacity, in vivo, of antigen and adjuvant co-loaded into microparticles made from a novel diaminosulfide polymer, poly(4,4'-trimethylenedipiperdyl sulfide) (PNSN), and to assess their potential as cancer vaccine vectors. PNSN microparticles co-loaded with the antigen, ovalbumin (OVA), and adjuvant, CpG 1826, (PNSN(OVA + CpG)) were fabricated and characterized for size (1.64 μm diameter; PDI=0.62), charge (-23.1 ± 0.3), and loading efficiencies of antigen (7.32 μg/mg particles) and adjuvant (0.95 μg/mg particles). The ability of PNSN(OVA + CpG) to stimulate cellular and humoral immune responses in vivo was compared with other PNSN microparticle formulations as well as with poly(lactic-co-glycolic acid)(PLGA)-based microparticles, co-loaded with OVA and CpG (PLGA(OVA + CpG)), an adenovirus encoding OVA (Ad5-OVA), and OVA delivered with incomplete Freund's adjuvant (IFA(OVA)). In vivo OVA-specific IgG1 responses, after subcutaneous prime/boosts in mice, were similar when PNSN(OVA + CpG) and PLGA(OVA + CpG) were compared and the presence of CpG 1826 within the PNSN microparticles demonstrated significantly improved responses when compared to PNSN microparticles loaded with OVA alone (PNSN(OVA)), plus or minus soluble CpG 1826. Cellular immune responses to all particle-based vaccine formulations ranged from being negligible to modest with PNSN(OVA + CpG) generating the greatest responses, displaying significantly increased levels of OVA-specific CD8+ T lymphocytes compared to controls and IFA(OVA) treated mice. Finally, it was shown that of all vaccination formulations tested PNSN(OVA + CpG) was the most protective against subsequent challenge with an OVA-expressing tumor cell line, E.G7. Thus, microparticles made from poly(diaminosulfide)-based macromolecules possess promising potential as vaccine vectors and, as demonstrated here, may have impact as cancer vaccines in particular.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Cell Line, Tumor; Chemistry, Pharmaceutical; Drug Carriers; Freund's Adjuvant; Immunity, Cellular; Immunity, Humoral; Lactic Acid; Male; Mice, Inbred C57BL; Oligodeoxyribonucleotides; Ovalbumin; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Sulfides; Thymoma; Thymus Neoplasms; Time Factors; Transfection; Tumor Burden; Vaccination

Vaccine strategies aimed at generating CD8(+) T cell memory responses are likely to show augmented efficacy against chronic challenges like tumor. The abundance in variety of memory CD8(+) T cells behooves development of vaccine strategies that generate distinct memory responses and evaluate them for tumor efficacy. In this study, we demonstrate the ability of a variety of rapamycin treatment regimens to regulate virus vaccination-induced CD8(+) T cell memory responses and tumor efficacy. Strikingly, a short course of high-dose, but not low-dose, rapamycin treatment transiently blocks viral vaccination-induced mammalian target of rapamycin activity in CD8(+) T cells favoring persistence and Ag-recall responses over type 1 effector maturation; however, prolonged high-dose rapamycin administration abrogated memory responses. Furthermore, a short course of high-dose rapamycin treatment generated CD8(+) T cell memory responses that were independent of IL-15 and IL-7 and were programmed early for sustenance and greater tumor efficacy. These results demonstrate the impact a regimen of rapamycin treatment has on vaccine-induced CD8(+) T cell responses and indicates that judicious application of rapamycin can augment vaccine efficacy for chronic challenges.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Animals; Antigens, Neoplasm; Canarypox virus; Cancer Vaccines; CD8-Positive T-Lymphocytes; Clonal Selection, Antigen-Mediated; Immunologic Memory; Interleukin-15; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Multiprotein Complexes; Neoplasm Transplantation; Ovalbumin; Proteins; Sirolimus; T-Cell Antigen Receptor Specificity; Thymoma; Thymus Neoplasms; TOR Serine-Threonine Kinases; Vaccination

In experimental tumor immunotherapy, incomplete Freund's adjuvant (IFA) has been considered as the "gold standard" for T-cell vaccination in mice and humans in spite of its considerable adverse effects. Recently, we succeeded in eliciting strong CTL responses in mice after vaccination with biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres (MS). In our study, we compared the immune response to IFA and PLGA-MS containing ovalbumin (OVA) and CpG-oligodeoxynucleotide (MS-OVA/CpG) or we used a mixture of MS-OVA/CpG and MS-polyI:C. A single vaccination with MS-OVA/CpG elicited long-lasting titers of IgG1 and IgG2a, but only low IgE titers, and also the T-cell response was biased toward Th(1) differentiation. Antigen presentation to CD4(+) and CD8(+) cells and activation of a cytotoxic T-cell response in mice vaccinated with PLGA-MS and IFA lasted for over 3 weeks. Preconditioning of the injection site with TNF-α and heterologous prime-boost regimen further enhanced the cytotoxic response. PLGA-MS were as efficient or superior to IFA in eradication of preexisting tumors and suppression of lung metastases. Taken together, PLGA-MS are well-defined, biodegradable and clinically compatible antigen carrier systems that compare favorably with IFA in their efficacy of tumor immunotherapy in mouse models and hence deserve to be tested for their effectiveness against human malignant diseases.

Topics: Animals; CD8-Positive T-Lymphocytes; Disease Models, Animal; Freund's Adjuvant; Immunotherapy; Lactic Acid; Lipids; Melanoma; Mice; Mice, Inbred C57BL; Microspheres; Oligodeoxyribonucleotides; Ovalbumin; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; T-Lymphocytes, Cytotoxic; Thymoma; Thymus Neoplasms; Treatment Outcome

Protein-based vaccines have significant potential as infectious disease and anticancer therapeutics, but clinical impact has been limited in some applications by their inability to generate a coordinated cellular immune response. Here, a pH-responsive carrier incorporating poly(propylacrylic acid) (PPAA) was evaluated to test whether improved cytosolic delivery of a protein antigen could enhance CD8+ cytotoxic lymphocyte generation and prophylactic tumor vaccine responses. PPAA was directly conjugated to the model ovalbumin antigen via reducible disulfide linkages and was also tested in a particulate formulation after condensation with cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA). Intracellular trafficking studies revealed that both PPAA-containing formulations were stably internalized and evaded exocytotic pathways, leading to increased intracellular accumulation and potential access to the cytosolic MHC-1 antigen presentation pathway. In an EG.7-OVA mouse tumor protection model, both PPAA-containing carriers robustly inhibited tumor growth and led to an approximately 3.5-fold increase in the longevity of tumor-free survival relative to controls. Mechanistically, this response was attributed to the 8-fold increase in production of ovalbumin-specific CD8+ T-lymphocytes and an 11-fold increase in production of antiovalbumin IgG. Significantly, this is one of the first demonstrated examples of in vivo immunotherapeutic efficacy using soluble protein-polymer conjugates. These results suggest that carriers enhancing cytosolic delivery of protein antigens could lead to more robust CD8+ T-cell response and demonstrate the potential of pH-responsive PPAA-based carriers for therapeutic vaccine applications.

Topics: Acrylates; Animals; Antigen Presentation; Antigens; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Proliferation; Disease-Free Survival; Drug Delivery Systems; Endosomes; Female; Hydrogen-Ion Concentration; Immunoglobulin G; Lymphocyte Activation; Methacrylates; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Nylons; Ovalbumin; Polymers; Thymoma; Thymus Neoplasms; Treatment Outcome

We recently established a novel drug delivery system (DDS) using oligomannose-coated liposomes (OMLs) which are probably taken up by macrophages (Mvarphi) to carry anti-cancer drugs to milky spots known as preferential metastatic sites of gastric cancers [Y. Ikehara, T. Niwa, L. Biao, S.K. Ikehara, N. Ohashi, T. Kobayashi, Y. Shimizu, N. Kojima, H. Nakanishi, A carbohydrate recognition-based drug delivery and controlled release system using intraperitoneal macrophages as a cellular vehicle, Cancer Res. 66 (2006) 8740-8748]. In the present study, we applied this intraperitoneal DDS for systemic cancer immunotherapy employing ovalbumin (OVA) as a model antigen. The cells taking up the OMLs containing FITC-OVA injected into the peritoneal cavity were predominantly Mvarphi, as they showed adhesive characteristics and expressed F4/80 and CD11b almost exclusively. The phagocytic cells also took up bare OVA directly to the same extent as OML-enclosed OVA (OML-OVA), as it is a highly mannosilated protein. The phagocytic cells taking up OML-OVA, however, could activate OVA-specific CD8+ (from OT-I: H-2Kb/OVA257-264-specific) and CD4+ (from OT-II: H-2Ab/OVA323-339-specific) T cells much more effectively in vitro than those taking up bare OVA. Furthermore, only the mice pre-immunized with OML-OVA rejected E.G7-OVA (OVA-transfected EL4) but not EL4. These results indicate that the OMLs can also be used as an effective antigen delivery system for cancer immunotherapy activating both CTL and Th subsets.

Topics: Animals; Antigen Presentation; Antigens, Differentiation; CD11b Antigen; Cell Line, Tumor; Cell Movement; Drug Carriers; Drug Compounding; Egg Proteins; Female; H-2 Antigens; Histocompatibility Antigens Class II; Immunodominant Epitopes; Immunotherapy; Liposomes; Lymphocyte Activation; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Peptide Fragments; Phagocytosis; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; Thymoma; Thymus Neoplasms; Transfection; Trisaccharides

In the present study, the adjuvant capacity of oligomannose-coated liposomes (OMLs) was evaluated in mice, and an OML-based vaccine was shown to induce effective anti-tumor immunity. C57BL/6 mice were immunized subcutaneously with OML-encased ovalbumin (OVA) and challenged with OVA-expressing E.G7-OVA tumor cells. All mice that received OVA in OMLs completely rejected the E.G7-OVA tumor. Spleen cells from the immunized mice showed strong cytotoxic activity against E.G7-OVA cells, but not against the parental EL4 cells. The therapeutic efficacy of OML-encased OVA against established E.G7-OVA tumors was then investigated. When the tumor mass became palpable (8-10 mm in length), the mice were treated with a single injection of 1 microg of OML-encased OVA. Tumor growth was reduced significantly in mice treated with OML-encased OVA and tumors were completely eliminated in about 40% of these mice. Similar results were obtained using EL4 tumors, with the EL4 cell lysate used as an antigen. These results indicate that an OML-based vaccine with an encased tumor antigen might be useful clinically to raise an effective immune response against a tumor.

Topics: Adjuvants, Immunologic; Animals; Antigens; Cancer Vaccines; Cell Line, Tumor; Chemistry, Pharmaceutical; Drug Compounding; Injections, Subcutaneous; Lipids; Liposomes; Lymph Nodes; Mice; Mice, Inbred C57BL; Ovalbumin; Technology, Pharmaceutical; Thymoma; Thymus Neoplasms; Time Factors; Trisaccharides

The effector CD8(+) T cells recognize major histocompatibility complex (MHC) class I binding altered self-peptides expressed in tumour cells. Although the requirement for CD4(+) T helper type 1 (Th1) cells in regulating CD8(+) T cells has been documented, their target epitopes and functional impact in antitumour responses remain unclear. We examined whether a potent immunogenic peptide of Mycobacterium tuberculosis eliciting Th1 immunity contributes to the generation of CD8(+) T cells and to protective antitumour immune responses to unrelated tumour-specific antigens. Peptide-25, a major Th epitope of Ag85B from M. tuberculosis preferentially induced CD4(+) Th1 cells in C57BL/6 mice and had an augmenting effect on Th1 generation for coimmunized unrelated antigenic peptides. Coimmunization of mice with Peptide-25 and ovalbumin (OVA) or Peptide-25 and B16 melanoma peptide [tyrosinase-related protein-2 (TRP-2)] for MHC class I led to a profound increase in CD8(+) T cells specific for OVA and TRP-2 peptides, respectively. This heightened response depended on Peptide-25-specific CD4(+) T cells and interferon-gamma-producing T cells. In tumour protection assays, immunization with Peptide-25 and OVA resulted in the enhancement of CD8(+) cytotoxic cell generation specific for OVA and the growth inhibition of EL-4 thymoma expressing OVA peptide leading to the tumour rejection. These phenomena were not achieved by immunization with OVA alone. Peptide-25-reactive Th1 cells counteractivated dendritic cells in the presence of Peptide-25 leading them to activate and present OVA peptide to CD8(+) cytotoxic T cells.

Topics: Acyltransferases; Animals; Antigen Presentation; Antigens, Bacterial; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cells, Cultured; Cytotoxicity, Immunologic; Dendritic Cells; Epitopes, T-Lymphocyte; Immunization; Interferon-gamma; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Transplantation; Ovalbumin; T-Lymphocytes, Helper-Inducer; Thymoma; Thymus Neoplasms

Prior studies have shown that transfer of ovalbumin (OVA)-specific T helper type 1 (Th1) cells into mice bearing MHC class II+ OVA-expressing tumor cells (A20-OVA) causes complete tumor rejection. Here we show that, although Th1 cell therapy alone was not effective against MHC class II- OVA-expressing tumor cells (EG-7), treatment of mice bearing established EG-7 tumors by i.v. transfer of Th1 cells combined with i.t. injection of the model tumor antigen OVA induced complete tumor rejection. Transferred Th1 cells enhanced the migration of tumor-infiltrating antigen-presenting cells (APC) that had processed OVA into the draining lymph node (DLN). Although transferred Th1 cells were randomly distributed in DLN, distal LN, spleen, and tumor tissue, active proliferation of Th1 cells always initiated in DLN, where Th1 cells efficiently interacted with APC that presented OVA. In parallel, OVA-tetramer+ CTLs, showing EG-7-specific cytotoxicity, were highly induced in DLN and the local tumor site. The OVA-tetramer+ CTL functioned systemically because two bilateral tumor masses were both completely rejected on treatment of one tumor. Furthermore, either active proliferation of transferred Th1 cells or generation of tetramer+ CTL was not induced in MHC class II-deficient mice and LN-deficient Aly/Aly mice. These results indicate that DLN is an indispensable organ for initiating active APC/Th1 cell interactions, which is critical for inducing complete eradication of tumor mass by tumor-specific CTL.

Topics: Animals; Antigen-Presenting Cells; Antigens, Neoplasm; Cell Communication; Cell Movement; Histocompatibility Antigens Class II; Immunotherapy, Adoptive; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; T-Lymphocytes, Cytotoxic; Th1 Cells; Thymoma; Thymus Neoplasms; Transfection

Uric acid released from dying cells has been shown recently to act as a danger signal for the immune system, stimulating dendritic cell maturation and enhancing T-cell responses to foreign antigens. Stimulation of dendritic cell maturation by uric acid has been proposed as a mechanism by which the immune system could generate responses against tumors. We show here that uric acid levels are elevated in tumors undergoing immune rejection and that the inhibition of uric acid production, by systemic administration of allopurinol, or the removal of uric acid, by administration of uricase, delayed tumor immune rejection, whereas subcutaneous administration of crystalline uric acid enhanced the rejection process.

Topics: Allopurinol; Animals; Apoptosis; Cell Division; Chickens; Enzyme Inhibitors; Female; Graft Rejection; Immune System; Injections, Subcutaneous; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Ovalbumin; Thymoma; Thymus Neoplasms; Urate Oxidase; Uric Acid

Pseudomonas exotoxin (PE) is a 66 kDa bacterial toxin that is able to bind to mammalian cells, undergo receptor mediated endocytosis, and translocate its C-terminal catalytic domain into the cytosol. We investigated whether PE could be used in vivo to deliver CD8+ T-cell epitopes to the MHC-class I antigen presentation pathway to trigger a specific cytotoxic T-lymphocyte (CTL) response.. Amino acid 553 of PE was deleted to eliminate toxin catalytic activity, and amino acids 204-386 of ovalbumin were fused near the nontoxic PE C-terminus to produce PE(D)-OVA200. Mice were vaccinated with 100 microg of PE(D)-OVA200 3 times at 21 day intervals. Splenocytes were harvested 1 week later, and stimulated in vitro with ovalbumin expressing EG7 murine thymoma cells. In vivo tumor protection experiments were performed by vaccinating groups of mice as before, followed by a lethal dose of ovalbumin expressing tumor cells (MO5) injected subcutaneously.. Splenocytes from PE(D)-OVA200 vaccinated mice lysed (51)Cr labeled EG7 cells but not the untransfected EL4 parent cell line, whereas splenocytes from mice immunized with PBS, PE(D), or ovalbumin were unable to lyse EG7 cells. Cytotoxicity in vitro was mediated by CD8+ T-cells. PE(D)-OVA200 vaccinated mice survived (88%) a lethal subcutaneous challenge of ovalbumin expressing MO5 cells. Depletion of CD8+ cells from PE(D)-OVA200 vaccinated mice abolished this protection, indicating that this cell population is required for tumor rejection in vivo.. Our results indicate that PE(D) may be used as a vehicle to stimulate a protective CTL response to heterologous antigen in vivo.

Topics: ADP Ribose Transferases; Animals; Bacterial Toxins; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cloning, Molecular; Exotoxins; Female; Genetic Vectors; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Ovalbumin; Plasmids; Pseudomonas aeruginosa Exotoxin A; Skin Neoplasms; T-Lymphocytes, Cytotoxic; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured; Viral Fusion Proteins; Virulence Factors

Some exogenous antigens, such as heat shock proteins or apoptotic bodies, gain access to the MHC class I processing pathway and initiate CTL responses, a process called cross-priming. To be efficient in vivo, this process requires endocytosis of the antigen by dendritic cells via receptors which remain unidentified. Here, we report that scavenger receptors are the main HSP binding structures on human dendritic cells and identify LOX-1 as one of these molecules. A neutralizing anti-LOX-1 mAb inhibits Hsp70 binding to dendritic cells and Hsp70-induced antigen cross-presentation. In vivo, to target LOX-1 with a tumor antigen using an anti-LOX-1 mAb induces antitumor immunity. Thus, the scavenger receptor LOX-1 is certainly a promising target for cancer immunotherapy.

Topics: Animals; Antibodies, Monoclonal; Antigen Presentation; Antigens, Neoplasm; Biotinylation; Dendritic Cells; Endocytosis; H-2 Antigens; HSP70 Heat-Shock Proteins; Humans; Immunotherapy; Mice; Mice, Inbred C57BL; Ovalbumin; Protein Binding; Recombinant Fusion Proteins; Serum Albumin, Bovine; Thymoma; Thymus Neoplasms; Transfection

Necrotic cell death yields the release of cellular components that can function in the initiation of cellular immune responses. Given the established capacity of the endoplasmic reticulum chaperone GRP94 (gp96) to elicit CD8(+) T cell activation, we have investigated the cellular fate and antigenicity of GRP94 in differing scenarios of cell death. Virally induced cell death or mechanical cell death, elicited by freeze/thaw treatment of cell suspensions, yielded GRP94 release into the extracellular space; apoptotic cell death occurring in response to serum deprivation did not elicit GRP94 release. To assess the antigenicity of GRP94 released following virally induced cell death (lethal infection of cells with rVV ES-OVA(Met258-265), a recombinant, ovalbumin epitope-expressing vaccinia virus) or mechanical cell death (freeze/thaw of ovalbumin-expressing cells), tissue culture supernatant fractions were pulsed onto antigen-presenting cells, and antigen re-presentation was assayed as activation of an ovalbumin-specific T cell hybridoma. For both cell death scenarios, released GRP94 elicited a dose-dependent, ovalbumin-specific, hybridoma activation. In contrast, calreticulin derived from rVV ES-OVA(Met258-265)-infected cell extracts did not stimulate B3Z activity. These data identify GRP94 as an antigenic component released upon pathological, but not apoptotic, cell death and provide an assay system for the identification of cellular components of related activity.

Topics: Animals; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Cell Death; Chickens; Heat-Shock Proteins; Hybridomas; Mice; Ovalbumin; Receptors, Antigen, T-Cell; Recombinant Proteins; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured; Vaccinia virus

The mode of peptide-based cancer vaccine administration critically affects the ability to achieve a clinically relevant tumor-specific response. We have previously shown (Cole et al., Clin. Cancer Res., 3: 867-873, 1997) that a specific formulation of the polysaccharide poly-N-acetyl glucosamine (p-GlcNAc, designated as F2 gel) is an effective vehicle for sustained cytokine and peptide delivery in vitro. The purpose of this study was to evaluate the efficacy of F2 gel/peptide vaccination in the murine EG.7-OVA tumor model and to elucidate potential mechanisms involved in the observed cell-mediated response. C57BL/6 mice were given injections of 200 microl in the base of tail/footpad using either F2 gel alone or 200 microg of: SIINFEKL minimal peptide (OVA) in PBS, OVA peptide/endoplasmic reticulum insertion signal sequence fusion (ESOVA) in PBS, OVA in F2 gel, or ESOVA in F2 gel. Splenocytes were tested 10 days later for a secondary response using a Cr51 assay as well as a primary CTL response using the lactate dehydrogenase cytotoxicity assay. Splenocytes from immunized mice were harvested at specific time points and assayed for cell surface and intracellular markers. On day 10 postvaccination, animals were challenged with EG.7-OVA murine thymoma cells. Tumor size and appearance were recorded. Vaccination with F2 gel/peptide (either OVA or ESOVA) resulted in a primary T-cell response (up to 25% tumor cell-specific lysis) and no tumor growth in 69% of the mice. By 48 h, the proportion of splenic T cells had increased 4-fold compared with B cells. Presence of an increased Th1 CD4 helper population was demonstrated by IFN-gamma production. CD4 cells were activated at 24 and 48 h as shown by IL-2 receptor alpha chain expression (from 2% basal expression to 15.4% at 48 h). Activated splenic macrophages increased from 3 to 8% within 10 h, and their level of B7-2 expression doubled. Depletion of macrophages before vaccine injection abolished any tumor-specific primary CTL response. F2 gel/peptide tumor vaccine can prime the immune system in an antigen-specific manner by generating a measurable primary T-cell response with minimal peptide; this process involves macrophage presence and activation as well as induction of Th1 CD4 cells. This is the first demonstration of a primary CTL response generated with minimal peptide vaccination using a noninfectious delivery system. These results justify additional studies to better define the mechanisms involved in F2 gel/

Topics: Adjuvants, Immunologic; Amino Acid Sequence; Animals; Antigens, CD; Antigens, Neoplasm; B7-2 Antigen; Cancer Vaccines; Cytotoxicity Tests, Immunologic; Epitopes; Female; Interferon-gamma; Lymphocyte Activation; Macrophage Activation; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasm Transplantation; Ovalbumin; Peptide Fragments; Receptors, Interleukin-2; Spleen; T-Lymphocytes, Cytotoxic; Th1 Cells; Thymoma; Thymus Neoplasms; Vaccination

A tumor-specific CD8+ T cell response was studied using adoptive transfer of OT-I TCR transgenic cells. Upon i.p. challenge with E.G7 tumor, OT-I cells undergo CD4+ T cell-independent expansion at the tumor site and develop lytic function. Before tumor elimination, however, they leave the peritoneal cavity (PC) and appear in the LN and spleen where they exhibit "split anergy" and cannot further proliferate to antigen. Administering anti-CTLA-4 mAb early caused sustained OT-1 expansion in the PC, and late administration caused the OT-I cells to return to the PC and further expand; in both cases, tumor was controlled. These effects required CD4+ T cells and IL-2 and appear to result from reversal of the nonresponsive state of the CD8+ T cells.

Topics: Abatacept; Adoptive Transfer; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Chemotaxis, Leukocyte; Clonal Anergy; CTLA-4 Antigen; Drug Administration Schedule; Immune Tolerance; Immunoconjugates; Injections, Intraperitoneal; Interleukin-2; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasm Transplantation; Ovalbumin; Peptide Fragments; Peritoneal Cavity; Receptors, Antigen, T-Cell; Spleen; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured

Anticancer vaccine strategies can now target intracellular antigens that are involved in the process of malignant transformation, such as oncogene products or mutated tumor suppressor genes. Fragments of these antigens, generally 8-10 amino acids in length and complexed with MHC class I molecules, can be recognized by CD8+ T lymphocytes (TCD8+). To explore the possibility of using a genetically encoded, minimally sized fragment of an intracellular antigen as an immunogen, we constructed a recombinant vaccinia virus encoding an 8-residue peptide derived from chicken ovalbumin that is known to associate with the mouse H-2Kb molecule. Compared to standard methods of immunization, recombinant molecule. Compared to standard methods of immunization, recombinant vaccinia virus expressing the minimal determinant as well as full length ovalbumin were the only approaches that elicited specific primary lytic responses in C57BL/6 mice against E.G7OVA, a transfectant of the murine thymoma EL4 containing the ovalbumin gene. Stimulating these effectors in vitro with OVA257-264 peptide induced H-2Kb-restricted TCD8+ that not only lysed but also specifically secreted IFN-gamma in response to an antigen. Furthermore, when transferred adoptively, these anti-OVA257-264 TCD8+ cells significantly reduced the growth of established ovalbumin-transfected tumors in a pulmonary metastasis model system. Synthetic transfected tumors in a pulmonary metastasis model system. Synthetic oligonucleotides encoding minimal antigenic determinants within expression constructs may be a useful approach for treatment of neoplastic disease, thus avoiding the potential hazards of immunizing with full-length cDNAs that are potentially oncogenic.

Topics: Animals; Base Sequence; Chickens; Cytotoxicity, Immunologic; Epitopes; Immunotherapy; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Oligodeoxyribonucleotides; Ovalbumin; Recombinant Proteins; T-Lymphocytes, Cytotoxic; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured; Vaccines, Synthetic; Vaccinia virus

CD8+ T-lymphocytes (TCD8+) recognize minimal peptides of 8-10 residues which are the products of intracellularly processed proteins and are presented at the cell surface by major histocompatibility complex class I molecules. An important step in this process is the translocation of processed proteins from the cytosol across the endoplasmic reticulum membrane, mediated by transporter associated with antigen-processing proteins or alternatively by endoplasmic reticulum-insertion signal sequences located at the NH2-terminus of the precursor molecules. We report here that the addition of an endoplasmic reticulum-insertion signal sequence at the NH2-terminus of TCD8+ epitopes from chicken ovalbumin (amino acids 257-264) or a naturally occurring tumor antigen expressed by the murine mastocytoma P815 (P1A amino acids 35-43) significantly enhanced the priming of specific TCD8+ in vivo. The signal sequence did not enhance peptide immunogenicity by merely increasing the hydrophobicity of the peptide, since ovalbumin amino acids 257-264 peptide with the signal sequence at its COOH-terminus did not demonstrate enhanced efficacy. The signal sequence did not act as a helper epitope, since TCD8+ responses were not diminished in class II-deficient transgenic mice or in mice depleted of CD4+ T-cells in vivo. Importantly, a single immunization with the fusion peptide significantly prolonged survival of mice challenged with E.G7OVA, a thymoma transfected with the complementary DNA of chicken ovalbumin.

Topics: Amino Acid Sequence; Animals; CD4-Positive T-Lymphocytes; Endoplasmic Reticulum; Epitopes; Female; Immunotherapy; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Molecular Sequence Data; Ovalbumin; Recombinant Fusion Proteins; Signal Transduction; T-Lymphocyte Subsets; Thymoma; Thymus Neoplasms; Transfection; Tumor Cells, Cultured

Exogenous, nonreplicating protein antigens (Ags) are usually taken up by antigen-presenting cells (APCs) via endocytosis or pinocytosis and enter the major histocompatibility complex (MHC) class II processing and presentation pathway. Although exogenous Ags are not processed and presented in the class I pathway by most cells, soluble proteins can enter the class I processing and presentation pathway if they are introduced directly into the cytoplasm of APCs. The purpose of these studies was to determine whether exogenous proteins could be processed and presented to T cells if they were delivered into cells by electroporation. The conditions for electroporation were optimized so that the viability of the electroporated cells was high, and the majority of electroporated cells had protein incorporated. Electroporated B cells not only presented exogenous ovalbumin to CD8+, class I MHC-restricted T cells but also stimulated CD4+, class II MHC-restricted T cells. Electroporated cells also primed Ag-specific cytotoxic T lymphocytes (CTLs) in vivo, stimulated CTL precursors in vitro, and served as target cells for lysis by Ag-specific CTLs, indistinguishable from transfected cells. Thus, electropermeabilized cells were structurally intact, and the introduced exogenous protein was processed and presented in association with both class I and class II MHC molecules. This approach is as efficient and reproducible as other techniques of delivering exogenous proteins into the intracellular processing pathways. These studies suggest that electroporation could be employed for the study of cell-mediated immunity to various exogenous proteins.

Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Electroporation; Epitopes; Female; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Hybridomas; Interleukin-2; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Ovalbumin; T-Lymphocytes, Cytotoxic; Thymoma; Thymus Neoplasms; Tumor Cells, Cultured