ovalbumin and Lymphoma

Topics: Animals; Antibody Affinity; Antibody Formation; Blood Bactericidal Activity; Cell Transformation, Neoplastic; Chickens; Dose-Response Relationship, Radiation; Hematopoiesis; Hypersensitivity, Delayed; Immune Tolerance; Immunity, Cellular; Leukemia Virus, Murine; Lymphocytes; Lymphoma; Mice; Muramidase; Neutrophils; Ovalbumin; Rabbits; Radiation Chimera; Radiation, Ionizing; Rats; T-Lymphocytes; T-Lymphocytes, Regulatory; X-Rays

Checkpoint blockade therapy has provided noteworthy benefits in multiple cancers in recent years; however, its clinical benefits remain confined to 10-40% of patients with extremely high costs. Here, we design an ultrafast, low-temperature, and universal self-assembly route to integrate immunology-associated large molecules into metal-organic-framework (MOF)-gated mesoporous silica (MS) as cancer vaccines. Core MS nanoparticles, acting as an intrinsic immunopotentiator, provide the niche, void, and space to accommodate antigens, soluble immunopotentiators, and so on, whereas the MOF gatekeeper protects the interiors from robust and off-target release. A combination of MOF-gated MS cancer vaccines with systemic programmed cell death 1 (PD-1) blockade therapy generates synergistic effects that potentiate antitumour immunity and reduce the effective dose of an anti-PD-1 antibody to as low as 1/10 of that for PD-1 blockade monotherapy in E.G7-OVA tumour-bearing mice, with eliciting the robust adaptive OVA-specific CD8

Topics: Animals; Antibodies, Neutralizing; Antigens, Neoplasm; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cytotoxicity, Immunologic; Drug Compounding; Female; Humans; Immunity, Innate; Immunotherapy; Lymph Nodes; Lymphoma; Metal-Organic Frameworks; Mice; Mice, Inbred C57BL; Nanoparticles; Ovalbumin; Programmed Cell Death 1 Receptor; Silicon Dioxide; Survival Analysis; Tumor Burden; Xenograft Model Antitumor Assays

Codelivery nanovaccines of antigens and adjuvants have achieved positive therapy for cancer immunotherapy. The insufficient immunogenicity of these vaccines leads to the difficulty of eliciting robust immune effects for immune clearance due to the inadequate loading efficiency, complex preparation processes, low safety concerns, and weak immune responses. Herein, a visible codelivery nanovaccine of an antigen and adjuvant based on self-cross-linked antigen nanoparticles (ovalbumin nanoparticles (ONPs)) combined with the adjuvant (CpG) for cancer immunotherapy was prepared using antigens themselves as carriers. ONPs not only provide sufficient antigens for continuous simulation of the immune response with high antigen loading efficiency but also serve as natural carriers of CpG. In vitro and in vivo experiments proved that ONPs-CpG can elicit a robust immune response including DC maturity, T cell activation, and IFN-γ production. ONPs-CpG induced strong tumor-specific immunity and exhibited remarkable antitumor immunotherapy effects in vivo using mouse models of lymphoma. Furthermore, to perform the precise vaccine delivery, the dual fluorescent codelivery nanovaccine was monitored in real time in vivo by the visible imaging method. With regard to migration tracking, fluorescence imaging allowed for both high resolution and sensitivity of visible detection based on the fluorescence of ONPs and CpG. The multifunctional nanovaccine could function as a robust platform for cancer immunotherapy and a visible system for antigen-adjuvant tracking.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Cells, Cultured; Dinucleoside Phosphates; Disease Models, Animal; Drug Delivery Systems; Fluorescent Dyes; Immunotherapy; Lymphoma; Mice; Nanoparticles; Optical Imaging; Ovalbumin; Particle Size

Messenger RNA encoding tumor antigens has the potential to evoke effective antitumor immunity. This study reports on a nanoparticle platform, named mRNA Galsomes, that successfully co-delivers nucleoside-modified antigen-encoding mRNA and the glycolipid antigen and immunopotentiator α-galactosylceramide (α-GC) to antigen-presenting cells after intravenous administration. By co-formulating low doses of α-GC, mRNA Galsomes induce a pluripotent innate and adaptive tumor-specific immune response in mice, with invariant natural killer T cells (iNKT) as a driving force. In comparison, mRNA Galsomes exhibit advantages over the state-of-the-art cancer vaccines using unmodified ovalbumin (OVA)-encoding mRNA, as we observed up to seven times more tumor-infiltrating antigen-specific cytotoxic T cells, combined with a strong iNKT cell and NK cell activation. In addition, the presence of suppressive myeloid cells (myeloid-derived suppressor cells and tumor-associated macrophages) in the tumor microenvironment was significantly lowered. Owing to these antitumor effects, OVA mRNA Galsomes significantly reduced tumor growth in established E.G7-OVA lymphoma, with a complete tumor rejection in 40% of the animals. Moreover, therapeutic vaccination with mRNA Galsomes enhanced the responsiveness to treatment with a PD-L1 checkpoint inhibitor in B16-OVA melanoma, as evidenced by a synergistic reduction of tumor outgrowth and a significantly prolonged median survival. Taken together, these data show that intravenously administered mRNA Galsomes can provide controllable, multifaceted, and effective antitumor immunity, especially when combined with checkpoint inhibition.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Female; Galactosylceramides; Immunity, Cellular; Kaplan-Meier Estimate; Killer Cells, Natural; Liposomes; Lymphocyte Activation; Lymphoma; Melanoma; Melanoma, Experimental; Mice; Natural Killer T-Cells; Ovalbumin; RNA, Messenger; T-Lymphocytes

A polymersome system for delivering protein antigen ovalbumin (OVA) based on amphiphilic polyphosphazene grafting with N,N-diisopropylethylenediamine (DPA) and poly(ethylene glycol) (PEG) groups (poly[(DPA)

Topics: Animals; Cytoplasm; Drug Carriers; Drug Delivery Systems; Hydrogen-Ion Concentration; Lymphoma; Mice; Organophosphorus Compounds; Ovalbumin; Polyethylene Glycols; Polymers; Th1 Cells; Tumor Cells, Cultured

During human pregnancy, paternally inherited antigens expressed by the fetal-placental unit can elicit expansion of antigen-specific CD8+ T cells. These cells can persist for years as memory T cells, but their effects on long-term maternal health are unknown. Shared placenta/tumor-associated antigens are expressed by placenta and tumors, but are minimally expressed or absent in normal adult tissues. We hypothesized that maternal T cells elicited against these antigens can alter risk of cancers expressing the same antigen after pregnancy, and tested this in mice using chicken ovalbumin (OVA) as a surrogate shared placenta/tumor antigen. Hemizygous OVA transgenic males were bred to wild-type C57BL/6 females (H2b haplotype) such that the fetuses inherited and expressed OVA. Maternal OVA/H2Kb-specific CD8+ T cells became detectable during gestation, and persisted in some animals for up to 24 weeks. To determine whether these cells might influence growth of OVA-expressing tumors in OVA-bred females, E.G7-OVA thymoma cells were inoculated subcutaneously in OVA-bred, wild-type bred, and virgin females, and monitored for growth. OVA-bred mice had prolonged survival as compared to virgin mice and the progression of tumors was delayed in comparison to wild-type bred and virgin females. Thus, paternally inherited OVA antigen elicited a CD8+ T cell response during pregnancy that was associated with delayed growth of OVA-expressing tumors following pregnancy. These data suggest a possible role of antigen-specific T cells in protecting parous females against tumors bearing shared placenta/tumor antigens.

Topics: Animals; CD8-Positive T-Lymphocytes; Exosomes; Female; Lymphoma; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; Placenta; Pregnancy; Pregnancy, Animal

Ginsenoside, a natural triterpenoid saponin, exhibits immunomodulatory and anticancer activities. In the present study, we demonstrated that ginsenoside Rg1 induced secretion of cytokines, including interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), and IL-1β, and chemokines such as IL-8 and IP-10 in a dose-dependent manner by human peripheral blood mononuclear cell (PBMC)-derived dendritic cells. Rg1 stimulated the expression of the surface molecules CD83, CD80, and human leukocyte antigen - antigen D related (HLA-DR) and decreased the expression of CD14. In in vivo experiments, C57BL/6 mice were divided into four groups, immunized with ovalbumin (OVA), OVA plus Rg1, Rg1, and phosphate-buffered saline (PBS), respectively. Splenocytes from C57BL/6 mice immunized with OVA plus Rg1 produced more antigen-specific splenocyte proliferation activity. The level of IFN-γ and IL-4 in the splenocytes was also upregulated when in vitro stimulated with OVA

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Cell Line, Tumor; Cytokines; Dendritic Cells; Ginsenosides; Humans; Lymphoma; Male; Mice; Mice, Inbred C57BL; Ovalbumin

Dentritic cell (DC)-based cancer immunotherapy faces challenges in both efficacy and practicality. However, DC-based vaccination requires multiple injections and elaborates ex vivo manipulation, which substantially limits their use. Therefore, we sought to develop a chitosan nanoparticle (CH-NP)-based platform for the next generation of vaccines to bypass the ex vivo manipulation and induce immune responses via active delivery of polyinosinic-polycytidylic acid sodium salt (poly I:C) to target Toll-like receptor 3 (TLR3) in endosomes. We developed CH-NPs encapsulating ovalbumin (OVA) as a model antigen and poly I:C as the adjuvant in an ionic complex. These CH-NPs showed increased in vivo intracellular delivery to the DCs in comparison with controls after injection into tumor-bearing mice, and promoted DC maturation, leading to emergence of antigen-specific cytotoxic CD8+ T cells. Finally, the CH-NPs showed significantly greater antitumor efficacy in EG.7 and TC-1 tumor-bearing mice compared to the control (p < 0.01). Taken together, these data show that the CH-NP platform can be used as an immune response modulatory vaccine for active cancer immunotherapy without ex vivo manipulation, thus resulting in increased anticancer efficacy.

Topics: Adjuvants, Immunologic; Animals; Antigen Presentation; Antigens; Cancer Vaccines; Cell Line, Tumor; Cell Proliferation; Chitosan; Dendritic Cells; Female; Gene Expression; Immunomodulation; Immunotherapy; Lung Neoplasms; Lymphoma; Mice; Mice, Inbred C57BL; Nanoparticles; Nanotechnology; Ovalbumin; Poly I-C; T-Lymphocytes, Cytotoxic; Toll-Like Receptor 3; Transfection

During primary Ag encounter, T cells receive numerous positive and negative signals that control their proliferation, function, and differentiation, but how these signals are integrated to modulate T cell memory has not been fully characterized. In these studies, we demonstrate that combining seemingly opposite signals, CTLA-4 blockade and rapamycin-mediated mammalian target of rapamycin inhibition, during in vivo T cell priming leads to both an increase in the frequency of memory CD8(+) T cells and improved memory responses to tumors and bacterial challenges. This enhanced efficacy corresponds to increased early expansion and memory precursor differentiation of CD8(+) T cells and increased mitochondrial biogenesis and spare respiratory capacity in memory CD8(+) T cells in mice treated with anti-CTLA-4 and rapamycin during immunization. Collectively, these results reveal that mammalian target of rapamycin inhibition cooperates with rather than antagonizes blockade of CTLA-4, promoting unrestrained effector function and proliferation, and an optimal metabolic program for CD8(+) T cell memory.

Topics: Animals; Antibodies, Monoclonal; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Proliferation; CTLA-4 Antigen; Gene Expression Regulation; Immunologic Memory; Listeria monocytogenes; Listeriosis; Lymphocyte Activation; Lymphoma; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

In a previous study, we reported on the efficient delivery of an antigen to the cytosol and a specific-antigen presentation on MHC class I in dendritic cells by rationally controlling the intracellular trafficking of ovalbumin (OVA), a model antigen, with stearylated octaarginine-modified liposomes (R8-Lip/OVA). However, no significant difference in antitumor effects against E.G7-OVA, OVA expressed lymphoma, was observed between R8-Lip/OVA and an electrostatic complex of R8 and OVA (R8/OVA-Com). In this study, we hypothesized that use of adjuvants clarified the difference in immune responses between R8-Lip/OVA and R8/OVA-Com, and selected polyinosine-polycytidylic acid (polyI:C) as an adjuvant. Cytotoxic T lymphocyte (CTL) activity of the polyI:C and OVA encapsulated R8-Lip (R8-Lip/PIC/OVA) was drastically enhanced compared to R8-Lip/OVA and complete Freund's adjuvant with OVA. Moreover, the incorporation of polyI:C clearly was critical for the difference in antitumor effects and CTL activities between R8-Lip/OVA and R8/OVA-Com. These findings suggest that the carriers that are incorporated polyI:C has a great influence on the induction of cellular immunity in vivo.

Topics: Adjuvants, Immunologic; Animals; Cell Line, Tumor; Female; Freund's Adjuvant; Immunity, Cellular; Liposomes; Lymphoma; Mice; Mice, Inbred C57BL; Oligopeptides; Ovalbumin; Poly I-C; Static Electricity; T-Lymphocytes, Cytotoxic

Staphylococcal enterotoxins A (SEA) and B (SEB) are classical models of superantigens (SAg), which induce potent T-cell-stimulating activity by forming complexes with MHC class II molecules on antigen-presenting cells. This large-scale activation of T-cells is accompanied by increased production of cytokines such as interferon-γ (IFN-γ). Additionally, as we previously reported, IFN-γ-producing CD8(+) T cells act as "helper cells," supporting the ability of dendritic cells to produce interleukin-12 (IL-12)p70. Here, we show that DC pulsed with SAg promote the enhancement of anti-tumor immunity. Murine bone marrow-derived dendritic cells (DC) were pulsed with OVA(257-264) (SIINFEKL), which is an H-2Kb target epitope of EG7 [ovalbumin (OVA)-expressing EL4] cell lines, in the presence of SEA and SEB and were subcutaneously injected into naïve C57BL/6 mice. SAg plus OVA(257-264)-pulsed DC vaccine strongly enhanced peptide-specific CD8(+) T cells exhibiting OVA(257-264)-specific cytotoxic activity and IFN-γ production, leading to the induction of protective immunity against EG7 tumors. Furthermore, cyclophosphamide (CY) added to SAg plus tumor-antigens (OVA(257-264), tumor lysate, or TRP-2) pulsed DC immunization markedly enhanced tumor-specific T-cell expansion and had a significant therapeutic effect against various tumors (EG7, 2LL, and B16). Superantigens are potential candidates for enhancing tumor immunity in DC vaccines.

Topics: Animals; Antigen-Presenting Cells; Antineoplastic Agents, Alkylating; Carcinoma, Lewis Lung; CD8-Positive T-Lymphocytes; Cyclophosphamide; Cytokines; Dendritic Cells; Flow Cytometry; Histocompatibility Antigens Class II; Interferon-gamma; Interleukin-12; Lung Neoplasms; Lymphocyte Activation; Lymphoma; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Ovalbumin; Receptors, G-Protein-Coupled; Superantigens; Survival Rate; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Helper-Inducer; Tumor Cells, Cultured; Vaccines, Subunit

We previously showed that exposure to UV radiation after immunization suppresses Th1 and Th2 immune responses, leading to impaired Ab and allo-immune responses, but the impact of UV radiation after immunization on anti-tumor immune responses mediated by tumor-specific CD8(+) T cell responses remains less clear. Furthermore, the exact phenotypic and functional characteristics of regulatory T cell population responsible for the UV-induced immunosuppression still remain elusive. Using the MBL-2 lymphoma cell line engineered to express OVA as a surrogate tumor Ag, here we demonstrate that UV irradiation after tumor Ag-immunization suppresses the anti-tumor immune response in a manner dependent on the immunizing Ag. This suppression was mediated by interleukin (IL)-10 released from CD4(+) CD25(+) T cells, by which impaired the induction of cytotoxic T lymphocytes (CTL) able to kill Ag-expressing tumor cells. In addition, we generated a panel of T cell clones from UV-irradiated and non-irradiated mice, and all of the clones derived from UV-irradiated mice had a Tr1-type regulatory T cell phenotype with expression of IL-10 and c-Maf, but not Foxp3. These Tr1-type regulatory T cell clones suppressed tumor rejection in vivo as well as Th cell activation in vitro in an IL-10 dependent manner. Given that suppression of Ag-specific CTL responses can be induced in Ag-sensitized mice by UV irradiation, our results may imply that exposure to UV radiation during premalignant stage induces tumor-Ag specific Tr1 cells that mediate tumor-Ag specific immune suppression resulting in the promotion of tumor progression.

Topics: Animals; Blotting, Western; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Enzyme-Linked Immunosorbent Assay; Female; Immune Tolerance; Immunization; Immunosuppression Therapy; Interleukin-10; Lymphocyte Activation; Lymphoma; Mice; Mice, Inbred C57BL; Ovalbumin; Proto-Oncogene Proteins c-maf; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Th1 Cells; Th2 Cells; Ultraviolet Rays

While multiple pathways of dendritic cell (DC) maturation result in transient production of IL-12, fully mature DCs show reduced ability to produce IL-12p70 upon a subsequent interaction with Ag-specific T cells, limiting their in vivo performance as vaccines. Such "DC exhaustion" can be prevented by the presence of IFNgamma during the maturation of human DCs (type-1-polarization), resulting in improved induction of tumor-specific Th1 and CTL responses in vitro. Here, we show that type-1 polarization of mouse DCs strongly enhances their ability to induce CTL responses against a model tumor antigen, OVA, in vivo, promoting the induction of protective immunity against OVA-expressing EG7 lymphoma. Interestingly, in contrast to the human system, the induction of mouse DC1s requires the participation of IL-4, a nominal Th2-inducing cytokine. The current data help to explain the previously reported Th1-driving and anti-tumor activities of IL-4, and demonstrate that type-1 polarization increases in vivo activity of DC-based vaccines.

Topics: Animals; Cancer Vaccines; Cell Polarity; Dendritic Cells; Female; Humans; Immunotherapy; Interferon-gamma; Interleukin-12; Interleukin-4; Lipopolysaccharides; Lymphoma; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasms; Ovalbumin; T-Lymphocytes, Cytotoxic

Tumor-derived exosomes have been regarded as a new kind of cancer vaccine; however, their therapeutic efficacy needs to be further improved. Superantigen staphylococcal enterotoxin A (SEA)-coated tumor cells have been shown to potently induce tumor-specific T cell response. To increase efficacy of tumor-derived exosomes to induce antitumor immune response, we modified the exosomes by protein transfer of SEA tailed with a highly hydrophobic transmembrane domain (SEA-TM) and designated those SEA-TM-anchored exosomes as Exo/SEA-TM. We found the exosomes secreted from murine lymphoma E.G7-OVA cell line were round vesicles with the sizes of 40-100 nm limited by a bilayer membrane. Interestingly, the inner structure of the exosomes were visible under the transmission electron microscope; those "honeycomb-like" inner structure has not been described by other labs. Immunization with Exo/SEA-TM inhibited tumor growth and prolonged survival of the mice challenged with parental tumor cells more significantly than with exosomes (Exo) and even more than with the mixture of exosomes and SEA-TM. The results of mixed lymphocyte-tumor reaction (MLTR) showed that the increased IL-2, IFN-gamma secretion, and specific cytotoxic T lymphocyte (CTL) could be effectively induced from the splenic lymphocytes of the mice immunized with Exo/SEA-TM. In vivo depletion experiments showed that CD8(+) T cells are the main effector cells, and both CD4(+) T cells and NK cells are also involved in the antitumor effect of Exo/SEA-TM immunization. Therefore, tumor-derived exosomes surface anchored with SEA-TM can efficiently induce tumor-specific CTL thereby resulting in more potent inhibition of tumor growth. Our data provide an efficient and novel approach to tumor immunotherapy by protein modification of tumor-derived exosomes.

Topics: Animals; Cancer Vaccines; Cell Line, Tumor; Enterotoxins; Exocytosis; Feasibility Studies; Female; Immunization; Immunotoxins; Interferon-gamma; Interleukin-2; Killer Cells, Natural; Lymphoma; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Ovalbumin; Particle Size; Superantigens; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Transport Vesicles

Dendritic cells (DCs) were pulsed with the H-2K(b) binding OVA(257-264)-peptide (SIINFEKL), and used as one single-injection vaccine in combination with anti-CTLA-4 monoclonal antibody (mAb) to treat mice inoculated 3 days previously with 3x10(5) E.G7-OVA lymphoma cells. Neither DC vaccination nor CTLA-4 blockage alone prevented tumor growth in tumor challenged mice. In contrast, the combination of one vaccination and injection of anti-CTLA-4 mAb lead to rejection or retarded tumor growth in more than 60% of the mice. The OVA-transgene or the SIINFEKL-epitope was not lost in the progressing tumors of vaccinated mice, however, the highest degree of anti-SIINFEKL reactivity of host CTLs in an IFN-gamma ELISPOT assay was found only in mice showing complete tumor rejection. Vaccinated mice having rejected E.G7-OVA tumors were capable of rejecting subsequent challenges with 1x10(6) E.G7-OVA tumor cells, and later on these mice even rejected wild-type EL-4 tumor cells indicating that tumor epitope spreading takes place during the process of vaccination-induced E.G7-OVA rejection. In agreement with these observations, mice having rejected E.G7-OVA tumors showed long lasting CTL memory in spleen and bone marrow towards both the SIINFEKL-peptide and other EL-4-derived tumor rejecting epitopes.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation; Bone Marrow; Cancer Vaccines; CTLA-4 Antigen; Dendritic Cells; Drug Therapy, Combination; Egg Proteins; Epitopes; Female; H-2 Antigens; Lymphoma; Mice; Mice, Inbred C57BL; Ovalbumin; Peptide Fragments; Signal Transduction; Spleen; T-Lymphocytes, Cytotoxic; Thymoma; Transgenes; Vaccination

Aberrant gene expression can be caused by several different mechanisms at the transcriptional, RNA processing, and translational level. Although most of the resulting proteins may have no significant biological function, they can be meaningful for the immune system, which is sensitive to extremely low levels of Ag. We have tested this possibility by investigating the ability of CD8+ T cells (TCD8+) to respond to an epitope whose expression results from incidental ribosomal frameshifting at a sequence element within the HSV thymidine kinase gene. This element, with no apparent functional significance, has been identified due to its ability to facilitate escape from the antiviral compound acyclovir. Using a recombinant vaccinia virus expression system, we find that in vitro and in vivo TCD8+ responses to the frameshift-dependent epitope are easily discernible. Furthermore, the in vivo response is at a sufficient level to mediate protection from a tumor challenge. Thus, the targets of immune responses to infectious agents can extend beyond the products of conventional open reading frames. On a per-cell basis, responses to such minimally expressed epitopes may be exceedingly effective due to the selective expansion of high avidity TCD8+.

Topics: Animals; Antibodies, Monoclonal; Cytotoxicity, Immunologic; Egg Proteins; Epitopes, T-Lymphocyte; Female; Frameshifting, Ribosomal; Herpesvirus 1, Human; Lymphocyte Activation; Lymphoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Neoplasm Transplantation; Nucleocapsid Proteins; Nucleoproteins; Ovalbumin; Peptide Fragments; Receptors, Antigen, T-Cell; RNA-Binding Proteins; T-Lymphocytes, Cytotoxic; Thymidine Kinase; Viral Core Proteins

Tumor antigen-specific T-cell tolerance imposes a significant barrier to the development of effective therapeutic cancer vaccines. Bone marrow-derived antigen-presenting cells (APCs) are critical in the induction of this unresponsive state. Here we show that in vitro treatment of APCs with the tyrosine kinase inhibitor, imatinib mesylate (STI-571), enhances the activation of naive antigen-specific T cells and restores the responsiveness of tolerant T cells from tumor-bearing hosts. Furthermore, in vivo treatment with STI-571 not only prevented the induction of tolerance in tumor-specific CD4(+) T cells, preserving their responsiveness to a subsequent immunization, but also resulted in enhanced vaccine efficacy. These findings demonstrate that tolerance to tumor antigens is not an insurmountable obstacle and points to modulation of APC function as a promising strategy in the immunotherapy of cancer.

Topics: Amino Acid Sequence; Animals; Antigen-Presenting Cells; Antineoplastic Agents; Benzamides; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Dendritic Cells; Imatinib Mesylate; Immune Tolerance; Lymphoma; Macrophages; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Neoplasms; Ovalbumin; Peptide Fragments; Piperazines; Pyrimidines

Vaccine immunotherapy must induce helper and cytotoxic cell-mediated immunity to generate the powerful antitumor immune responses needed to suppress cancer progression. We reported previously that a 16-amino acid peptide analogue derived from pigeon cytochrome c can bind broad ranges of MHC class II types and activate helper T cells in mice. To determine whether DNA encoding the Pan-MHC class II IA peptide (Pan-IA) can increase the efficacy of tumor suppression by DNA vaccine immunotherapy targeting tumor antigens, Pan-IA DNA was administered with ovalbumin (OVA) DNA to C57BL/6 mice bearing the OVA-expressing tumor cell line E.G7. Specific proliferative responses to and cytotoxic activities against OVA-expressing targets were induced in mice vaccinated with both OVA and Pan-IA DNA but not in those vaccinated with OVA DNA alone or control DNA plus Pan-IA DNA. Growth of E.G7 cells was suppressed only by combined vaccination with OVA and Pan-IA DNA, and tumors in five of the nine mice that received this combined vaccination were eradicated completely. In those mice, the frequency of CD8-positive T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased in the tumor site. Furthermore, immunofluorescent study of the inoculated tumors revealed increased accumulation of both CD4- and CD8-positive T cells producing IFN-gamma in the tumor only by this vaccine protocol. The data suggest that Pan-IA DNA can augment suppressive effects of DNA vaccines on tumor growth by increasing numbers of antigen-specific CTLs and helper T cells. This is the first study in which established tumors have been eradicated successfully by vaccination with DNA corresponding to CTL epitopes and helper T cell epitopes. Our animal model may contribute to the development of therapeutic DNA vaccines against cancer.

Topics: Amino Acid Sequence; Animals; Antibodies, Neoplasm; Antigens, Neoplasm; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Division; Cell Line, Tumor; Female; Histocompatibility Antigens Class II; Immunotherapy, Active; Interferon-gamma; Lymphocytes, Tumor-Infiltrating; Lymphoma; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Ovalbumin; Vaccines, DNA

Recently, human dendritic cells (DCs) pulsed with mRNA encoding a broad range of tumor antigens have proven to be potent activators of a primary anti-tumor-specific T-cell response in vitro. The aim of this study was to improve the mRNA pulsing of murine DC. Compared to a standard lipofection protocol and passive pulsing, electroporation was, in our hands, the most efficient method. The optimal conditions to electroporate murine bone marrow-derived DCs with mRNA were determined using enhanced green fluorescent protein and a truncated form of the nerve growth factor receptor. We could obtain high transfection efficiencies around 70-80% with a mean fluorescence intensity of 100-200. A maximal expression level was reached 3 hours after electroporation. A clear dose-response effect was seen depending on the amount of mRNA used. Importantly, the electroporation process did not affect the viability nor the allostimulatory capacity or phenotype of the DC. To study the capacity of mRNA-electroporated DCs to present antigen in the context of MHC classes I and II, we made use of chimeric constructs of ovalbumin. The dose-dependent response effect and the duration of presentation were also determined. Together, these results demonstrate that mRNA electroporation is a useful method to generate genetically modified murine DC, which can be used for preclinical studies testing immunotherapeutic approaches.

Topics: Animals; Antigen Presentation; Cell Division; Chickens; Dendritic Cells; DNA Primers; Electroporation; Female; Flow Cytometry; Gene Transfer Techniques; Genes, MHC Class I; Genes, MHC Class II; Green Fluorescent Proteins; Humans; Luminescent Proteins; Lymphocyte Activation; Lymphoma; Mice; Mice, Inbred C57BL; Ovalbumin; Peptide Fragments; Polymerase Chain Reaction; Receptor, Nerve Growth Factor; RNA, Messenger; T-Lymphocytes; Transcription, Genetic; Ubiquitin

Although the importance of CD4+ T-cell help for generation of an effective CD8+ effector cytotoxic T cell (CTL) response is well established, the role of T-cell help in the activation of memory T cells to become fully functional tumor killer cells is undefined. Using synthetic peptide immunizations corresponding to the major CTLs and T-helper epitopes of ovalbumin, adoptive transfers of ovalbumin-specific memory CTLs (mCTLs), and ovalbumin as the tumor-specific antigen in a mouse tumor model, we have determined that T help is essential for the activation of mCTLs to kill tumors. Our data show that T-helper cells specific for the tumor-associated antigen are required for the reactivation of mCTLs by antigen presented indirectly from tumor. In contrast, effector CTLs do not need T help to kill tumors. These results have implications for induction of tumor immunotherapy by immunization.

Topics: Amino Acid Sequence; Animals; Antigens, Neoplasm; Egg Proteins; Epitopes, T-Lymphocyte; Female; Immunologic Memory; Immunotherapy, Adoptive; Lymphocyte Activation; Lymphoma; Mice; Mice, Inbred C57BL; Mice, Transgenic; Molecular Sequence Data; Ovalbumin; Peptide Fragments; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Tumor Cells, Cultured

Mouse fibroblasts (H-2(b)) were genetically engineered to express a co-stimulatory B7.1 and an IFN-gamma (Fb/IFN-gamma/B7.1). The Fb/IFN-gamma/B7.1 cells were then pulsed with an ovalbumin epitope (amino acids 257-264, SIINFEKL, H-2K(b)-restricted) as a model antigen (Fb/IFN-gamma/B7.1/OVA) and tested for the induction of OVA-specific cytotoxic T lymphocytes (CTLs) in C57BL/6 mice (H-2(b)). Genetically engineered fibroblasts lacking either IFN-gamma or B7.1 were constructed and used as controls. Immunization with the Fb/IFN-gamma/B7.1/OVA cells induced strong cytotoxic activity against OVA-expressing EL4 (EG7) tumor cells but not against other H-2(b) tumor cells, such as EL4, C1498, and B16F1. The magnitude of the cytotoxic response in mice with the Fb/IFN-gamma/B7.1/OVA cells was significantly higher than that in mice immunized with any other cell construct. CD8(+) T cells with OVA-specific cytotoxic activity were predominant in mice immunized with Fb/IFN-gamma/B7.1/OVA cells. Furthermore, treatment with Fb/IFN-gamma/B7.1/OVA cells significantly prolonged the survival period of EG7 tumor-bearing mice. Anti-tumor CTL immunity by the Fb/IFN-gamma/B7.1/OVA cells could be induced without the help of host antigen-presenting cells, CD4(+) T cells, or NK1.1(+) cells. Our results suggest that fibroblasts can be genetically modified into efficient antigen-presenting cells for the induction of antigen-specific CTL response in cancer immunotherapy.

Topics: Animals; Antibodies, Monoclonal; Antigen Presentation; Antigen-Presenting Cells; Antigens, Differentiation; B7-1 Antigen; Complement System Proteins; DNA, Complementary; Epitopes; Female; Fibroblasts; Genetic Therapy; H-2 Antigens; Immunization; Immunotherapy; Interferon-gamma; Lymphoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Transplantation; Ovalbumin; Peptide Fragments; Recombinant Fusion Proteins; Recombinant Proteins; T-Lymphocytes, Cytotoxic; Transfection

Following subcutaneous implantation, the murine lymphoma E.G7 [a variant of EL-4, transfected with the chicken ovalbumin (OVA) gene] up-regulates the CD4 molecule. We previously showed that the administration of an anti-CD4 monoclonal antibody (mAb) to EG.7-bearing mice leads to a rapid and complete regression of large established tumors. This tumor regression was shown to require both CD8+ cells and functional Fcgamma receptors (FcgammaR), as it failed to occur in mice depleted of CD8 cells, or mice genetically deficient in FcgammaI/III (gamma-/-mice). Using adoptive transfer, we now show that the FcgammaR+ cells required for this regression are the CD11b+ (phagocytic) cells. Furthermore, experiments using peptide tolerization demonstrated that the critical CD8 CTL population in this model is tumor specific. Analysis of tumors at various stages of regression revealed a massive CD11b+FcgammaR+ and a marginal CD8 infiltration. In the presence of the CTL determinant OVA-8 on tumor cells and of the antitumor mAb, this CD8 infiltration became remarkable, and correlated with tumor regression. These results identify the specific cellular effectors essential for the mAb-mediated tumor regression, and suggest that FcgammaR-activated macrophages induced an expansion of tumor-eliminating CTL in situ.

Topics: Animals; Antibodies, Monoclonal; CD4 Antigens; CD8-Positive T-Lymphocytes; Chickens; Hematopoietic Stem Cells; Kinetics; Lymphocytes, Tumor-Infiltrating; Lymphoma; Macrophage-1 Antigen; Mice; Mice, Knockout; Ovalbumin; Receptors, IgG; T-Lymphocytes, Cytotoxic; Transfection

We investigated the effector mechanisms operating during the rejection of a transplantable solid lymphoma E.G7 (H-2b) which expresses the gene encoding chicken ovalbumin (OVA). Anti-OVA cytotoxic T lymphocytes (CTL) completely and specifically protected animals from the onset of, but could not eradicate established, E.G7 tumors. The growth of the same lymphoma was also effectively prevented by the antibody GK1.5, whose target molecule, CD4, was expressed on E.G7 cells in vivo. Furthermore, GK1.5 was able to eradicate established solid E.G7 tumors. GK1.5-mediated tumor elimination was due to its antitumor activity, and not to the elimination of regulatory CD4+ cells, based on unimpaired tumor growth in the absence of GK1.5 in animals that genetically lack CD4 T cells. In vitro, GK1.5 did not kill tumor cells: complement activation or apoptosis induction were not evident. In vivo, GK1.5-mediated tumor regression did not depend on natural killer cells, but it absolutely required CD8+ cells and intact Fcgamma receptor. We conclude that, in the E.G7 model, the collaboration of antibody and CTL immunity was crucial for the successful immunotherapy of established tumors. The mechanism of this collaboration is discussed.

Topics: Animals; Antibodies, Neoplasm; Antibody-Dependent Cell Cytotoxicity; CD4 Antigens; CD8 Antigens; Cytotoxicity, Immunologic; Female; Graft Rejection; Killer Cells, Natural; Lymphoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Ovalbumin; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

It has previously been shown that bone marrow-generated dendritic cells (DC) are potent stimulators in allogeneic mixed leukocyte reactions and are capable of activating naive CD4+ T cells in situ in an antigen-specific manner. In this study we have investigated whether bone marrow-generated DC are capable of inducing antigen-specific CD8+ T cell responses in vivo. Initial attempts to induce specific cytotoxic T lymphocyte (CTL) responses in mice injected with bone marrow-generated DC pulsed with ovalbumin (OVA) peptide were frustrated by the presence of high levels of nonspecific lytic activity, which obscured, though not completely, the presence of Ag-specific CTL. Using conditions that effectively differentiate between antigen-specific and nonspecific lytic activity, we have shown that bone marrow-generated DC pulsed with OVA peptide are potent inducers of OVA-specific CTL responses in vivo, compared with splenocytes or RMA-S cells pulsed with OVA peptide, or compared with immunization with free OVA peptide mixed with adjuvant. Antibody-mediated depletion experiments have shown that the cytotoxic effector cells consist primarily of CD8+ cells, and that induction of CTL in vivo is dependent on CD4+ as well as on CD8+ T cells. These results provide the basis for exploring the role of bone marrow-generated DC in major histocompatibility class I-restricted immune responses, and they provide the rationale for using bone marrow-generated DC in CTL-mediated immunotherapy of cancer and infectious diseases.

Topics: Adjuvants, Immunologic; Animals; Antigen Presentation; Antigens; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Dendritic Cells; Epitopes; Female; H-2 Antigens; Immunization; Immunophenotyping; Lymphoma; Mice; Mice, Inbred C57BL; Ovalbumin; Peptide Fragments; Spleen; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Persistence of the underlying malignancy remains the main obstacle to the successful treatment of human malignancies with high-dose chemoradiotherapy and bone marrow transplantation.. The aim of this study was to determine whether antigen-specific antitumor immune responses, elicited in normal donor mice by immunization with the soluble form of a surrogate tumor antigen (i.e., ovalbumin [OVA]), can be transferred via bone marrow transplantation into lethally irradiated, syngeneic recipient mice. An additional goal was to evaluate the ability of these adoptively transferred bone marrow cells to eradicate established recombinant OVA-expressing lymphomas that recurred after lethal-dose total-body irradiation (TBI).. Female C57BL/6 donor mice were immunized twice with OVA emulsified in a muramyl-dipeptide-containing adjuvant. Syngeneic mice bearing a day-10 or day-11, approximately 1-cm subcutaneous E.G7-OVA tumor (E.G7-OVA tumor cells were derived from transfection of EL-4 thymoma tumor cells using the coding sequence of chicken OVA gene complementary DNA) were treated with TBI and reconstituted with bone marrow from nonimmune or OVA-immunized mice. In subsequent experiments, tumor-bearing mice, treated with TBI and OVA-immune bone marrow, were given additional therapy either with a single OVA immunization or by the adoptive transfer of 1 x 10(7) in vitro activated spleen cells derived from OVA-immune donor mice and cultured 5 days with irradiated E.G7-OVA cells before transfer.. E.G7-OVA tumor-bearing mice given TBI and OVA-immune bone marrow showed a significantly increased cure rate when compared with that among controls reconstituted with nonimmune bone marrow after TBI (logrank, P < .01). The antitumor effect of immune bone marrow was abrogated by T-cell depletion of the marrow graft (P < .016). The antitumor effect of immune marrow was enhanced by the addition of OVA immunization of tumor-bearing recipients (P < .015). OVA-specific cytotoxic T-lymphocyte (CTL) activity was recovered from tumor-bearing recipients of immune marrow 14 days after bone marrow transplantation. The antitumor effect observed following the adoptive transfer of immune marrow was further augmented by the addition of 1 x 10(7) splenic E.G7-OVA-specific in vitro activated CTLs derived from OVA-immune mice (P < .03).. These studies establish the principle that antigen-specific T-cell immunity against a defined tumor-specific antigen can be transferred with bone marrow from an immune donor.. Active immunization of normal human bone marrow or T-cell donors with a refined, safe tumor antigen and transfer of immunity to the patient may represent a novel strategy for circumventing the obstacle of host immune suppression associated with the tumor-bearing state.

Topics: Animals; Antigens, Neoplasm; Bone Marrow Transplantation; Epitopes; Female; Gene Expression Regulation, Neoplastic; Immunotherapy, Adoptive; Lymphoma; Mice; Mice, Inbred C57BL; Neoplasms, Radiation-Induced; Ovalbumin; Survival Analysis; T-Lymphocytes; Tumor Cells, Cultured

During the last 15 years several laboratories have attempted to generate rabbit monoclonal antibodies, mainly because rabbits recognize antigens and epitopes that are not immunogenic in mice or rats, two species from which monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To obtain a rabbit plasmacytoma cell line that could be used as a fusion partner we generated transgenic rabbits carrying two transgenes, c-myc and v-abl. These rabbits developed plasmacytomas, and we obtained several plasmacytoma cell lines from which we isolated hypoxanthine/aminopterin/thymidine-sensitive clones. One of these clones, when fused with spleen cells of immunized rabbits, produced stable hybridomas that secreted antibodies specific for the immunogen. The hybridomas can be cloned and propagated in nude mice, and they can be frozen without change in their ability to secrete specific monoclonal antibodies. These rabbit-rabbit hybridomas will be useful not only for production of monoclonal antibodies but also for studies of immunoglobulin gene rearrangements and isotype switching.

Topics: Animals; Antibodies, Monoclonal; Antigens; Antigens, Neoplasm; Blood Proteins; Cell Fusion; Cell Line; Enzyme-Linked Immunosorbent Assay; Epitopes; Fluorescent Antibody Technique; Humans; Hybridomas; Immunologic Techniques; Lymph Nodes; Lymphocytes; Lymphoma; Mice; Ovalbumin; Peyer's Patches; Plasmacytoma; Rabbits; Rats; Spleen; Tumor Cells, Cultured

We have exploited the pore forming activity of listeriolysin, the hemolysin of Listeria monocytogenes, to activate CD8+ T cells with soluble proteins in vivo and in vitro. Immunization with soluble, hemolytically active listeriolysin induces both cytotoxic CD8+ T cells and CD4+ T cells, and the CD8+ T cells can be propagated with soluble listeriolysin in vitro. Moreover, conventional antigens like ovalbumin mixed together with listeriolysin are also efficiently introduced into the MHC class I pathway in vitro and in vivo. Hence, listeriolysin effectively directs itself and passenger molecules into the intracellular compartment that leads to the cytotoxic T cell response. In this way, we circumvent the bias of CD8+ T cells to recognize intracellular antigens presented by major histocompatibility complex class I molecules. As cytotoxic CD8+ T cells are of pivotal importance in eliminating viral and microbial pathogens, the findings reported here could prove to be useful in vaccine development.

Topics: Animals; Antigen Presentation; Bacterial Toxins; CD4-Positive T-Lymphocytes; Female; H-2 Antigens; Heat-Shock Proteins; Hemolysin Proteins; Histocompatibility Antigens Class II; Immunization; Interleukin-2; Lymphocyte Activation; Lymphoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Solubility; T-Lymphocytes, Cytotoxic; Tumor Cells, Cultured

Using a B lymphoma, A20-HL, bearing IgM receptors for TNP, we have shown that presentation of an Ag taken up through the receptors is highly sensitive, whereas that of an Ag taken up nonspecifically is resistant to inhibition of protein synthesis or protein transport from the endoplasmic reticulum. To analyze the difference, we have examined the effect of protein synthesis inhibition on A20-HL cells in terms of internalization and fragmentation of a specific Ag, TNP-OVA, and distribution of MHC class II molecules. Inhibition of protein synthesis in A20-HL cells with emetine, an irreversible protein synthesis inhibitor, did not decrease the surface expression of anti-TNP receptors, or the kinetics of internalization of 125I-TNP-OVA. To detect fragmentation of TNP-OVA, A20-HL cells were incubated at 37 degrees C in the presence of 125I-TNP-OVA, and the cell lysate was analyzed in SDS-PAGE. The number of detectable fragments increased with the incubation period, and inhibition of protein synthesis did not change the electrophoretic pattern. Expression of MHC class II molecules on the surface of A20-HL cells was not affected by inhibition of protein synthesis. However, intracellular MHC class II molecules markedly decreased in amount in the emetine-treated cells. Thus, presentation of an Ag taken up through Ag receptors seems to be dependent on intracellular MHC class II molecules, whereas that of an Ag taken up nonspecifically does not, suggesting that the Ag-processing pathway in B cells for a specific Ag is different from that for a nonspecific one, at least partly.

Topics: Antigen Presentation; B-Lymphocytes; Emetine; Epitopes; Histocompatibility Antigens Class II; Humans; Lymphoma; Ovalbumin; Protein Synthesis Inhibitors; Receptors, Immunologic; Trinitrobenzenes; Tumor Cells, Cultured

Small peptides, derived from endogenous proteins bind within the antigen binding groove created by the beta-pleated sheets and alpha helices of the alpha 1 and alpha 2 domains of the class I molecule of the major histocompatibility complex (MHC). However, the precise role of peptide in class I MHC conformation remains unclear. Here, we have shown that, in at least some instances, changes induced in the MHC molecule by the binding of distinct peptides can be identified as specific alterations in serological epitopes expressed on the class I protein. The nature of specific peptides expressed by class I-bearing cells may, therefore, have a dramatic influence on T cell development, self-tolerance, and alloreactivity.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Binding Sites; Cell Transformation, Viral; Histocompatibility Antigens Class I; Humans; Lymphoma; Major Histocompatibility Complex; Molecular Sequence Data; Mutagenesis, Site-Directed; Ovalbumin; Peptides; Protein Conformation; Rauscher Virus; Tumor Cells, Cultured

The binding of a lysozyme and ovalbumin peptide to purified class II major histocompatibility molecules in detergents was increased by the addition of certain lipids. Natural lipids from B lymphoma cells enhanced the binding and so did phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylethanolamine, sphingomyelin, and cholesterol had no effect. There was no major difference between the effects of a phospholipid and its lyso derivative. As studied with phosphatidylcholine, the increase in peptide binding was also dependent on the fatty acid composition of the lipid. The binding affinity was increased 10- to 50-fold in the presence of lipid as a result of an increase in the association rate while the off-rate remained essentially unchanged. Our results suggest that lipids, directly or indirectly, induce conformational changes in class II molecules that favor their peptide-binding property.

Topics: Amino Acid Sequence; B-Lymphocytes; Cell Line; Histocompatibility Antigens Class II; Humans; Kinetics; Lymphoma; Molecular Sequence Data; Ovalbumin; Peptides; Phospholipids; Protein Binding; Structure-Activity Relationship

The L3T4+, Lyt-2-, cloned BALB/c T cell lines 5.9.24 and 5.8.6 are cytotoxic for the BALB/c B cell tumor line A20/2J. The T cell cytotoxicity against A20/2J cells could be triggered either by the specific antigen ovalbumin (OVA), which is recognized by the T cell clones in association with I-Ad determinants, or by the T cell mitogens Con A and rabbit anti-mouse brain (RaMBr) antiserum. Repeated exposure of A20/2J cells to 5.9.24 and 5.8.6 T cell cytotoxicity selected variant cell lines that had developed resistance to cytotoxicity. The variant lines could be classified into four different variant phenotypes of which three were stably maintained in vitro. The type of variant obtained appeared to be related to the nature of the ligand used to trigger T cell cytotoxicity during selection. Cytotoxicity triggered by the antigen OVA generated type 1 variants that expressed abnormally low levels of I-Ad determinants at the cell surface. Type 1 variants were resistant to OVA-triggered 5.9.24 T cell cytotoxicity, but were fully susceptible to cytotoxicity triggered by Con A or RaMBr antiserum. RaMBr-triggered cytotoxicity generated two unique types of variant cell lines: type 3 variants that were deficient in cell surface Fc receptors and resistant to 5.9.24 cytotoxicity only when triggered by RaMBr antiserum, and type 4 variants that were resistant to cytotoxicity triggered by all three ligands. One type 4 variant, the IC-1 cell line, appeared to be resistant to soluble cytotoxic factors released by 5.9.24 T cells after activation by antigen. All of these variant lines retained sensitivity to cytotoxicity by classic Lyt-2+ cytotoxic T lymphocytes (CTL), a finding that indicates that L3T4a+ T cells and Lyt-2+ CTL use different molecules to attack their target cells. The variant phenotypes were inherited by clones derived from the original cell lines. Because the variants were generated without mutagenesis, they are thought to have been derived by the immunoselection of pre-existing variant cells that arose spontaneously in the parental A20/2J cell line. It is postulated that inheritable variation of A20/2J cells may represent changes that normally occur during B cell differentiation in response to T cell signals. The variant A20/2J cell lines described here provide material for the investigation of B cell surface structures that may regulate T-B cell interactions.

Topics: Animals; Antigens, Surface; B-Lymphocytes; Cell Line; Clone Cells; Cytotoxicity, Immunologic; Epitopes; Histocompatibility Antigens Class II; Immune Sera; Immunity, Innate; Lymphocyte Function-Associated Antigen-1; Lymphoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Phenotype; Preservation, Biological; Rabbits; Receptors, Fc; T-Lymphocytes

Immunoreactivity for S-100 protein was investigated immunohistochemically in a series of 49 fixed and paraffin-embedded normal, reactive, and neoplastic human lymphoid tissue specimens. The avidin-biotin complex immunoperoxidase method was used, with overnight (12-hour) incubation with a commercially available antiserum to S-100 protein. In addition, cryostat sections were tested with DRC 1 monoclonal antibody to dendritic reticulum cells (DRCs) in three cases and with OKT6 antibody to interdigitating reticulum cells (IRCs) in nine cases. All tissues, including lymph nodes, tonsils, adenoid, spleens, appendices, thymuses, and tissues containing nodular reactive lymphoid infiltrates, demonstrated a consistent immune staining pattern. A striking network composed of dendritic processes that showed finely granular S-100 protein immunoreactivity was observed in most of the follicular germinal centers; a similar dendritic pattern was observed in the follicular centers when the corresponding frozen sections were immunostained with DRC 1. In the extrafollicular areas, the S-100-positive cells topographically and morphologically resembled the IRCs that were demonstrated by OKT6 antibody in the corresponding frozen sections. The results seem to indicate that cells topographically and morphologically similar to IRCs and DRCs in human lymphoid tissues from different sites share immunoreactivity for S-100 protein. The present study confirms the unexpected presence of S-100 protein in dendritic cells of follicular germinal centers by a simple and currently available method.

Topics: Antibodies, Monoclonal; Avidin; Biotin; Humans; Hyperplasia; Immune Sera; Immunologic Techniques; Lymph Nodes; Lymphatic Diseases; Lymphoid Tissue; Lymphoma; Ovalbumin; S100 Proteins; Staining and Labeling; Tissue Distribution

Antigen non-specific activities in culture supernatants from clones of a radiation leukemia virus transformed, ovalbumin-specific helper T lymphoma were studied. After stimulation with ovalbumin-pulsed macrophages, these clones release factor(s) capable of providing carrier-specific help for anti-hapten antibody responses. It was found that in addition to IL 2-like activity, one clone constitutively released a suppressive factor that could inhibit polyclonal growth and differentiation of B cells stimulated with LPS. The factor inhibited activation of both syngeneic and allogeneic B cells directly, rather than via activation of accessory suppressor T lymphocytes. However, upon dilution of the supernatant, B cell growth and differentiation activities were detected. A role for a polyclonal B cell suppressor factor in the overall helper activity of the clone is proposed.

Topics: Animals; Antibody Formation; B-Lymphocytes; Cell Line; Culture Media; Female; Immune Tolerance; Immunoglobulin G; Immunoglobulin M; Lymphocyte Activation; Lymphokines; Lymphoma; Male; Mice; Mice, Inbred Strains; Ovalbumin; T-Lymphocytes; T-Lymphocytes, Helper-Inducer

A 17-amino acid tryptic peptide of chicken ovalbumin, designated P323-339, that substituted for processed antigen when presented by glutaraldehyde prefixed accessory cells to specific I-restricted T hybridomas was characterized. The peptide antigen could not be demonstrated to have any specific or stable interactions with accessory cell Ia antigens by either direct binding or functional assays for inhibition of specific T cell activation. In addition, the T cell receptor for I-restricted antigen had no affinity for free antigen alone. A rabbit antibody specific for the antigenic peptide inhibited presentation when introduced before but not after binding of the peptide to accessory cells. These results extend our earlier finding that accessory cell-mediated processing of chicken ovalbumin can be completely explained by the fragmentation of the native molecule into smaller m.w. peptides, and suggests that if an antigen/Ia complex is important in T cell activation, it forms significantly only in the presence of the T cell receptor for I-restricted antigen.

Topics: Amino Acid Sequence; Animals; Antigens; B-Lymphocytes; H-2 Antigens; Histocompatibility Antigens Class II; Immune Sera; Lymphocyte Activation; Lymphoma; Mice; Mice, Inbred BALB C; Ovalbumin; Peptide Fragments; Rabbits; T-Lymphocytes; Trypsin

Proliferation of antigen-specific T-cell populations was induced in cultures stimulated with antigen and a suitable source of antigen-presenting cells. Soluble (keyhole limpet hemocyanin) and particulate (horse red blood cells) antigens were presented by irradiated spleen cells and by a variety of B-lymphoma-cell lines, providing support for antigen-specific H-2-restricted T-cell responses. A marked heterogeneity was demonstrated, however, in the capacity of T-cell lines to proliferate in response to antigen presented by the B-lymphoma cells. T-cell populations were prepared from the lymph nodes of antigen-primed mice and restimulated in vitro in the presence of antigen and irradiated spleen cells. During the first six in vitro restimulations, these T-cell populations maintained the capacity to respond to antigen presented either by irradiated spleen cells or by B-lymphoma cells. Continued growth of these T-cell populations, again in the presence of antigen and irradiated spleen cells, resulted in the generation of T-cell lines which had lost the ability to respond to antigen presented by B-lymphoma cells. These lines however, fully retained the capacity to proliferate in the presence of antigen and irradiated spleen cells. T-cell clones derived from one of these lines were also unable to respond to antigen presented by B-lymphoma cells but again proliferated in the presence of antigen and irradiated spleen cells. Supernatants containing high levels of IL-1, IL-2, or IL-3 activity failed to reconstituted the antigen-specific response of T-cell lines which had lost the capacity to respond to antigen presented by B-lymphoma cells. Furthermore, titrated numbers of irradiated spleen cells, while having the capacity to support T-cell proliferation themselves, failed to synergize with B-lymphoma cells in the support of antigen-specific T-cell proliferation. Thus we have defined populations of antigen-specific, H-2-restricted T cells which do not recognize antigen presented by B-lymphoma cells and can therefore discriminate between different antigen-presenting cell types.

Topics: Animals; Antigen-Presenting Cells; B-Lymphocytes; Female; Hemocyanins; Interleukin-1; Interleukin-2; Lymphocyte Activation; Lymphoma; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Spleen; T-Lymphocytes

We examined the ability of a set of cloned chicken ovalbumin (cOVA)-specific, Id-restricted, T cell hybridomas to produce interleukin-2 in response to cOVA presented by the Ia+ B cell lymphoma line, A20-2J. Although viable A20-2J cells presented native, denatured, and fragmented cOVA more or less equally well, A20-2J cells that were glutaraldehyde-fixed could present only enzymatically or chemically fragmented cOVA. These results suggest that antigen fragmentation may be both necessary and sufficient to define accessory cell processing of soluble antigens so that they may be recognized in association with I-region molecules by T cells.

Topics: Animals; Antigens; B-Lymphocytes; Cell-Free System; Chickens; Epitopes; Fixatives; Glutaral; H-2 Antigens; Hybridomas; Immunologic Techniques; Lymphocyte Cooperation; Lymphoma; Mice; Mice, Inbred BALB C; Ovalbumin; Peptides; T-Lymphocytes

Topics: Animals; Antigens, Neoplasm; Cell Transformation, Neoplastic; Clone Cells; Epitopes; Female; Haptens; Lymphoma; Male; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Ovalbumin; Retroviridae; T-Lymphocytes

Topics: Animals; Cell Line; Epitopes; Erythrocytes; Female; Hybrid Cells; Lymphoma; Male; Mice; Ovalbumin; Rabbits; Receptors, Antigen, T-Cell; Rosette Formation; Spleen; T-Lymphocytes; T-Lymphocytes, Regulatory

Topics: Animals; Antibodies, Neoplasm; Antibody Formation; Antigens, Neoplasm; Chlorambucil; Female; Lymphoma; Mice; Ovalbumin; Protein Binding; Receptors, Antigen, B-Cell