Cytotoxic immune cells with specificity for defined soluble antigens. II. Chasing the killing cells

Cells from mice immune against soluble antigens were tested for their in vitro cytotoxicity against chicken red blood cells (CRBC) coated with these antigens. In parallel, cells from mice immune against allogeneic P815 mastocytoma cells were tested for their in -vitro cytotoxicity against P815 cells. Before the cytotoxicity test, the immune cell populations were fractionated, using four different types of techniques, to check the impact of the removal of given subpopulations of cells on cytotoxic activity.Fractionation on anti-immunoglobulin coated columns did not affect the anti P815 cytotoxicity, but markedly decreased the cytotoxicity against antigen-coated CRBC. The same results were obtained by incubation on a plastic surface and/or an “ironplus-magnet” technique. Preincubation of the cytotoxic cell populations with homologous anti-θ or heterologous anti-T antiserum, plus complement, abolished both types of cytotoxicity. However, preincubation with anti-θ or anti-T antiserum alone, without complement, also blocked the cytotoxicity against antigen-coated CRBC, but not the anti P815 cytotoxicity. In vivo injection of heterologous anti-lymphocyte gammaglobulin completely abolished the anti P815 cytotoxicity, but not the cytotoxicity against antigen-coated CRBC.These results confirm that T cells (thymus-processed lymphocytes) can kill autonomously in the anti P815 system. They also suggest that, in the cytotoxicity against antigen-coated CRBC, as effector cells, (1) non-T cells are involved, (2) T cells might not be involved.     

Ontogeny and expression of chicken A blood group antigens

Expression of chicken red blood cell (RBC) surface antigens was studied by using a monoclonal antibody (ISU-cA) specific for chicken A blood group antigens. Erythrocytes were examined from embryos of 3-18 days of incubation and from chicks at hatch up to 21 weeks of age. Specific antigens were detected on embryonic RBC surfaces by immunofluorescence as early as 3 days of incubation. Antigenic expression was examined by both haemagglutination and immunofluorescence and found to increase with age from embryos to mature birds. The antigen concentration on the cell surface was found to be affected by genotype; heterozygotes had an intermediate level of antigen between that of the two parental genotypes. These data confirm the co-dominance that is observed with most blood group antigens. Flow cytometric analysis allowed confirmation that the entire erythrocyte population gradually increased in antigenic expression over time, rather than having an antigen-negative subpopulation being replaced by a positive subpopulation.     

Definition of human cytomegalovirus-specific target antigens recognized by cytotoxic T cells generated in vitro by using an autologous lymphocyte system

We developed an in vitro system for the generation of human cytomegalovirus (CMV)-specific cytotoxic T cells (CTL) that avoids the necessity of constituting a panel of HLA-typed fibroblasts. Autologous donor leucocytes were coated with CMV antigens and were used as both stimulator and target cells. With the use of this system, CMV-specific effector cells were efficiently generated from seropositive but not seronegative donors. These CMV-specific effectors were HLA-restricted and had characteristics of T cells. Maximum lymphoproliferation preceded the appearance of maximum CTL activity by 3 to 4 days, and a close correlation was seen between both activities. Mouse anti-CMV monoclonal antibodies were used in blocking experiments in an attempt to define target antigens recognized by CMV-specific cytotoxic lymphocytes. Monoclonal antibodies directed against an early CMV membrane antigen, against neutralization epitopes, or against nuclear inclusion body protein all specifically inhibited CMV-sensitized effector cell activity but did not affect influenza virus-specific lysis. Monoclonal antibodies directed against a normal cell determinant or against poliovirus did not affect CMV-specific CTL activity. CMV-immune cytotoxic T cells could be consistently and specifically inhibited in their lytic activity by pretreating antigen-coated target cells with monoclonal antibodies directed against CMV-related proteins.     

鸡红细胞融合最适条件的探讨

目的:探讨快速、有效的细胞融合条件。方法:用鸡红细胞为材料,聚乙二醇(Mw=4000)为诱导剂,诱导鸡红细胞融合。结果:鸡红细胞融合的最适温度为39℃,最适时间为15min。结论:在该条件下,同时用Giemsa染液对融合细胞染色,实验观察效果明显。     

Antibody formation by one or both lymphoid populations present in chimeric marmosets

Development of an isoimmune serum capable of identifying a specific leukocyte antigen in the marmoset, Saguinus fuscicollis illigeri, permitted detection of lymphoid cell chimerism in this species by the cytotoxic test. This reagent was then used to identify the cell population in the chimera responsible for antibody production against a test antigen, sheep red blood cells. Primary in vitro antibody formation as measured by plaque-forming cells with blood leukocytes or splenic lymphocytes of six animals chimeric for the leukocyte antigen, MLA-1, revealed an immune response by both cell types of the chimeric population from three animals and a response by only one cell type in the other three.     

Dendritic cells pulsed with gp96-peptide complexes derived from human hepatocellular carcinoma (HCC) induce specific cytotoxic T lymphocytes

Dendritic cells (DCs) are one of the most potent antigen-presenting cells (APCs) capable of activating immune responses. Different forms of tumor antigens have been used to load DCs to initiate tumor-specific immune responses. Heat shock proteins (HSPs) are considered natural adjuvants which have the ability to chaperone peptides associated with them presented efficiently by interaction with professional APCs through specific receptors. In the present study, we used HSP, gp96-peptide complexes, derived from human hepatocellular carcinoma (HCC) cells as antigens for pulsing DCs. We found that gp96-peptide complexes derived from HCC cells induced the maturation of DCs by enhancing expression of human leukocyte antigen class II, CD80, CD86, CD40, and CD83. The matured DCs stimulated a high level of autologous T cell proliferation and induced HCC specific cytotoxic T lymphocytes, which specifically killed HCC cells by a major histocompatability complex (MHC) class I restricted mechanism. These findings demonstrate that DCs pulsed with gp96-peptide complexes derived from HCC cells are effective in activating specific T cell responses against HCC cells.     

Influence of endogenous Thy1.1 cells upon the efficacy of an anti-Thy1.1 antibody-diphtheria toxin conjugate.

The chemical conjugation of antibodies to protein toxins results in cell-specific cytotoxic agents that can be defined in terms of in vitro potency and efficacy; however, it is the in vivo utilities that are largely being pursued in clinical trials. The nature of in vivo target cell depletion by toxin conjugates is largely unknown. The anti-murine Thy1.1 antibody-diphtheria toxin conjugate possesses high in vitro efficacy, and because mice are remarkably resistant to the native toxin, the conjugate possesses in vivo efficacy. When administered intravenously, the conjugate is shown to deplete peripheral blood Thy1.1+ target cells in a concentration-dependent fashion. When the log kill of Thy1.1+ tumor cells was analyzed by the life span extension method, it was determined, however, that the log kill is inversely proportional to the number of target cells. That is, the presence of an endogenous cell population, which is expressing the same surface antigen targeted by the antibody conjugate as on the pathological cell, may drastically lower the clinical efficacy of the immunotoxin. Thus, the greatest potential for antibody-toxin conjugates will be for low target cell burdens and for pathogenic cell populations expressing unique surface antigens. These are important considerations in the design of bioconjugates to insure high in vivo efficacy in elimination of intended target cells.     

CTP-BCR-ABL抗原肽负载树突状细胞体外致敏T淋巴细胞靶向杀伤CML细胞

BCR-ABL为慢性髓细胞白血病特异胞质抗原,为良好的免疫治疗靶标.该研究选择BCR-ABL融合位点的两段抗原肽SSKALQRPV(SS)、GFKQSSKAL(GF)为靶点,与胞质转导肽融合表达,负载小鼠骨髓源性树突状细胞.在胞质转导肽介导下,SS、GF短肽进入树突状细胞并定位于内质网,具备了被树突状细胞识别为内源性抗...     

Chicken red blood cells as a substrate for direct polymerase chain reaction

A protocol is presented for direct polymerase chain reaction (PCR) amplification of DNA from chicken nucleated red blood cells. Chicken blood in EDTA was found to have a strong inhibitory effect on the PCR. Consequently, PCR using this protocol should be performed only on a narrow range of blood volumes, from 0125 to 025 |ll.     

Chicken A blood groups system antigens: molecular characteristics and lack of expression on lymphocytes

The molecular structure of two antigens (A2 and A4) of the chicken A blood group system was determined by using an A4-specific monoclonal antibody (ISU-cA) and several alloantisera specific for chicken A blood group antigens. Molecules immunoprecipitated from erythrocytes were separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under either reducing or non-reducing conditions. Molecules of relative molecular weights 53.0 and 54.5 Kd were identified under reducing conditions for A2 and A4 antigens, respectively, and non-reduced molecules had a relative molecular weight of 44.5 Kd for both antigens. Two-dimensional electrophoresis showed a similar, single, diffuse band near pH 6.5 for each antigen. The data are consistent with a glycosylated molecule with one or more intrachain disulphide bonds. Allelic differences between A2 and A4 antigens seem to be due to an additional moiety on A4 antigen with a net neutral charge. Binding to chicken lymphocytes of antibody specific for A antigens was not detected by enzyme-linked immunosorbent assay (ELISA). Immunoprecipitations of radiolabelled peripheral blood lymphocyte-surface molecules using ISU-cA and A-specific alloantisera also did not detect A blood group antigens. Thus, chicken A blood group antigens are not indicated to be present on lymphocytes.     

Resistance to tumor growth mediated by Listeria monocytogenes. Destruction of experimental malignant melanoma by LM-activated peritoneal and lymphoid cells.

A murine experimental model of nonspecific tumor destruction mediated by cells activated by Listeria monocytogenes (LM) is described. B16 melanoma growth is prevented or suppressed in the syngeneic host when tumor cells are inoculated in contact with viable LM. In vitro, cultured B16 cells are destroyed by LM immune peritoneal or splenic cells in the presence of the bacterial antigen(s). Activation of LM immune cells in vitro is immunologically specific. Replacement of LM by sheep red blood cells or bovine serum albumin in the in vitro cultures aborts the cytotoxic effect. Further, no tumor cell killing is obtained when thioglycollate-induced or normal peritoneal cells are substituted for LM immune cells in the in vitro cultures. Normal spleen cells in the presence of LM are weakly cytotoxic for B16 cells. Normal peritoneal cells plus LM or LM alone are not. Elimination of thymus derived "T" cells by anti-theta C3H or rabbit anti-mouse brain serum (RAMB) abrogated the cytotoxic effect. Therefore, LM-induced tumor destruction probably occurs through nonspecific mechanism(s) consequent to activation of host "T" cells by specific immune reactivity to LM antigen(s).     

Characterization of tumor antigen peptide-specific T cells isolated from the neoplastic tissue of patients with gastric adenocarcinoma

Gastric cancer is a significant cause of morbidity and mortality worldwide. Surgical resection remains the primary curative treatment for gastric adenocarcinoma, but the poor (15–35%) survival rate at 5 years has prompted many studies for new therapeutic strategies, such as specific immunotherapy. The aim of this study was to analyze the functional properties of the T cell response to different antigen peptides related to gastric cancer in patients with gastric adenocarcinoma. To this purpose, we have cloned and characterized tumor-infiltrating T cells (TILs) isolated from the neoplastic gastric tissue samples. A T cell response specific to different peptides of gastric cancer antigens tested was documented in 17 out of 20 patients, selected for their HLA-A02 and/or -A24 alleles. Most of the cancer peptide-specific TILs expressed a Th1/Tc1 profile and cytotoxic activity against target cells. The effector functions of cancer peptide-specific T cells obtained from the peripheral blood of the same patients were also studied. The majority of peripheral blood peptide-specific T cells also expressed the Th1/Tc1 functional profile. In conclusion, in most of the patients with gastric adenocarcinoma, a specific type-1 T cell response to gastric cancer antigens was detectable and would have the potential of hamper tumor cell growth. However, in order to get tumor cell killing in vivo, the activity and the number of cancer peptide-specific Th1/Tc1 cells probably need to be enhanced by vaccination with the appropriate cancer antigenic peptides or by injection of the autologus tumor peptide-specific T cells expanded in vitro.     

Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin

The skin accommodates multiple dendritic cell (DC) subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, E. coli beta-galactosidase (betaGal) and chicken ovalbumin (OVA) under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin⁺ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC) as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin⁺ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation.     

Molecular characterization of fetal antigens on red blood cells of chickens,Japanese quail,and quail-chicken hybrids

The molecular nature of chicken fetal antigen (CFA) and quail fetal antigen (QFA) was studied on embryonic red blood cells (RBCs) of the chicken, the Japanese quail, and the quail-chicken hybrid. Specific immunoprecipitation of radiolabeled membrane proteins followed by electrophoretic separation and autoradiography were used to identify the protein molecules carrying these fetal antigens. CFA was found on molecules of 24, 50, 88, 99, 130, 170, and 220 kd (kilodaltons) in the chicken and hybrid and on molecules of 24, 50, 99, and 170 kd in the Japanese quail. Similarly, quail fetal antigen was associated with 24-, 50-, 99-, and 170-kd molecules in the quail and hybrid and was not detected in the chicken. Partial proteolytic digestion of the 50- and 170-kd molecules isolated from RBCs of all sources showed remarkably similar peptide patterns. Likewise, two-dimensional separation of the CFA-positive and QFA-positive 50-kd molecules from quail RBCs revealed a similar pattern of at least nine isomorphic variants. Sequential depletions of quail embryonic RBC extracts with either anti-CFA or anti-QFA followed by immune precipitation with the reciprocal antiserum suggested that most of the cell surface proteins carrying QFA also have CFA on the same molecules. It is suggested that specific glycosylations of a variety of distinct molecular weight proteins determines the antigenic phenotype characterized as "fetal antigens."     

Heterogenous populations of cytotoxic cells in the peritoneal cavity of BALB/c mice immunized with allogeneic EL4 leukemia cells

Adherent cells, presumably macrophages, obtained from the peritoneal cavity shortly after rejection of the allogeneic leukemia EL4, produced effective cell-mediated cytotoxicity (CMC) in vitro. These cytotoxic cells were sensitive to anti-macrophage serum and resistant to anti-thymocyte serum and 10,000 roentgen irradiation. In contrast, a second population of specifically cytotoxic cells were nonadherent, sensitive to x-rays and anti-thymocyte serum, but not to anti-macrophage serum.The two cell populations had a cooperative cytotoxic effect in vitro against allogeneic tumor cells.     

Canine blood groups. 2. Description of a new allele in the Tr blood group system

A previously unrecognized canine red cell antigen, tentatively named O, was found after absorption analysis of an alloimmune antiserum and absorption of several immune or naturally occurring cross-reacting heteroantibodies from man, cattle and swine. The serological results and genetic analysis of a limited number of complete dog families indicated that the new factor probably belongs to the Tr blood group system. No individual possessed both factors Tr and O, and a large proportion of animals was negative for both factors. The serological pattern for the new Tr system obtained was consistent with similar systems observed in sheep (A-O), pig (R-O) and man (Bombay) in which the gene for one factor was dominant over and masked the gene for the second factor in the system. The negative phenotype was accounted for by the actions of an epistatic gene.     

Allogeneic tumor-specific cytotoxic T lymphocytes

Summary We report the development of cytotoxic T lymphocytes specific for an allogeneic brain tumor in a rat model. DA strain cytotoxic T cell precursors stimulated by an allogeneic tumor (9L gliosarcoma) from the Fischer rat could generate a population of cytotoxic T lymphocytes that lysed the allogeneic 9L tumor but failed to lyse other targets, including Fischer concanavalin-A(ConA)-stimulated lymphoid blast targets. DA T cells depleted of reactivity to the Fischer haplotype (DA-f) retained reactivity to the 9L tumor, demonstrating that T cell precursors with specificity for normal Fischer alloantigens were not required for the generation of a response to the 9L Fischer tumor. The preferential lysis of the tumor target did not simply reflect a higher density of Fischer target antigens on the tumor than that found on normal Fischer ConA blast targets. First, the relative densities of class I antigen on the 9L tumor and normal Fischer ConA blasts were comparable. Second, cytotoxic T cells could not be generated from DA-f precursors when Fischer ConA blasts were used as stimulators. If DA-f T cells were simply responding to the higher density of Fischer antigen found on 9L tumor, it would have been expected that the ConA blasts expressing comparable levels of antigen to that found on the tumor would have generated cytotoxicity for both the 9L and ConA targets. We conclude that the cytotoxic T cells are specific for a determinant expressed only by the tumor. Such tumor-specific cytotoxic T cells could be useful in vivo for adoptive immunotherapy of brain tumors.     

Functional properties of lymphocyte subpopulations in hepatitis B virus infection. II. Cytotoxic effector cell killing of targets that naturally express hepatitis B surface antigen and liver-specific lipoprotein

Cytotoxic effector cell responsiveness to host and/or virus-determined hepatocyte surface membrane antigens has been postulated as an important pathogenetic determinant of hepatocellular injury in hepatitis B virus infection. Assuming that such effector cell populations would be detectable in peripheral blood, the present study was designed to examine 2 questions: first, whether target cells that normally express liver-specific protein (LSP) and hepatitis B surface antigen (HBsAg) are selectively destroyed by peripheral blood effector cells from patients with viral hepatitis; second, whether cytotoxic effector cell function emerges coincident with the development of defective suppressor cell activity in the same patients. No evidence of increased HBsAg or LSP specific cytotoxic effector cell activity was found in the peripheral blood natural killer (NK) or T killer cell populations of patients with acute or chronic viral hepatitis.     

Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus.

The phenomenon of antibody-dependent cell-mediated cytoxicity (ADCC) has been extended to include target cells acutely infected with herpes simplex type 1 virus (HSV-1) or herpes simplex type 2 virus (HSV-2) in an in vitro system that employs immune human serum and human blood mononuclear cells. The cytotoxic reaction was detectable after 1 hr of incubation and was complete between 4 and 8 hr. The amount of ADCC noted was directly proportional to the logarithm(10) of the effector: target cell ratio (E:T), and ADCC was noted at E:T as low as 1:1. The mononuclear effector cell was present in the blood of both HSV immune and non-immune individuals. The immune serum factor was demonstrated to be an antibody with specificity for HSV membrane antigen(s) and was reactive with target cells infected with either of the two HSV types. The antibody rendered the mononuclear cell cytotoxic by sensitization of the target cell rather than by direct attachment to or "arming" of the mononuclear cell. The physiochemical properties of the antibody as well as its presence in cord blood demonstrated that it is an immunoglobulin on the IgG class.     

Selective MHC expression in tumours modulates adaptive and innate antitumour responses

Progress towards developing vaccines that can stimulate an immune response against growing tumours has involved the identification of the protein antigens associated with a given tumour type. Epitope mapping of tumour antigens for HLA class I- and class II-restricted binding motifs followed by immunization with these peptides has induced protective immunity in murine models against cancers expressing the antigen. MHC class I molecules presenting the appropriate peptides are necessary to provide the specific signals for recognition and killing by cytotoxic T cells (CTL). The principle mechanism of tumour escape is the loss, downregulation or alteration of HLA profiles that may render the target cell resistant to CTL lysis, even if the cell expresses the appropriate tumour antigen. In human tumours HLA loss may be as high as 50%, inferring that a reduction in protein levels might offer a survival advantage to the tumour cells. Alternatively, MHC loss may render tumour cells susceptible to natural killer cell-mediated lysis because they are known to act as ligands for killer inhibitory receptors (KIRs). We review the molecular features of MHC class I and class II antigens and discuss how surface MHC expression may be regulated upon cellular transformation. In addition, selective loss of MHC molecules may alter target tumour cell susceptibility to lymphocyte killing. The development of clinical immunotherapy will need to consider not only the expression of relevant CTL target MHC proteins, but also HLA inhibitory to NK and T cells. Received: 20 March 1999 / Accepted: 3 May 1999