Induction of syngeneic cytotoxic T lymphocytes against a B cell tumor. II. Characterization of anti-idiotypic CTL lines and clones.

In an earlier communication we showed that idiotypic immunoglobulin (Id+ Ig) of a B cell hybrid, 2C3, can induce cytotoxic T lymphocytes (CTL) in the spleens of mice that are hyperimmunized with the irradiated tumor cells. To understand the extent of heterogeneity in the splenic CTL population, stable anti-idiotypic CTL lines and clones were established from 2C3-primed splenocytes. One representative CTL line A102 which exhibited the phenotype of CD3+, CD4-, and CD8+, has been maintained in long-term culture for more than 18 months. Cytotoxic specificity of A102 was determined by cold target inhibition assay using a panel of syngeneic and allogeneic B cell tumors. The CTL line A102 was highly cytotoxic to 2C3, only weakly to other syngeneic tumors, but not at all to allogeneic B cell tumor CH12. Furthermore, CTL-mediated cytolysis was significantly abrogated by blocking 2C3 cells with anti-idiotypic monoclonal and polyclonal antibodies. These results clearly show that 2C3 Id represents the immunodominant epitope(s) recognized by the CTL line A102. To isolate a highly Id-specific effector population, A102 was repeatedly subcloned by limiting dilution. One such clone 102.F5 exhibited considerable specificity toward Id+ 2C3 while another clone 102.E10 showed no such specificity in a competitive cytotoxicity assay. This was further confirmed by the inhibition studies with anti-Id mAb. Thus, hyperimmunization with irradiated 2C3 cells evokes a spectrum of anti-2C3 cytotoxic effector cells, of which a major population is reactive to the idiotypic determinants associated with 2C3 Ig.     

In vitro reactivity of splenic lymphocytes from normal and UV-irradiated mice against syngeneic UV-induced tumors.

Skin tumors induced in mice by chronic ultraviolet (UV) irradiation are highly antigenic and are frequently immunologically rejected upon transplantation to normal syngeneic recipients. In this study we characterized this immune response with an in vitro microcytotoxicity test. Cytotoxic activity was present in the spleen cells of mice given a single injection of syngeneic UV-induced fibrosarcoma cells. After removal of adherent spleen cells, the remaining splenic lymphocytes were specifically cytotoxic for the immunizing tumor and showed no cross-reactivity with other syngeneic UV-induced or methylcholanthrene-induced tumors of similar histologic type. The level of cell-mediated reactivity against UV-induced tumors was quite high compared to that obtained with syngeneic tumors induced by methylcholanthrene, and the cytotoxicity was attributable to a population of theta antigen-bearing lymphocytes. With this in vitro test, we compared the response of normal mice, which reject a syngeneic tumor challenge, with that of UV-irradiated mice, in which the syngeneic UV-induced tumors grow progressively. After tumor cell inoculation, lymphocytes form the unirradiated (regressor) mice showed a high degree of cytotoxicity that reached a maximum level 8 days after injection. In contrast, no reactivity could be detected in the spleens of tumor-challenged UV-irradiated (progressor) mice.     

Anti-idiotypic antibodies induce neutralizing antibodies to rabies virus glycoprotein.

Rabbit anti-idiotypic antibodies (alpha Id Ab) were prepared against five murine monoclonal antibodies (mAb) specific for the rabies virus glycoprotein. Four of the mAb were directed against three known, type-specific, neutralizing sites on the glycoprotein, and the other mAb was directed against a topographically uncharacterized, nonneutralizing epitope. An absence of significant cross-reactivity among the alpha Id Ab for heterologous mAb suggested that the alpha Id Ab were highly specific for unique variable region determinants. The binding of three of the five alpha Id Ab to their homologous mAb could be inhibited by rabies virus-soluble glycoprotein, suggesting that the alpha Id Ab possessed subpopulations similar or adjacent to the antigen-binding site of the mAb. Two of the five alpha Id Ab injected into mice elicited a specific virus-neutralizing antibody response. Mechanisms to account for the induction of the virus-neutralizing antibody by alpha Id Ab are discussed.     

Induction of antibodies against Newcastle disease virus with syngeneic anti-idiotype antibodies in mice

Anti-idiotype antibodies were induced by injecting BALB/c mice with syngeneic antibody against the hemagglutinin of Newcastle disease virus (NDV). These anti-idiotype antibodies were purified and injected into syngeneic mice. Anti-anti-idiotype sera thus prepared contained antibodies against the hemagglutinin of NDV. This NDV-mouse experimental system might provide a good experimental model for investigation of basic problems of idiotype vaccine.     

Requirement for recognition of class II molecules and processed tumor antigen for optimal generation of syngeneic tumor-specific class I-restricted CTL

The roles of Class II-restricted L3T4+ T cells and of accessory cells (AC) during the in vitro generation of Class I-restricted Lyt-2+ cytotoxic T cells (CTL) specific for a Class II-negative syngeneic tumor cell line, FBL, was examined. Treatment of responder FBL-immune spleen cells with alpha L3T4 plus complement before culture, as well as the direct addition of alpha L3T4 to cultures, diminished the generation of FBL-specific CTL. The contribution of L3T4+ cells could be completely replaced by the addition of exogenous cytokines. The data demonstrate that the optimal generation of FBL-specific Lyt-2+ CTL requires the presence of L3T4+ cells, presumably to provide necessary lymphokines. FBL-specific CTL could not be generated from purified FBL-immune T cells in the absence of AC. Syngeneic Ia+ macrophages (M phi), added at the initiation of culture, restored the response of purified T cells. Pretreatment of M phi with ammonium chloride or chloroquine, or the addition of monoclonal alpha I-Ab antibody at the initiation of culture, inhibited the ability of M phi to reconstitute the CTL response. Finally, the addition of exogenous helper factors could replace M phi and reconstitute the FBL-specific response of AC-depleted immune T cells. These results suggest that during the generation of Lyt-2+ CTL to a syngeneic tumor expressing only Class I MHC antigens, Ia+ AC are required to biochemically process antigen released from the tumor cells and present this modified antigen to Class II-restricted T helper cells.     

Expression of anti-idiotypic clones against auto-anti-DNA antibodies in normal individuals

A serum pool from 280 blood donors and individual samples from blood donors were assayed for anti-idiotypic activity to auto-anti-DNA antibodies by competitive radioimmunoassays. We found that serum from several normal blood donors inhibited the binding activity of anti-DNA antibodies affinity purified from the sera of patients with systemic lupus erythematosus. This inhibition was due to immunoglobulin molecules but was not due to rheumatoid factor activity. Antiallotypic antibodies were not responsible for the anti-anti-DNA activity detected. Because this inhibition was blocked by DNA molecules, the observed reactivity was probably caused by idiotype-anti-idiotype interactions. These results provide evidence that anti-idiotype antibodies against anti-DNA autoantibodies are present in certain normal human sera. Anti-anti-DNA antibodies could play a role in the regulation of autoimmunity to DNA.     

UV-induced lethal sectoring and pure mutant clones in yeast

The induction of lethal sectoring and pure mutant clones by ultraviolet light has been studied in a homogeneous G₁ population of Saccharomyces cerevisiae grown in a normal growth medium. At the lowest UV dose of 250 ergs, which corresponds to a shoulder in the survival curve, all mutants appeared as pure clones. At higher doses the frequency of mosaic mutants progressively increased. These results indicate a relationship between the highest frequency of complete mutants and the maximum repair activity. In addition, the frequency of lethal sectoring at all doses tested was too low to account for the origin of pure mutant clones.     

Idiotypic intramolecular help. Induction of tumor-specific antibodies by monoclonal anti-idiotypic antibody with the help of Fc-specific T helper clones

In this study, the mechanism of anti-Id vaccination was investigated by using cloned Th cells and an anti-idiotypic mAb. 2F10, an anti-idiotypic mAb derived from an Igh1-e allotype mouse strain, which induces protection against the L1210/GZL DBA/2 tumor, was used to prime DBA/2 mice. An Fc (Igh1-e)-specific syngeneic Th clone was cocultured in the presence of 2F10 anti-Id with 2F10-Fab-primed B cells. The Th clone responded with proliferation and also provided help for 2F10-Fab-primed B cells to produce antibodies that bind to L1210/GZL and not to P815 tumor cells. Intact 2F10 anti-Id was presented to Fc-specific Th cells by Fab (or Id) primed B cells more efficiently than the fragment mixture (Fab plus Fc) of 2F10 anti-Id, indicating that 2F10-Fab (or Id)-primed B cells capture 2F10 anti-Id through surface Ig receptors. Presenting B cells are sensitive to treatment with chloroquine and must come from H-2 matched mice, indicating that the Ag presentation by Fab-primed B cells to Fc-specific Th cells requires processing and is MHC restricted. Collectively these results outline a mechanism that may operate in anti-Id therapy of tumor-bearing animals by using tumor Ag mimicking anti-idiotypic antibodies. A similar mechanism could be effective in tumor patients immunized with xenogeneic anti-idiotypic antibodies operating under the "intra(Ag) molecular help."     

幽门螺杆菌尿素酶单克隆抗体的研制及鉴定

应用杂交瘤技术 ,建立稳定分泌抗幽门螺杆菌尿素酶单克隆抗体 (HPU McAb)的细胞系。用纯化幽门螺杆菌尿素酶 (HPU)抗原加福氏佐剂免疫BALB/c小鼠 ,按常规方法进行细胞融合 ,以纯化HPU抗原包被 ,间接ELISA方法筛选 ,并经多次有限稀释法克隆。获得 6株抗HPU的杂交瘤 ,腹水效价达 1∶6 .4× 10 4～ 1∶2 .5 6× 10 5,特异性专一 ,并对其进行体内外连续传代 3个月 ,分泌抗体能力稳定。IgG亚类分型主要为IgG1型。该细胞系能稳定分泌幽门螺杆菌尿素酶单抗。     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. III. Analysis of viral glycoproteins recognized by CTL clones by using recombinant herpes simplex viruses

Human cytotoxic T cell (CTL) clones specific for herpes simplex virus (HSV) type 1- and type 2-infected cells were generated and were analyzed with regard to the viral glycoproteins they recognize on autologous HSV-infected cells. By use of target cells infected with wild-type HSV strains, a gC deletion mutant of HSV-1, and HSV-1 X HSV-2 intertypic recombinants, some HSV-1-specific CTL clones were found to be directed against L region-encoded gA/B-1, and others against S region-encoded glycoproteins (gD-1 or gE-1). Some HSV-2-specific clones were found to be directed against L region-encoded gC-2, whereas others were directed against S region-encoded glycoproteins (gD-2, gE-2, or gG). These findings provide direct evidence that several HSV glycoproteins that are expressed on the surface of HSV-infected cells serve as recognition structures for human HSV-specific CTL.     

Anti-idiotypic antibodies induce formation of anti-H-2 sera and of idiotypes.

Alloantisera could be induced in parental strain recipients either by means of alloantigen (skin allografting) or by injecting anti-idiotypic (anti-T cell receptor) sera. The resulting sera displayed activity against H-2 antigens; they also contained soluble isiotypic structures. With these sera anti-idiotypic sera could be provoked in F1 hybrid hosts. If antibody cycles initiated with alloantigen were carried through four runs, specificity appeared to be preserved and titers obtained corresponded to those found in conventionally raised anti-idiotypic sera and in alloantisera.     

Cutting edge: rapid cloning of tumor-specific CTL suitable for adoptive immunotherapy of melanoma.

Adoptive immunotherapy using CTL has provided some clinical benefit to patients with metastatic melanoma. Use of cloned CTL of known specificity might improve clinical effect, but technical difficulties have limited exploration of this possibility. We have used fluorescence-driven cell sorting to clone tumor-specific CTL after staining with tetrameric MHC class I/peptide complexes. CTL specific for the melanoma Ags melan-A, tyrosinase, and MAGE3 were cloned from the peripheral blood, tumor-infiltrated lymph nodes, and skin metastases of five patients. Clones were isolated and characterized in as little as 6 weeks, much faster than is possible with previous techniques. We show that these CTL clones express markers compatible with immunotherapeutic use in melanoma, including the cutaneous lymphocyte Ag, which is associated with homing to skin.     

Adoptive immunotherapy of syngeneic murine leukemia is enhanced by the combination of recombinant IFN-gamma and a tumor-specific cytotoxic T cell clone

Mice with an established syngeneic T cell tumor (RBL5) received short term adoptive chemoimmunotherapy with CTL clone 1.B6 and murine rIFN-gamma. In comparison with treatment with either agent alone, the combination of 1.B6 and rIFN-gamma was associated with a dramatic increase in long term survival. No direct effects of rIFN-gamma on tumor cell proliferation, MHC Ag expression, or susceptibility to CTL-mediated lysis could be demonstrated to explain the prolongation of survival. However, rIFN-gamma induced a distinct increase in broad-spectrum cytolytic capacity of peritoneal exudate cells and further increased class II MHC expression on peritoneal macrophages. The explanation for enhanced adoptive chemoimmunotherapy after combined short term administration of a CTL clone and rIFN-gamma is uncertain. Potential mechanisms include direct tumor lysis by activated cells, indirect tumor lysis via sensitization to other lymphokines or monokines, improved Ag-specific activation of transferred CTL clones, and/or more effective development of de novo host anti-tumor immunity.     

抗酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris)单克隆抗体制备

【背景】基于本实验室已经建立的脂环酸芽孢杆菌检测和鉴定方法工作基础之上,以期建立具有很好的经济价值和实用价值,且更为方便、快捷、准确、特异、灵敏的检测方法。【目的】实现对果汁生产中脂环酸芽孢杆菌从原料到成品的快速检测和鉴定。【方法】采用杂交瘤细胞技术,以Alicyclobacillus acidoterrestris(ATCC49025)免疫BALB/c小鼠,用建立的间接ELISA方法筛选杂交瘤细胞,得到了3株能稳定分泌A.acidocaldarius抗体的杂交瘤细胞株,其中两株为Ig G1亚类,并对其进行生物学特性的鉴定。【结果】得到的两株单抗3F7和9C4是针对不同的抗原位点,且多次传代后稳定性基本保持不变;特异性实验表明两株单抗均不与A.acidocaldarius(NCIMB11725)、Bacillus cereus(ATCC11778)、Bacillus subtilis(ATCC11774)、A.cycloheptanicus(ATCC49029)等发生交叉反应。【结论】3F7和9C4这两株单抗可以进一步用于检测胶体金试纸条的研制。     

Increased tumor-specific CTL activity in human tumor-infiltrating lymphocytes stimulated with autologous tumor lines

Two long-term tumor-infiltrating lymphocyte (TIL) lines and their autologous tumor lines have been established from solid tumors derived from different patients with metastatic melanoma. In 4-hr 51Cr release assays, each TIL culture lysed only the autologous cryopreserved fresh or established melanoma line, but failed to lyse other melanoma tumors or K562 cells. Repeated stimulation of TIL with the autologous melanoma lines resulted in significant increases in anti-tumor CTL activity with no apparent loss in specificity. Stimulated cells have retained cytotoxic activity for up to 5 months in culture. Tumor cell CTL activity for both long-term TIL lines is inhibited by several mAbs, including those against CD3, CD8, and class I MHC molecules, indicating that the effector cells are class I-restricted CD8+, CTL. Furthermore, recognition of Ag on one of the established melanoma lines by TIL is restricted by HLA A-2. The availability of autologous tumor lines may prove clinically useful for the selective stimulation and expansion of cells with anti-tumor activity within a heterogeneous TIL population.     

Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.     

Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo

Bispecific antibodies with specificity for CD3 and a tumor antigen can redirect cytolytic T cells to kill tumor targets, regardless of their natural specificity. To assess the clinical potential of bispecific antibodies for treatment of human cancers we have, in the present study, adapted a totally syngeneic mouse model to the targeting of mouse T cells against mouse tumors in immunocompetent mice. We show that gp52 of the mouse mammary tumor virus (MTV) can serve as a tumor-specific antigen for redirected cellular cytotoxicity. Chemically crosslinked and genetically engineered bispecific antibodies with specificities for gp52 and murine CD3 -chain induced activated mouse T cells to specifically lyse mouse mammary tumor cells from cultured lines and primary tumors from C3H-MTV⁺ mice. Retargeted T cells also blocked the growth of mammary tumors in vitro as well as their growth in syngeneic mice. These findings identify murine MTV-induced mammary adenocarcinomas as a solid-tumor, animal model for retargetin T cells with bispecific antibodies against syngeneic breast cancer.