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Stimulation of butyrophilin 3 molecules by specific agonistic mAbs results in down-regulation of proliferation and cytokine secretion in CD4 and CD8 T lymphocyte subsets. The BTN molecule consists of three subfamilies, BTN1, BTN2. and BTN3, and possesses interesting properties for biological regulation. Although the biological significance of BTN1 and BTN2 has been progressively clarified, the receptor function of BTN3 remains to be elucidated as a result of the absence of appropriate agonists. To clarify the participation of BTN3 in immune regulation, BTN3-specific mAb, referred to as 34-7 and 232-5, were generated from BTN3 gene-immunized mice. The 232-5 mAb, specific to the extracellular domain of the BTN3 molecule, stained almost all populations of human PBMCs, including T, NK, NKT, and B cells. Notably, treatment with the 232-5 mAb resulted in phosphorylation of BTN3A3 molecules, leading to attenuated proliferation and cytokine secretion by CD4+ and CD8+ T cells in a CD4+ CD25+ Treg cell-independent manner, demonstrating the agonistic property of the 232-5 mAb in BTN3-mediated negative signal transduction. The magnitude of the cell surface expression of BTN3 molecules correlated inversely with lymphocyte activity, suggesting that BTN3 molecules contribute to the maintenance of the immune system. Taken together, our findings provide new insights for the role of BTN3 as an inhibitor of excessive cellular immune responses.

Q5 antigens are expressed on the surface of various experimental murine tumor cells. They share partially common antigenicity with Qa-2 alloantigens expressed on normal lymphocytes. For that reason we tested the immunoprotection by anti-Qa-2 immunization of mice against a Q5+ tumor. Nerve fibrosarcoma (NSFA) tumor, which specifically develops metastasis in the lung, has been reported to be poorly immunogenic. However, expression of the Q5 antigen was evident on the surface of NFSA cells. After immunizing (C3H/He x B6.K1)F1 (Qa-2-) mice with B6 (Qa-2+) lymphocytes, the protection against the proliferation of the semi-syngeneic NFSA tumor was examined. First, immunization of normal mice induced resistance to NFSA cell transplants. Second, when the tumor cells were transplanted to the hind foot of a mouse and the resulting tumor was removed by amputating the leg, the mice were protected against the development of lung metastasis after immunization by intraperitoneal inoculation of B6 cells 3 days after tumor removal. Immunization with attenuated NFSA cells in this system failed to protect the mice from lung metastasis. On the other hand, inoculation of the mice with B6 cells without removal of the original tumor on the foot showed little effect on the progression of the tumor. Thus, cytotoxic T lymphocytes (CTL), which seemed to be present in an inactive form in the mice from which the tumor had not been removed, were induced in the mice after the removal of the major tumor followed by immunization with B6 lymphocytes. The induction of CTL by the immunization was suppressed in mice bearing large tumors. Cells stimulated by the tumor antigen seemed to be involved in the suppression. It was also shown that the Q5 antigen is the direct recognition target of the CTL since the activity of Q5-specific CTL clones in lysing tumor cells was inhibited by a monoclonal antibody specific for the Q5 antigen. In contrast to immunization with attenuated tumor cells, our novel allogeneic lymphocyte immunization procedure offers high CTL activation, by-passing the induction of T cell unresponsiveness.

Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa-arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate-buffered saline. However, almost all the R8-conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary-K1, Cos-7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8-conjugated β-galactosidase and R8-conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen-presenting cells and will benefit research and innovation in vaccine design and discovery.

Serum from C3H/He mice, which show regression of MM2 tumor cells after transplantation and removal (regressor serum, RS) contains non-gammaglobulin components that cross-react with various tumor cells of mice [22, 23]. In addition to tumor cells, various allogeneic lymphocytes are also susceptible to an RS-dependent lymphocyte-mediated cytotoxic reaction. To identify tumor cell surface antigens that cause the cross-reactive host response, the serum components were analyzed by absorption of RS with allogeneic lymphocytes. RS components were found to recognize allogeneic lymphocyte antigens including Qa-2 and Ly6.2. Specificity for the Qa-2 antigen was further tested using Qa-2-congenic mice. The expression of Qa-2 antigen was detected on the surfaces of MM2 and other tumor cells derived from H-2k mice (seven among nine cell lines tested) by a membrane immunofluorescence method using a Qa-2-specific mAb. Physical characteristics of the Qa-2-specific component in RS were determined and found to differ from those of regular IgGs but to be similar to those of IgDs. Using an enzyme-linked immunosorbent assay with an IgD-specific mAb and Qa-2-lacZ fusion protein, the existence of IgD in RS with specificity for Qa-2 was confirmed. These results suggest that the RS component with Qa-2 specificity is an IgD, the specificity and physiological role of which are unknown.

Immunological and biochemical characteristics of the Qa-2 murine nonclassical histocompatibility class 1 antigen expressed on tumor cells derived from H-2k (Qa-2-) mice were studied. It was found that the Qa-2 antigen on normal H-2b lymphocytes reacted with Qa-2-specific monoclonal antibodies (mAbs) 34-1.2, 59 (both specific to the alpha 1/alpha 2 region) and 141-15.8 (specific to the alpha 3 domain), and the Qa-2 antigen on H-2k tumor cells (Qa-2k antigen) reacted with mAbs 59 and 141-15.8, but not with 34-1.2. The normal Qa-2 antigen was susceptible to treatment with phosphatidylinositol-specific phospholipase C, but the Qa-2k antigen was insensitive to it. By Northern hybridization, polymerase chain reaction (PCR) studies on cDNA, Southern hybridization, Western blotting, and nucleotide sequence analysis, the Q5k gene was identified as the gene encoding the Qa-2k antigen expressed on BW5147 lymphoma cells derived from a mouse of AKR strain (H-2k, Qa-2-). The nucleotide sequence of PCR-amplified BW5147 Q5k cDNA showed complete agreement with the reported sequence of exons 1-5 of the Q5k gene of C3H/He. It also showed complete deletion of the region corresponding to exons 6 and 7, and a very short coding region in exon 8, resulting in very short cytoplasmic domain of the product compared with regular class 1 antigens. These characteristics were expected from the reported Q5k genomic sequence. These results revealed that the Qa-2k antigen was distinct from the normal Qa-2 antigen expressed on H-2b lymphocytes although it cross-reacted with some Qa-2-specific mAbs.

A drawback of cancer gene therapy is the failure of toxic gene introduction into a proportion of the tumor cells, resulting in re-progression of the disease. Cancer cell-specificity of the gene introduction has also been problematic.Previously defined promoter/enhancer DNA of Q5 tumor antigen gene was used for the purpose of transcriptional targeting. A replication defective adenovirus carrying H-2D(d) cDNA placed downstream of the Q5 promoter/enhancer (Ad/Q5-H-2D(d)) was constructed and administered systemically to C57Bl/6 (H-2(b)) mice bearing pre-established hepatic metastasis of the syngeneic M5076 tumor.5 of the 10 mice showed complete regression of the tumor. Combination of the therapy with low dose cyclophosphamide (CY) administration augmented the therapeutic effect. The effect was brought about by tumor cell-specific allograft rejection reactions and by tumor antigen-specific reactions induced as "bystander effect" by the former reactions.The results provide an experimental basis of a highly efficient novel strategy of cancer gene therapy.

Regulatory DNA that would induce tumor cell-specific gene expression of arbitrary genes in human cells has been sought. We previously reported that the transcription of mouse Q5 gene was tumor-selective and hypothesized that Q5 5'-flanking DNA had a key role in tumor-selective transcription.Isolation of 3.9 kb Q5 5'-flanking DNA was carried out and the sequence characteristics determined. Reporter plasmids, recombinant reporter adenoviruses and recombinant reporter lentiviruses were prepared that contained the Q5 5'-flanking DNA fragments of various lengths. Expression of these reporter genes was examined in various murine cells in vitro and in vivo and in various human cells in vitro.Adenovirus vectors that had Q5 5'-flanking DNA contained a regulatory region and induced tumor cell-specific gene expression not only in mouse cells but also in human cells.Our newly constructed vector system could be utilized in gene therapy of human cancer.

We previously reported that the Q5 gene product (Q5 antigen) was expressed on the surface of various tumor cells derived from H-2k (Qa-2-) mice. The Q5 antigen has tumor-protective antigenicity in the syngeneic mice.Transcripts of the Qa region genes were analyzed by the RT-PCR method. Cell fractionation was performed with the MACS method and the phenotypes were estimated by flow cytometry analysis.Transcripts of Q5 were produced by all tumor cell lines. The Q5 transcription was detected only in thymocytes and PBMCs of H-2k (AKR and C3H/He) mice. The phenotype of the PBMCs in which Q5 transcription takes place seems to be, at least partly, CD3-48 cells that might be related to CD3-4-8- spontaneous thymoma cells derived from an H-2k mouse.The expression of Q5 in tumor cells in general is a result of a change of transcriptional regulation associated with malignant transformation.

Previous studies of CTL responses to influenza peptides in HLA single transgenic mice resulted in the identification of at most one immunodominant epitope. Since HLA-B*3501 is known to present multiple HIV-1-specific T cell epitopes we tested the cellular immune response of HLA-B*3501 transgenic mice to synthetic HTLV-1 peptides mixed with the lipohexapeptideN-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteinyl-seryl-lysyl-lysyl-lysyl-lysine, which is a biocompatible, Th-epitope-independent adjuvant. Eleven of 37 tested HLA-B*3501 binding peptides mounted a CTL response after threein vitrostimulations. The HLA-B*3501 affinity of peptides correlated with their ability to induce CTL in HLA-B*3501 transgenic mice. Seven peptides derived from env-gp46 (VPSPSSTPLL, VPSSSSTPL, YPSLALAPH, and YPSLALAPA), pol (QAFPQCTIL), gag-p19 (YPGRVNEIL), and tax (GAFLTNVPY) proteins induced peptide-specific CTL. Bulk CTL generated by four peptides derived from env-gp46 (SPPSTPLLY, VPSPSSTPLLY, and VPSPSSTPLL) and pol (QAFPQCTILQY) killed peptide-pulsed and recombinant vaccinia-infected target cells. The latter peptides therefore present T-cell epitopes and are vaccine candidates for our transgenic mouse model.

In an attempt to obtain monoclonal antibodies specific to tumor-associated antigens, C3H/He mice were immunized with syngeneic MM2 tumor cells, and the primed spleen cells were fused with P3-X63-Ag8.653 myeloma cells. The outgrowth of hybridomas, however, was extremely low and monoclonal antibodies were not obtained. The reason for the low hybridoma growth was studied. It was found that MM2 cells used as the immunogen, the fusion partner myeloma cells and the resulting hybridomas shared at least one tumor-associated antigen, namely Q5 antigen. Because of this common antigen, cytotoxic cells, presumably cytotoxic T lymphocytes, which were lytic to the hybridomas, were induced during the culture for generation of the hybridomas. Removal of lysosome-rich cells, including cytotoxic T lymphocytes, from the primed spleen cells before the fusion by treatment with leucine methyl ester, a lysosomotropic agent, drastically improved the outgrowth of hybridomas. By this method, seven stable hybridoma clones producing monoclonal antibodies specific to tumor-associated antigens were obtained. Two of the seven clones were found to secrete monoclonal IgM species, which reacted with the extra-cellular region of the Q5 antigen. This procedure will be an option when production of monoclonal antibodies specific to cell-surface antigens is intended and outgrowth of hybridomas is unexpectedly low.

The major surface glycoprotein (MSG) of Pneumocystis carinii plays a crucial role in the pathobiology of P. carinii, which often causes fatal pneumonia in AIDS patients. The cDNAs encoding MSG antigens were cloned from a lambda gt11 expression library of rat-derived P. carinii by immunoscreening. The cloned cDNAs constituted a gene family containing approximately 70% amino acid identity between subtypes. The diversity of MSG cDNAs was high and reflected the genomic structure of MSG genes clustered in the P. carinii chromosomes. These multiple genes may account for the high-level expression of MSG that could generate potential variations in the cell surface. Moreover, the MSG sequences have significant sequence homology to tropomyosins and myosins, suggesting physical or functional association with the membrane cytoskeleton.

Tumor-specific expression of Qa-2k antigen coded by the Q5k gene on various mouse tumor cells and immunological response of the host mice to the antigen have been demonstrated [Seo et al. (1992) J Exp Med 175: 547; Tanino et al. (1992) Cancer Immunol Immunother 35: 230]. The possibility was examined that Qa-2 antigen is one of the recognition target molecules of immunopotentiator-induced, H-2-nonrestricted tumoricidal lymphocytes of Qa-2-mice. Lymphocytes stimulated in vivo with P. acnes or culture-induced anomalous killers of B6.K1 mice did not exhibit significant in vitro cytotoxicity against B6.K1 lymphoblasts but lysed their Qa-2,3-congenic counterpart B6 lymphoblasts. To demonstrate the Qa-2 specificity of such cytotoxic cells more precisely, an L cell transformant clone (LQ7b/Kb), which expressed the alpha 1 and alpha 2 domains of the Qa-2 antigen (Q7b gene product), was generated by transfecting a cloned plasmid DNA containing a hybrid gene constructed from the 5' half of the Q7b gene and the 3' half of the H-2Kb gene (pQ7b/Kb). Using LQ7b/Kb cells as the target cells and the nylon-wool-nonadherent fraction of lymphocytes from P. acnes-stimulated (C3H/He x B6.K1)F1 mice (H-2k, Qa-2-) as the effector cells of the in vitro cytotoxicity reaction, the presence of cytotoxic cells that recognize the alpha 1/alpha 2 region of the Q7b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the gamma/delta type (TCR gamma/delta). The (C3H/He x B6.K1)F1 effector cells, as well as the B6.K1 effector cells also lysed BW5147 lymphoma cells (Qa-2k+) derived from AKR mice (Qa-2-, H-2k). By target-competition experiments it was shown that some of the effector cells lytic to BW5147 were identical to those that lysed LQ7b/Kb. Therefore some of the tumoricidal cells induced by the immunopotentiator interact with the target tumor cells through recognition of the alpha 1/alpha 2 region of the Qa-2k tumor antigen by TCR gamma/delta.

A method of in vitro agglutination of murine ascitic tumor cells which had been used to detect and to semiquantitate components with saccharide-binding properties in sera from cancer patients was applied on sera from patients with various autoimmune diseases. Sera from 36 of 56 patients with such diseases, after removal of their cytocidal activity on the murine tumor cells by absorption with liver tissue from normal adult mice, agglutinated the tumor cells which had been harvested while they were rapidly proliferating in vivo. The agglutination activity was absorbed completely by embryonic tissue of mouse. The agglutination was inhibited by perchloric acid extract of the tumor cells and also by monosaccharides such as galactose or N-acetyl-D-glucosamine. The inhibition activity of the perchloric acid extract was affected by treatment with glycosidases but not trypsinization. These results showed that carbohydrate moieties of murine tumor cell surface components were involved in the agglutination and therefore that there was an increase of components with saccharide-binding properties in the sera of patients with autoimmune diseases compared to the sera of normal controls. This increase in turn suggested by analogy to tumor-bearing animals that a sort of humoral reaction took place in the patients against changes related to autoimmune diseases of carbohydrate moieties of the cell surface substances.

Components on the surface of MM2 ascites mammary carcinoma cells induce agglutination factors in the serum of syngeneic host C3H/He mice, and bind the factors in vitro. These components have been classified into three groups: MM2-specific substances, mammary tumor virus (MTV)-associated substances and tumor-associated embryonic materials. The substances contained saccharide moieties and their terminal sugar residues were essential for the binding of the serum factors. These terminal saccharides were exposed during cell proliferation, but masked in stationary cells, at least partly, due to elongation of the saccharide moieties. The terminal structures of these polysaccharide moieties of growing and stationary cells were studied by semiquantitative tumor cell agglutination using the agglutinating activities against MM2 cells of MM2-regressor serum and of FM3A/R- and Ehrlich-regressor sera which had partial cross-agglutination activities. Agglutination by phytohemagglutinins, inhibition of the agglutination by saccharides or with isolated cell surface components and treatment of the cells with glycosidases were also used for this purpose.

Immunity against transplantation of MM2 cells, an ascites cell line of mouse mammary carcinoma, was induced in syngeneic C3H/He mice by intraperitoneal injection of a complex formed between a MM2-specific cell surface component isolated from the cells and a serum factor found in the serum from MM2-regressor and MM2-bearing mice. The induced immunity was transferable to other mice with the spleen cells or with the serum from the immunized animals. This cell surface component was not immunogenic by itself. A new immunogenicity was induced in the protein moiety of this substance when the serum factor was bound to the polysaccharidecontaining part of the molecule. On the basis of these results, a biphasic mechanism for the induction of the humoral immunity against tumor antigens was proposed. The first phase takes place in tumorbearing animals and gives rise to the serum factor. The second phase, which is blocked in tumor-bearing animals, consists of the induction of the humoral immunity by the complex in tumor-free animals.

Sera from 72 of 94 patients with various malignancies and those from seven of nine pregnant womens agglutinated mouse ascitic tumor cells in vitro. Sera from 15 of 17 benign disease controls also agglutinated the cells, but none of the sera from 21 normal controls did. The agglutination activity of the sera from cancer patients was studied in detail. The activity, which remained after absorption with liver tissue from normal adult mice and also with blood group A substance, was absorbed completely by embryonic tissue of the mouse and partially by perchloric acid soluble fraction (PCAS) of the target cells or by carcinoembryonic antigen (CEA) from human cancer, and therefore was related to cross-reacting tumor-associated embryonic materials. The agglutination was inhibited by monosaccharides such as galactose or N-acetyl glucosamine. The receptor activity of PCAS for the agglutination factors in the sera from cancer patients was also affected by treatment with glycosidases, but not by trypsinization. These results indicated that carbohydrate moieties of the cell surface components were involved in the agglutination. It was thus evident that a sort of humoral reaction took place in cancer patients against tumor-associated embryonic materials on the tumor cell surface, that a considerable part, at least, of such reactions were directed to their carbohydrate moieties and that their teminal sugar residues played the most important role in the reaction. The humoral factors induced by this reaction was located in β-globulin fraction of the serum protein. The extent of such humoral reaction by the hosts was semiquantitated by the simple in vitro agglutination method. Cancer 40:693–699, 1977.

Cell-line specific cell surface components were demonstrated on cells of various hypotetraploid ascites cell lines, but not on hyperdiploid cells derived from a mammary carcinoma induced by mammary tumor virus (MTV) in syngeneic C3H/He mice. These substances caused cell-line-specific transplantation antigenicity and were identified, together with MTV-associated substances and tumor-associatied embryonic materials, as being the binding sites for tumor cell agglutinating factors found in sera from tumor-bearing and regressor animals. Particles containing the cell line-specific substances were released from these cells by hypnotic treatment. They could be purified as a single peak in a sucrose density gradient. They were, however, not dissociated from the MTV-associated substances. Absorption studies of agglutination activity were extensively used for demonstration of specificity.

Binding of 125I-labelled epidermal growth factor (EGF) to C3H/2K cells and the effect of a tumor promotor, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of a tumor promotor antagonist, retinoic acid, on the binding was studied. Scatchard plot analysis of the binding showed the presence of two types of binding sites with different affinity to EGF. Treatment of the cells with retinoic acid for 1 h resulted in elevation of the affinity of both sites without changing their number per cell. Prolonged exposure to retinoic acid abrogated this elevation of the affinity and caused cycloheximide-sensitive increase of the number of the binding sites of both types. TPA inhibited binding of EGF to the cells by abolishing the binding to the high affinity sites, whereas retinoic acid, in the presence of TPA, enhanced it by increasing the number of the low affinity sites.

From whole nucleolar RNA of Novikoff hepatoma cells, a single symmetrical peak of 23 s RNA was obtained by repeated sucrose density-gradient centrifugations. Hybridization experiments showed that nucleolar 23 s RNA is composed of 18 s ribosomal RNA and other RNA segments. Further, nucleolar 45 s RNA is composed of nucleolar 23 s RNA and other RNA segments. The purified nucleolar 23 s RNA contained almost the same percentages and numbers of each of the alkali stable dinucleotides as 18 s rRNA; these differ markedly from the corresponding values for the alkali stable dinucleotides of 28 s rRNA. The 5′-terminal nucleotide of both nucleolar 23 s RNA and 18 s rRNA is pGp. However, the major 3′-terminal nucleosides of 18 s rRNA and 23 s nucleolar RNA are adenosine and uridine, respectively. From these results, it is concluded that nucleolar 23 s RNA contains ribosomal 18 s RNA in its 5′-terminal portion and is a cleavage product of nucleolar 45 s RNA.