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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.

Dendritic cells (DCs) are potent antigen presenting cells that are able to initiate and modulate immune responses and are hence exploited as cellular vaccines for immunotherapy. In particular DCs generated from peripheral blood monocytes (Mo-DCs) have been used with promising results as a new approach for the immunotherapy of cancer. In this study, we have analyzed the changes in the pattern of expression molecules on Mo-DCs during DC maturation using different maturation cytokine combinations and the expansion capacity of an antigen specific CD8+T cells monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. These analyses revealed that the expansion of antigen specific CD8+T cells is the most effective when T cells were activated by fully maturated DCs by culturing monocytes for 5 days in the presence of GM-CSF and IL-4, followed by 2-3 days of maturation with pro-inflammatory mediators including TNFalpha, IL-6, IL-1beta and PGE2. These results pave the way to a more effective immunotherapy using DCs for patients with malignancy, as well as infectious diseases.

In vitro proliferation and functional activation of V alpha 24NKT cells following stimulation with alpha -galactosylceramide (alpha -GalCer)-pulsed dendritic cells (DCs) have been observed. Because little is known about the molecular events on DCs following interaction with alpha -GalCer, we performed gene expression profiling of 2400 genes in monocytes and monocyte-derived immature DCs pulsed with alpha -GalCer (alpha -GalCer-imDCs). Overall, the expression levels of 48 genes were up-regulated and 28 were down-regulated in alpha -GalCer-imDCs. Semiquantitative RT-PCR analysis on monocytes, imDCs, alpha -GalCer-imDCs, and mature DCs confirmed the up- and down-regulation of the mRNA expression levels of 28 selected genes. Notably, we identified the specific up-regulation of mRNA expression levels of ribonuclease A and collapsin response mediator protein upon the stimulation of imDC with alpha -GalCer, suggesting a novel immunomodulating effect of alpha -GalCer on imDCs. In this study, we used imDCs prepared by culturing of monocytes with GM-CSF and IL-4 for 5 days and mDCs prepared by further culturing of imDCs with TNF alpha for two extra days. (C) 2001 Elsevier Science.

It has been demonstrated that γδ T cells accumulating in early tumor lesions and those purified from spleen cells of tumor-bearing mice attenuate the activity of CTLs and NK cells. We, therefore, investigated whether depletion of γδ T cells from early lesions of tumors results in restoration of CTL and NK cell activities and subsequent regression of tumors. A daunomycin-conjugated anti-γδTCR mAb UC7-13D5 (Dau-UC7) was prepared to efficiently deplete γδ T cells. An in vitro study revealed that Dau-UC7 specifically lysed γδTCR+ cells and effectively inhibited splenic γδ T cells from tumor-bearing mice to produce cytotoxic cell-suppressive factors. Furthermore, intralesional injections of Dau-UC7 at an early stage of tumor development led to augmentation of tumor-specific CTL as well as NK cell activities and to the resultant regression or growth inhibition of the tumors. On analysis of cytokine profile, γδ T cells transcribed mRNAs for IL-10 and TGF-β, but not IL-4 or IFN-γ, suggesting the T regulatory 1-like phenotype. Finally, a blocking study with mAbs showed that the inhibitory action of γδ T cells on CTLs and NK cells was at least partly mediated by IL-10 and TGF-β. These results clearly demonstrated the novel mechanism by which T regulatory 1-like γδ T cells suppress anti-tumor CTL and NK activities by their regulatory cytokines in early tumor formation.

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.

Spleen cells derived from tumor-bearing mice prove useful for the elucidation of the mechanism determining how tumor cells evade cytotoxic T lymphocytes (CTL) in tumor-bearing hosts. Our data indicate that inactive CTL or precursor CTL specific for tumor antigens are present among lymphocytes of tumor-bearing mice. However, their activity is inhibited by a soluble factor produced by other cells present in the same source. Inhibition of the cytolytic reaction was also detected in the culture supernatant of spleen cells obtained from normal mice, precultured in the presence of tumor cell culture supernatant and interleukin-2 (IL-2). Cell-depletion and cell-purification studies let us conclude that cells that produced the CTL-inhibitory factor (CTL-IF) were γ/δ T cells. The γ/δ T cells that were activated in vivo in tumor bearers were able to produce CTL-IF after isolation and in vitro culture. Maximum activation of γ/δ T cells was achieved by antigenic stimulation and by suppression of cells that interfered with the activation of γ/δ T cells. CTL-IF, which was assayed by use of CTL clones, did not show antigen specificity. Inhibition depended on a relatively heat- and acidstable, but alkali-labile molecule with a molecular mass of less than 10 kDa. The latter characteristics imply that CTL-IF does not resemble any of the known lymphokines produced by γ/δ T cells. These observations emphasize the crucial role of the γ/δ T cells in the escape of tumor cells from the attack of tumorspecific CTL.

Two HLA-B*3501 binding self-peptides, LPFDFTPGY (37F) and LPGPKFLQY (28H), were isolated from HLA-B*3501 molecules expressed by cultured human B lymphoid cells. Both sequences were consistent with previously reported motifs of HLA-B*3501 binding peptides which carry proline at position 2 and tyrosine at position 9 as anchor residues. Direct binding of these peptides to HLA-B*3501 molecules was quantitated by flow cytometry analysis of RMA-S cells. transfected with the HLA-B*3501 gene (RMA-S-B*3501). Both 37F and 28H peptides bound effectively to HLA-B*3501 molecules. Substitution of amino acids at position 2 and/or 9 of HLA-B*3501 binding peptides markedly reduced their binding to HLA-B*3501 molecules. These results indicate that two anchor residues, proline at position 2 and tyrosine at position 9 are critical in binding of peptides to HLA-B*3501 molecules. Insertion of up to four glycine residues at position 8 of the peptide 37F did not affect its binding affinity to HLA*3501 molecules. These results indicate that long peptides can effectively bind to HLA class I molecules provided that anchor residues are conserved.

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.

The immuno-cell therapy of cancer in Japan is in the phase of transition from therapeutic research to a more generalized option for all patients. A short history of cancer immunotherapy in Japan and its present status are summarized. In 1999, the first private clinic with a cell processing facility and specializing in immuno-cell therapy was established. Since then, the number of such clinics has increased. As a result, the number of patients who undergone the therapy has markedly increased. A summary of the clinical results of the therapy in 1401 patients treated in two leading clinics is also presented.

For successful dendritic cell (DC)-based immunotherapy, it is critical to identify the most potent stage of human DCs, including immature DCs (imDCs) and mature DCs (mDCs).imDCs were obtained by culturing monocytes in the presence of GM-CSF and IL-4 for 5- 7 days and imDCs were further cultured for 24-48 h in the presence of TNFalpha, IL-6, IL-1beta and PGE2 to obtain mDCs. Melan-A- and EBV (BRF1) peptides were used and the frequency of antigen-specific CD8+ T cells was assessed using appropriate tetramers.mDCs were potent antigen-presenting cells for the induction and proliferation of antigen-specific naive and memory CD8+ T cells and may overcome regulatory functions that suppress antigen-specific CD8+ T cells.Our findings that mDCs can efficiently expand antigen-specific naive and memory CD8 + T cells have important implications in the development of vaccination strategies and support the use of antigen-loaded mature DCs in human clinical trials

We previously reported that a Q5 gene product (Q5 antigen) on the surface of tumor cells caused humoral immune reaction in syngeneic tumor-bearing mice. Transcripts of the Q5 gene were observed in various tumor cells. The human counterpart of the Q5 gene has not been clarified yet.Transcripts of human class Ib genes in various human tumor cell lines were analyzed by the RT-PCR method. Whether anti-HLA-F antibodies were present in sera from cancer patients was investigated through Western blotting with recombinant HLA-F as an antigen.Transcripts of the HLA-F gene were produced by various tumor cell lines. In addition, anti-HLA-F IgG was present in the sera derived from various types of cancer patients (26 positives / 42 tested), but not in the sera derived from healthy donors (0 / 20).Anti-HLA-F antibodies should be further investigated as possible diagnostic tumor markers.

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.

Autologous immuno-cell therapy of cancer has been mainly carried out in university hospitals as therapeutic research. Recently, private clinics specialized in the therapy are appearing in Japan. At present, cell processing necessary for the therapy is carried out not by pharmaceutical companies but by medical institutions. In this case, there is no government regulation on the safety assurance of the procedure. For the wholesome popularization of the therapy, however, it is of utmost importance to carry out cell processing under safety assurance, which could be done as self-regulation by medical institutions. A guideline for safety assurance tentatively set by Seta Clinic Group, which is a group of clinics specialized in the therapy and its usefulness in protecting patients from hazardous accidents are shown.

It has been widely recognized that chemotherapy of cancer has profound toxic side-effects and suffers limitations in efficacy but, as yet, no solution has been found to this conflict. Consequently, many patients with advanced cancer desire treatment by less toxic therapies even if the technique is not established as a conventional cancer therapy. Thus the establishment of less toxic therapies should be considered as a tentative requirement in clinical practice. Adoptive immunotherapy is one such therapy which has enjoyed some popularity in Japan. Clinical experience accumulated over the last 15 years indicates benefits to be gained from the therapy. In 1998, the Ministry of Health, Labor and Welfare of Japan (MHLW) approved portions of adoptive immunotherapy practices in their definition of highly-advanced medical technology. In April 1999, we established a private out-patient clinic specializing in adoptive immunotherapy; this clinic has administered 200 infusions of activated lymphocytes a month. Out of the 188 patients treated, we evaluated the tumors of 57 cases (2 CR, 4 PR, 20 SD and 31 PD). The response rate was 11%. It is noteworthy that in 11 of the 20 SD patients, the disease remained stable for more than six months. The follow-up periods for these 11 patients ranged from 11 to 24 months. During this period tumor progressed in 2, but the remaining 9 were still alive and without noticeable disease progression. Our present experience of adoptive immunotherapy administered to a large number of patients indicates that this non-toxic therapy has some beneficial role for the treatment of refractory cancer.

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.

Recent studies have demonstrated directly that alloreactive mouse CTL recognize peptides presented by MHC class I molecules. However, there is no direct evidence that human alloreactive CTL recognize peptides presented by HLA class I molecules. We have isolated an HLA-B51 alloreactive CTL clone, 2B3, that did not kill the TAP defective cell lines T2 and .174, whereas it killed the TAP-positive cell line T1 and .174 cells transfected with TAP genes. These findings suggested that this clone recognizes a TAP-dependent allo-peptide. We attempted to isolate the human allo-peptide recognized by the 2B3 clone from HLA-B51 molecules. A naturally occurring HLA-B*5101 binding peptide isolated from T1 cells was recognized by the 2B3 clone. The peptide was also isolated from HLA-B*5101 molecules purified from C1R-B*5101 cells. In the present study, we directly demonstrated that a human alloreactive CTL clone recognizes peptide presented by HLA class I molecules.

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.

Incompatibility of human minor histocompatibility (hmH) antigens induces rejection of grafts in organ transplantation and graft versus host disease in bone marrow transplantation if donor and recipient are matched for human leukocyte antigen (HLA) genes. These antigens are recognized only by T cells. We describe here the isolation of hmH peptides recognized by a hmH antigen specific, HLA-B35 restricted CTL clone which was derived from a patient who rejected the kidneys from two HLA-identical sisters. Naturally occurring hmH peptides were isolated from a donor derived B cell line and an HLA-B35 transfected human B cell line by acid elution. Analysis of various HLA class I transfectant cells demonstrated that MHC class I molecules themselves determine the peptides which are naturally processed and presented to T cells.

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.

Phagocytosis of Staphylococcus aureus by human peripheral polymorpho-nuclear leucocytes in vitro in the presence of fetal calf serum was inhibited by D-galactose, N-acetyl-D-glucosamine and asialofetuin but not by fetuin. Absorption of opsonin in the serum with the bacteria, inhibition of the absorption by sugars and the effect of glycosidase treatment of the bacteria on phagocytosis showed that terminal structures of polysaccharides on the surface of bacteria play an important role in binding of opsonin to the target.

1. 1. A novel lectin (Achatinin) was isolated from the body-surface mucus of African giant snail, Achatina fulica Ferussac. 2. 2. The lectin recognizes N-acetylneuraminic acid preferentially and also other N-acetyl aminosugars to some extent, while it does not bind to N-glycolylneuraminic acid. 3. 3. The molecular weight of the lectin estimated by SDS polyacrylamide gel electrophoresis was 78,000. 4. 4. The lectin requires Ca2+ for its haemagglutinating activity. 5. 5. The isolated lectin was free of antibacterial activity which has been detected in the body-surface mucus.

Various solvents (guanidine hydrochloride, normal propanol, urea, and sodium carbonate), previously demonstrated to solubilize Trichoplusia ni granulosis virus capsules, produced alterations which were characteristic for a given solvent. All solvents, with the exception of guanidine hydrochloride (G·HCl) caused longitudinal fragmentation of the proteinic crystal early in the solubilization process. G·HCl, in contrast, appeared to be capable of penetrating and producing small cavities within the crystal. Intact free virions were obtained only from carbonate-solubilized capsules. Capsules seemed to dissolve in carbonate by a sequential process. Evidence consistent with the occurrence of normally distributed differences in carbonate solubility among capsules is presented. The pattern of carbonate solubilization of the proteinic crystal and release of free virions most closely resembled the process in vivo as observed within the midgut lumen of larval Trichoplusia ni.

Methods for the solubilization of an insect granulosis virus capsule from Trichoplusia ni were studied using turbidity measurements. The solubilization in the solvents employed was most strongly influenced by pH. Various alcohols and hydrogen bond cleaving agents also enhanced solubilization. The most effective solvent tested at pH′s close to neutrality was 60% n-propanol saturated with guanidine. HCl. The effectiveness of this solvent was minimal at pH 5.2. It was shown that the time courses of the solubilization were generally composed of three phases. A plot of the third phase levels versus pH indicated a normal distribution of those capsules or capsular fragments in resisting solubilization. Validity of the calculation of rate constants for the dissociation reaction in the second phase was discussed. Logarithm of the rate constant showed a linear relationship with the reciprocal of absolute temperature. The activation energy for the dissociation calculated from this relationship using the Arrhenius equation indicated that hydrophobic and hydrogen binding forces play the major roles for the stability of the crystalline structure of the capsular protein. Inic binding forces were estimated to be of minor importance.

The protein solubilized from the proteinic crystalline structure surrounding the granulosis virus of Trichoplusia ni by use of a carbonate buffer (pH 10.7) gives a major component, as analyzed by ultracentrifugation, with a molecular weight of 180,000. This protein has heterogeneous subunit structure as demonstrated by estimates of molecular weights by use of gel electrophoresis, amino-, and carboxy-terminal analyses, and peptide mapping of enzyme digests of the protein. The amino acid composition shows that the protein is acidic with a high percentage of amino acids with hydrophobic side groups. Optical rotatory dispersion studies reveal the presence of β-structure in the protein complex. The conversion of the β-structure to α-helix with sodium lauryl sulfate and to a random coil state with strong alkaline treatment are observed.

The mutant nusA DNAs (nusA11 and nusA1) were sequenced. Single base substitutions caused by these mutations were found in the coding region of nusA. The nusA11 mutation, which is conditionally lethal, substituted Thr for the 181st Ala. Also, nusA1, which restricts lambda growth, substituted Ala for the 183rd Ser. These two positions were located in the same hydrophobic amino acid cluster. This cluster seemed to be an essential region in the functional domain of NusA. In the course of these experiments, several mistakes in the published nusA nucleotide sequence were found. These errors are revised in this article. The molecular weight of NusA is accordingly revised to 54,430.

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.