Takae Tanino

Detection of allogeneic Qa/Tl and Ly specificities on murine tumor cells with IgD in tumor-regressor serum

SummarySerum 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.

Recognition of the Qa-2k tumor antigen by T cell receptor γ/δ of an immunopotentiator-induced tumoricidal T cell of mice

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 withP. 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 α1 and α2 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 fromP. acnes-stimulated (C3H/He × 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 α1/α2 region of the Q7b gene product was demonstrated. The cytotoxic activity was dependent on T cells bearing T cell receptors of the γ/δ type (TCRγ/δ). The (C3H/He × 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 α1/α2 region of the Qa-2k tumor antigen by TCRγ/δ.

Protection against metastasis by immunization with an allogeneic lymphocyte antigen

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.

Agglutination of murine tumor cells by sera from various cancer patients

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.

Agglutination of murine tumor cells by sera from patients with autoimmune diseases.

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 cance