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A spontaneously arising murine plasmacytoma, HPC-202, derived from a BALB/c.H-2b congenic mouse that lacks any detectable H-2 determinants on its cell surface is described. However, the expression of H-2 determinants is inducible by interferon-gamma. The H-2 negative cell surface phenotype permits the HPC-202 tumor to escape H-2 allospecific cytotoxic cell lysis but not NK cell lysis, as well as to grow, to varying degrees, in some H-2 incompatible hosts. In those strains which exhibit a resistance to HPC-202 growth, resistance does not map to a single gene within the major histocompatibility complex of the mouse. Resistance is also radiosensitive and is therefore presumably due to a rapidly dividing cell population. The utility of this tumor as a model system to study both the non-H-2-restricted natural resistance to tumor growth, and the mechanism by which H-2 genes are regulated by cells is discussed.

Previous studies have clearly established that murine plasmacytomas suppress B-cell responses, but no effects on T-cell responses have been noted by most investigators. However, in our investigation of the tumor-associated antigens of plasmacytomas, we have found that immunosuppression plays an important role in the complex interaction between host immune T cells and tumor cells. In these investigations, varying the number of plasmacytoma cells added to in vitro induction systems separated two different effects which these cells had on the generation of cytotoxic T cells. On the one hand, when appropriate numbers were present, tumor cells acted as immunogens and stimulated the generation of specific killer cells against tumor-associated antigen or alloantigen which they expressed. In contrast, greater numbers of tumor cells exerted a profound inhibitory effect on the generation of cytotoxic activity when cells inactivated with irradiation were used. Mitomycin-C inactivation of plasmacytoma cells abrogated this inhibitory activity. In further experiments, MPC-II cells that had been inactivated with Mitomycin C were able to prime T cells in vivo, whereas priming by live tumor cells could not be detected in the same situation. It is suggested that immunosuppression by the live tumor cells may account for their inability to prime T cells in vivo. These results indicate that immunosuppression by plasmacytomas may be one of the important variables which influence the generation of cytotoxic T cells against tumor-associated antigens both in vivo and in vitro.

Tumor-associated antigens (TAA) were demonstrated on plasmacytomas derived from BALB/c, NZB, and C3H mouse strains, by in vivo and in vitro techniques. By immunizing the appropriate F1 hybrid mice with these tumors, it was possible to show that all the plasmacytomas expressed cross-reactive tumor-associated transplantation antigens. When cytotoxic lymphocytes (CL) were induced in vitro by the coculturing of syngeneic or F1 hybrid spleen cells with irradiated plasmacytoma cells, "shared" and "unique" plasmacytoma TAA were demonstrable. This was accomplished by inducing CL in vitro against one syngeneic plasmacytoma and assaying for lytic activity on a range of 51Cr-labeled BALB/c, NZB and C3H plasmacytoma cells in vitro. In a second in vitro assay, unlabeled plasmacytoma cells were tested for their ability to inhibit the lysis of a particular 51Cr-labeled plasmacytoma, with the use of CL induced in vitro against it. The possibility that these TAA were "self" antigens was excluded by demonstrating in the inhibition assay that they were not present on T lymphomas and spleen cells of the same strain, and that CL "autosensitized" in vitro could not significantly lyse 51Cr-labeled plasmacytoma cells in vitro. From both in vivo and in vitro studies of immunity to these tumors, it was concluded that any one plasmacytoma line possesses multiple TAA of both shared and unique types.

Cytotoxic T-lymphocytes (Tc) were induced in vitro to plasmacytoma tumor-associated antigens (TAA) by coculture of irradiated cells of plasma cell tumors (PCT) from NZB mice and viable nonimmune or PCT-immune spleen cells from NZB. (NZB X C57BL)F1, (NZB X B10.D2)F1, and (NZB X B10,BR)F1 mice. When nonimmune spleen cells were used, major histocompatibility complex (MHC)-linked genetic control of te primary in vitro induction of Tc to NZB PCT was demostrated for a shared TAA expressed on NZB and BALB/c PCT. Evidence was also obtained for a non-MHC-linked genetic control of the primary in vitro induction of Tc to a second TAA that ws expressed on both PCT and T-lymphomas. When the spleen cells were obtained from mice preimmunized to an NZB PCT, a secondary in vitro Tc response was observed, and a PCT-specific and strain-specific TAA or NZB mice was identified. In addition, results with the F1 hybrids also indicated an MHC-linked genetic control of the in vitro Tc response to this strain-specific TAA.

A tumour-associated antigen has been identified which is expressed on both BALB/c plasmacytomas and T lymphomas, but which does not induce a detectable cell-mediated immune response in the strain of origin of the tumours. The (BALB/c x C57BL) F1 hybrid, however, makes a readily detectable response both in vivo and in vitro in vitro experiments with various BALB/c H-2 F1 cogenic mice indicated that the genetic control of the response was not H-2 linked, and was associated with other C57BL genes.

In previous studies with in vitro activated cytotoxic T lymphocytes, we have demonstrated the presence of oncofetal antigens (OFA) on a range of murine tumor cells. The present studies with the same tumor lines attempt to determine whether these antigens are also capable of activating lymphocyte responses in vivo. Several experimental designs were followed, each being performed many times (1). Preimmunization of mice with irradiated fetal liver cells and followed by challenge with viable tumor cells did not consistently produce a state of tumor resistance. However, injection of live fetal cells frequently led to enhanced tumor growth (2). The growth of tumors in multiparous mice can be inhibited, in contrast to controls, but this effect was relatively short lived after parturition (3). Preimmunization with fetal cells in vivo did not result in augmented secondary cytotoxic T cell responses in vitro (4). Immunization of mice with irradiated tumor cells frequently led to a state of resistance to the clonal growth of hemopoietic fetal cells, although again the level of resistance was usually relatively weak. From the overall results, we conclude that OFA are relatively poor immunogens on tumor cells or fetal cells in vivo, and in contrast to in vitro responses, do not act as potent tumor transplantation antigens.

Continuous cytotoxic T cell lines against murine tumor-associated antigens (TAA) were generated from normal BALB/c and (C57BL/6 X BALB/c)F1 spleen lymphocytes by sensitizing these cells in vitro to the BALB/c plasmacytoma (PCT) MPC-11. These cytotoxic T cell lines (CTLL) were maintained for periods of over 1 yr in T cell growth factor from cultures of mitogen-stimulated rat spleen cells. The lines were highly cytotoxic against the immunizing tumor, as well as against a select number of other tumor cell lines, and the patterns of reactivity of 3 of these lines against the various tumor cell targets indicated that these lines each recognized different PCT TAA. The antigens recognized included an MHC-restricted PCT antigen, an antigen shared by PCT and T lymphomas, and an MPC-11 unique PCT antigen. phenotypic characterization of the CTLL populations for cell surface marker expression indicated that they were largely homogeneous with respect to their differentiation state, and expressed Thy-1.2, Lyt-2, Lyt-3.2, and T30 antigens on their cell surface. In addition, 1 of the lines demonstrated variable low-level expression of Lyt-1. Thus, the populations were characteristics of mature T cells, and did not express cell surface markers characteristic of other lymphocyte subpopulations.

The relationship between cell surface antigen expression and function in cytotoxic T cells directed against a non-H-2-restricted plasmacytoma antigen was examined using Lyt-2- variants of a continuous T cell line. This cytotoxic cell line originally had expressed Lyt-2 on all the cells as detected by flow microfluorometry, but after several months of culture, Lyt-2- variants spontaneously developed. Using cell sorting and limiting dilution cloning, Lyt-2- and Lyt-2+ cell lines were obtained, cultured and analyzed. These analyses indicated that such cytotoxic cells had similar activity regardless of Lyt-2 phenotype. This observation supports the concept that the Lyt-2 molecule is not obligatory for recognition and killing by cytotoxic T cells and emphasizes the need to clarify the precise relationship between cell surface phenotype and T cell function.

Many recent studies have demonstrated that cytotoxic T lymphocytes (CL) activated to various antigens other than those of the H-2 complex, will lyse target cells only when H-2 compatibility exists between the CL and target cell. From these observations, it has been inferred that T lymphocytes might only be capable of responding to H-2 antigen or antigens that become associated with H-2 region gene products. Our results suggest that this is not the case, and that in some situations, cytotoxic T lymphocytes can specifically lyse target cells of different H-2 types. Two in vitro systems are described where primary induction of cytotoxic T lymphocytes to oncofetal and plasmacytoma antigens results in CL capable of lysing suitable targets bearing these antigens, of either syngeneic or allogeneic derivation. Thus it is proposed that although interaction antigens involving H-2 components may preferentially activate T lymphocytes, this does not imply a restriction on the recognition potential of T lymphocytes.

In this study, a large series of murine B lymphomas and plasmacytomas were examined by quantitative flow cytometry analysis using a panel of monoclonal antibodies against murine differentiation antigens. These cell lines appear to represent subpopulations of lymphoid cells arrested at distinct stages of differentiation. In general, the pattern of reactions of these monoclonal antibodies with the B cell neoplasms was comparable to the reactions seen with normal cells in the same lineage. The Thy-1.2, Lyt-2, and T-30 differentiation antigens were not detected on any B lymphoma or plasmacytoma. However, certain surface Ig-positive B lymphomas do express the Lyt-1 antigen. With respect to other differentiation markers examined, including E2, F1, ThB, Lgp-100 (Ly 9.1), G2, and T-200 (Ly 5), the reaction of the B cell tumors reflected the expression of these markers on comparable normal cells. This investigation also emphasized the marked intratumor and intertumor heterogeneity that is observed when cloned cell lines are analyzed quantitatively for a large number of surface markers. Thus, this approach may be particularly useful in defining heterogeneity in maturation states within cloned tumor cell lines.

As a part of our continuing investigations directed toward defining the nature of the antigens on murine plasmacytomas (PCT), MPC-11 and two immunoglobulin (Ig) synthesis variants of this cell line were examined for H-2 and tumor-associated antigen (TAA) expression, and for the possible role of H-2 in the cytotoxic T lymphocyte response to TAA. The tumor lines studied were MPC-11 (an IgG2b producer), MPC-11,662 (a variant which produces only light chains), and NP.2 (a variant which produces neither Ig chain). Both H-2 expression, which was analyzed by flow cytometry and by cold target cell blocking of the activity of cytotoxic T lymphocytes (CTL) generated in allogeneic cultures, and TAA expression, which was analyzed by cold target cell blocking of the activity of tumor-specific CTL generated in syngeneic cultures, were found to be quantitatively similar on the three lines. When a possible association between H-2 and TAA expression was examined by testing the ability of anti H-2 sera to block tumor-specific CTL directed against MPC-11, no association between H-2 and TAA was detected. The results of these studies provide new information about H-2 and TAA expression on murine PCT. Furthermore, they suggest that changes in H-2 and TAA expression which have been reported to sometimes accompany loss of Ig synthesis may represent independent genetic, phenotypic, or metabolic variations, rather than alterations linked to the loss of Ig synthesis. In addition, these results, in agreement with out previous studies on MPC-11, support the concept that there is not an obligatory association of TAA with H-2 on the surface of murine PCT. Because these results differ from those reported in some other systems in which an association between H-2 and TAA on murine PCT was demonstrated, it must be emphasized that the TAA of PCT appear to be quite heterogeneous, and more investigations will be necessary in order to clearly define these antigen and the T-cell response to each of them.