MHC class-I antigen deficiency, malignancy and susceptibility of P815 mastocytoma to NK and macrophage killing.

A subline of the murine P815 mastocytoma passaged for a long period of time in histoincompatible hosts was found to be deficient in surface MHC class-I antigens by serological and biochemical methods. In agreement with the lack of restriction elements, this variant cell line was no longer susceptible to killing by cytotoxic T cells stimulated by and directed against the parental tumor cell line in syngeneic DBA-2 hosts. We did not observe the appearance of susceptibility to NK killing concomitantly with H-2 loss. We thus could not confirm the hypothesis of a regulatory function of H-2 structures in NK recognition/killing. Both cell lines were also resistant to lysis by mouse macrophages. The parental cell line was sensitive to rat macrophage killing, whereas the variant line had lost such sensitivity. In spite of resistance in vitro to various defense mechanisms, the variant H-2 loss tumor cell line was less tumorigenic in syngeneic hosts and exhibited a lower metastatic capacity than the parental cell line. We propose that in the H-2 loss subline, survival of cells in vivo is impeded since positive signals mediated by H-2 structures are missing, which are necessary for cell-cell contact and survival.

Role of CD8 (Lyt-2) in cytotoxic T-cell function. Analysis of variants of a cytotoxic T-cell clone with reduced Lyt-2 expression.

The role of CD8 (Lyt-2) in the function of a long-term cytotoxic T-cell (CTL) clone (Cl96) was analysed. Previous studies had shown that C196 cells utilize the alpha beta T-cell receptor and depend on Lyt-2 to recognize and lyse P815 mastocytoma target cells. Recognition is H-2Kd-restricted, presumably involving a P815 specific antigenic structure. Here we analyse a number of variants selected from Cl96 that have reduced or virtually abolished expression of Lyt-2, alone or in combination with other deficiencies. We studied the ability of these variant cells to lyse either P815, a process requiring both specific antigen recognition and triggering of cytolytic function, or to lyse the anti-CD3 hybridoma 145-2C11, a process that requires the triggering of cytolytic function only. The results shows that in the case or a permanently activated CTL effector cell such as Cl96, Lyt-2 is required for antigen recognition but is not essential for the triggering of cytotoxicity.

Sequential determination of circulating immune complexes during progressive and regressive phases of mouse mastocytoma.

Circulating immune complexes were determined by 125I-C1q-binding and Raji cell-binding sequentially during distinct phases of progression and regression of a weakly immunogenic murine tumour. No increase in levels of circulating immune complexes was found at any time during tumour development, although reference complexes formed between tumour cell membrane antigens and a murine histocompatibility antigen-directed alloantibody were easily detected by both tests. These findings parallel the absence of humoral antibody during tumour development but are in some contradiction to the pronounced B cell proliferation which was observed in this tumour model. The results add evidence for a more critical evaluation of the role which levels of circulating immune complexes might play in tumour diagnosis and prognosis.

Proliferative patterns of lymphocytes in lymph nodes during tumour development: involvement of T and B cell areas.

DNA-synthetizing lymphocytes were identified in the lymph nodes regional and more distal to the site of developing P-815 tumours by incorporation of [3H]-thymidine followed by autoradiography of lymph node sections. It appeared that not only T but also B cell areas of draining and to a lesser extent of distal lymph nodes were stimulated by the growing tumour. This result was unexpected since neither humoral nor tumour cell-bound antibody could be identified so far as a functional correlate of B cell stimulation. In general the proliferative response of lymphocytes followed a biphasic pattern with an early peak of reactivity on days 2-3 and a second peak around day 12-15 after tumour cell inoculation. In the draining (axillary) lymph node the second peak of reactivity was suppressed, possibly as a consequence of metastatic tumour cells in this node when tumour cells were inoculated in the flank. The pattern of lymphocyte stimulation revealed larger individual variations after tumour cell inoculation in the flank than the foot pad. These results were associated with a slower and less regular drainage of carbon particles from the flank to the axillary and exceptionally the brachial lymph node than from the foot pad to the popliteal node after injection of India ink.

Stimulation of different pathways of T-cell functions by syngeneic tumor cells and soluble membrane proteins.

Cells from the draining lymph nodes of DBA/2 mice bearing syngeneic intradermal P-815 tumors represent an excellent responding cell population for secondary stimulation in vitro, despite the virtual absence of response by spleen cells of the same animals. Cytotoxicity is a result of stimulation with intact mitomycin-treated tumor cells. Isolated tumor cell membranes, in the form of small vesicles, stimulated cytotoxicity to a very limited extent and inhibited the development of cytotoxic T lymphocytes over a wide range of concentrations. Membrane proteins solubilized with deoxycholate and papain had only a suppressive effect. Soluble proteins exerted their effect during the induction of cytotoxic T lymphocytes and were ineffective when present during the effector phase of cytotoxic T lymphocytes. The suppressive capacity was shown to reside in a cell population which was sensitive to treatment with monoclonal Lyt-2.1 antibody and complement but not with Lyt-1.1 antibody. This phenotype was compatible with a specific rather than a promiscuous type of suppressor effector cell.

Suppressor T cells and the regression phase of syngeneic intradermally developing p-815 mastocytomas.

Thymus cells obtained 6, 8 or 10 days after the intradermal injection of P-815 tumor cells into syngeneic DBA/2 mice were, upon adoptive transfer into new hosts, incapable of abrogating the normally observed tumor regression phase. Transfer of cells from day 8 revealed a tendency toward suppression of regression phases; cells from day 6 or 10 were inert or had a rather immunopotentiating effect. Pretreatment of tumor hosts with cyclophosphamide did not result in more pronounced regression phases, nor did this treatment raise the percentage of surviving animals. Injection of cyclophosphamide after tumor cell inoculation resulted in a slight delay of tumor growth, but not in more pronounced regression nor in higher survival rates. Taken together the data suggest that in a weakly immunogenic and highly malignant tumor, suppressor T cells may play a minor role in the subversion of the host's immune response.

The stimulation of a T cell response in the syngeneic host by the P-815 mastocytoma cell and the isolation of a tumor-associated antigen which has some H-2 antigen characteristics.

Although not obviously immunogenic when developing intraperitoneally or subcutaneously, the P-815 mastocytoma cell induces a significant immune response when injected intradermally into the syngeneic host. In some animals the immune reaction leads to spontaneous tumor regression and survival of the animals, but in most cases the effectively initiated immune-reaction is abrogated by as yet unknown mechanisms. The T cell is identified as the killer cell both in vivo and in vitro during the described period of immune reactivity. By means of two antibodies from two different species, rendered specific for the P-815 tumor cell by in vivo absorption in DBA/2 mice, two distinct antigens have been identified, isolated and partially purified from P-815 cell membranes. One of them is obviously different from H-2 antigens, the other one shows striking similarities with H-2 antigens (molecular weight, lectin binding properties), but does not coprecipitate with H-2 antigens in an indirect precipitation assay, using Staph. aureus Cowan I as the Fc-binding agent.

Problems relating to immunoselection of leukemias.

Problems relating to immunoselection of neoplastic, in particular leukemic, cell lines are reviewed. Since there is ample evidence that specific immune reactions of the host against malignant neoplastic cells do occur, it becomes important to consider the effectiveness and the relevance of immunity in suppression or elimination of neoplastic growth. Emphasis is placed on experimental results obtained in syngeneic tumor-host combinations, because they more closely resemble the situation of spontaneous tumorigenesis or leukemogenesis than xenogeneic or allogeneic model systems. Studies of types of neoplasia observed in cases of human immunodeficiency syndromes offer an important insight into problems involved in immunoselection of leukemic cell lines: the marked predominance of leukemias and lymphoreticular neoplasias in immunodeficient patients invites speculation on both the mechanisms of leukemogenesis and the relative importance of the immune system in eliminating malignant neoplastic cells.