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In vitro lymphocyte stimulation by mitomycin-blocked tumor cells can be used to measure tumor-specific immune responses. In order to determine the responding cell type(s) in this reaction, lymph node and spleen cell populations were specifically depleted of thymus- or bone marrow-derived cells by the use of the appropriate antisera and complement or by immunoadsorption of the Fc receptor-bearing cells to antibody-coated sheep red blood cell monolayers. The compositions of both the original and the modified lymphocyte populations were determined by (a) viability counting following treatment with antisera and complement, (b) direct and indirect immunofluorescence, (c) antibody-coated erythrocyte rosette formation, and (d) response to thymus- and bone marrow-derived cell mitogens. In the lymph node cell populations, only the thymus-derived cells were stimulated by the tumor cells. However, both bone marrow- and thymus-derived cells from tumor-immune spleens underwent stimulation when exposed to tumor cells in culture.

In vitro lymphocyte stimulation by mitomycin-C-blocked tumor cells has been used to demonstrate tumor-specific antigens in syngeneic murine systems and to follow the evolution of tumor immunity with the tumor-bearing state. Mitomycin-blocked tumor cells stimulated syngeneic lymphocytes from normal mice, from those bearing small tumors (less than 1 cm in diameter) and from tumor-immune mice, sensitized by tumor-cell inoculation and subsequent tumor removal, to undergo increased DNA synthesis as measured by the incorporation of tritiated thymidine. However, lymph-node cells from mice bearing tumors over 1 cm in diameter appeared to be maximally stimulated in vivo and incapable of further stimulation by the same tumor cells in vitro. This was reflected by the progressively increasing background levels of nucleic acid synthesis with the length of tumor-bearing and the size of the tumor. Although lymph-node cells from mice with large tumors did not respond to the same tumor cells in vitro, they did have normal responses to PHA. Within 7-14 days of surgical removal of the tumor, specific lymphocyte responsiveness and background activity returned to previous normal levels, but reinoculation with 10-6 tumor cells resulted in progressive tumor growth and loss of specific in vitro responsiveness when the second tumor had reached the critical size of 1 cm in diameter. Brief exposure of tumor-immune lymph-node cells to a soluble antigen extract of the same tumor resulted in a marked increase in DNA synthetic activity compared to that obtained after exposure to a different tumor extract, muscle extract or medium alone underwent stimulation when cultured with mitomycin-blocked tumor cells. However, normally responsive tumor-immune lymph-node cells, after brief exposure to a soluble antigen extract of the same tumor, initially underwent increased DNA synthesis, but were incapable of further stimulation by mitomycin-blocked tumor cells. Tumor antigen, alone or complexed with antibody, was also demonstrated in the sera of mice bearing large tumors and is thought to be responsible for the refractoriness of lymph-node cells from these mice to further stimulation in vitro. These experiments demonstrate that tumor size and the consequent antigen load to which the tumor-bearing animals is subjected have a profound effect on tumor-specific lymphocyte responsiveness.