Cyclophosphamide-inducedAppearanceof ImmunopotentiatingT-Cells in the Spleens of Mice Bearinga Large MOPC-315Tumor1

Following low-dose cyclophosphamide (CY) therapy (15 mg/ kg) of mice bearing a large MOPC-315 tumor, the suppressive activity of their Sephadex G-10-adherent spleen cells (primarily macrophages) is overcome. Accordingly, when Sephadex G-10-adherent spleen cells from CY-treated tumor-bearing mice are added to the in vitro immunization culture of normal spleen cells, they do not suppress but actually bring about the generation of an augmented level of antitumor cytotoxicity. The ability to enhance the generation of antitumor cytotoxicity appears in the Sephadex G-10-adherent spleen cell population by Day 5 post-CY therapy of tumor-bearing mice and persists for at least 55 days; no such immunopotentiation is observed following admin istration of a low dose of CY to normal mice. In order for the immunopotentiating cells from CY-treated tumor-bearing mice to be effective in enhancing the generation of antitumor cytotoxicity, they must be added to the immunization culture of normal spleen cells no later than Day 3 (out of the 5 days) post-culture initiation. The CY-induced immunopotentiating activity resides in the T-cells, as is evident from the following observations. The immu nopotentiating activity was abolished when the Sephadex G-10-adherent spleen cell population from CY-treated tumor-bearing mice was depleted of T-cells by anti-Thy 1.2 plus complement but not when this adherent spleen cell population was depleted of macrophages by carbonyl iron and magnet. Moreover, the immunopotentiating activity was also present in a population of CY-treated tumor-bearer spleen cells highly enriched for T-cells by passage through nylon wool columns. Thus, low-dose CY therapy overcomes the immunosuppressive activity of macro phages and induces the appearance of T-cell-mediated immu nopotentiating activity, thereby leading to the development of an augmented level of antitumor cytotoxicity that can cooperate effectively with the tumoricidal activity of CY in the eradication of a late-stage, large s.c. tumor and extensive mÃ©tastases. of an augmented level of antitumor cytotoxicity. These results indicate that the immunopotentiating activity of the adherent spleen cells from CY-treated tumor-bearing mice is not due to addition of mature cytotoxic cells, and the adherent cells have to be present in the culture for at least 2 days to bring about the generation of an augmented level of antitumor cytotoxicity. It is possible, however, that the adherent spleen cells from the CY-treated tumor bearers contain memory cells for which 2 days of in vitro immunization [instead of the 5 days needed for optimal generation of antitumor cytotoxicity by normal spleen cells are sufficient for the generation of a potent secondary antitumor cytotoxic response It should be emphasized, however, that a greatly aug mented level of antitumor cytotoxicity was generated even when the adherent, CY-treated tumor bearer spleen cells that were to the immunization culture of normal spleen cells consti tuted only 10% or less of the spleen cell mixture. Since the concentration of T-cells in the adherent spleen cell population is 10% even if we assume that all of the T-cells in the adherent population are immunopotentiating cells, the adherent T-cells added to the in vitro immunization culture of normal spleen cells represent, at best, just 1% of the total spleen cell mixture. In light of the low frequency of the adherent splenic T-cells from CY-treated tumor bearers in the in vitro immunization culture of normal spleen cells, it is more likely that they act as