Experiments were carried out to study the sequential events that follow the immunization of mice. To accomplish this goal, a reliable in vitro lymphoid-cell, spontaneous-proliferation assay was established. Mice were immunized with 2,4-dinitrophenylbovine serum albumin (DNP-BSA) or DNP-OVA albumin (OVA) in saline and subsequently received a secondary immunization at various intervals thereafter. 24 h after challenge, popliteal lymph node cells (PLN) from these mice were cultured in vitro for 24 h with 1 μCi of 3H-thymidine (3H-TdR). Kinetic studies revealed that carrier-specific, peak spontaneous proliferation occurred on day 6 following immunization, whereas no proliferation occurred on day 10. The proliferating cells were T cells. Prior treatment of mice with cyclophosphamide (CY) 3 days before immunization prolonged the ability of PLN CY from immunized mice to proliferate on day 10 after immunization. Addition of 5 × 103 thymocytes from day-10 DNP-BSA or DNP-OVA immunized mice to 1 × 105 PLN CY cells from day-10 immunized mice with homologous antigen caused a marked inhibition of the uptake of 3H-TdR, in contrast to thymocytes from day-6 immunized mice or from day-10 immunized mice that had been treated with 100 mg/kg CY 3 days before immunization. Our results suggest that the natural sequence of events following immunization with either DNP-BSA or DNP-OVA without adjuvant involves proliferation of T-lymphoid cells, which are subsequently turned off by a separate subset of thymus-derived suppressor T cells. These suppressor cells were found to be carrier-specific and CY-sensiti
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