Thymic anti-tumor effectors in mice cured of lymphoma by cyclophosphamide plus TNF-alpha therapy: phenotypic and functional characterization up to 20 months after initial tumor inoculation.

As reported previously, cyclophosphamide plus tumor necrosis factor-alpha treatment of C57BL/6 mice bearing advanced EL4 lymphoma induced approx. 60% long-term (i.e., >60 days) survivors. These mice developed protective immunity, as evidenced by 1) rejection (100% survival) of EL4 tumor re-implanted on day 60 (day 0 = initial tumor implantation); and 2) development of significant levels of specific EL4 tumor cell killing activity by both splenocytes and thymocytes. Using this model, age-related changes in functionally and phenotypically definable thymocyte subsets were assessed. In thymocytes from 90 to 308 day survivors, specific immune memory was long term; both CD4+ and CD8+ cells were required for the ex vivo stimulation of lytic activity, but the specific anti-EL4 cytotoxic effector was CD4-CD8+. On day 520, the surviving mice were randomized into 2 groups. One group received a second re-challenge with EL4 tumor cells and all survived. The other group was sacrificed on day 520. Their thymocytes, exposed to X-irradiated EL4, developed anti-EL4 lytic activity and, in comparison with thymocytes of young and age-matched control mice, were markedly enriched in CD4-CD8+CD44+ cells. On day 625, thymocytes from the survivors of the day 520 re-challenge were evaluated and were found to have developed specific anti-EL4 lytic activity. Phenotypically, they had returned toward the pattern seen in age-matched control mice although CD4-CD8+CD44+ cells remained increased. These mice were > or = 2 years old, the median life span of C57BL/6 mice. Thus, mice cured of tumor by an immuno-modulating regimen rejected re-implanted primary tumor and maintained specific thymic anti-tumor immune memory for life.

Cyclophosphamide plus tumor necrosis factor-alpha chemoimmunotherapy cured mice: life-long immunity and rejection of re-implanted primary lymphoma.

Changes in functionally and phenotypically definable splenocyte subsets in aging mice which had been rendered tumor-free in early life by immunochemotherapy (cyclophosphamide plus tumor necrosis factor-alpha) were studied in the syngeneic EL4 lymphoma-C57BL/6 murine model. Treatment-induced long-term survivors (LTS) surviving rechallenge are termed "immune-LTS". On day 120 (day 0, initial tumor inoculation), splenocytes from day 60 rechallenged immune-LTS developed significantly greater specific anti-EL4 cytolytic activity in an ex vitro assay than those from non-rechallenged LTS. Splenocytes from combination-treated groups developed significantly higher activity than those from cyclophosphamide-induced immune-LTS. The splenic effector precursor was a CD8+ T cell. The specific anti-EL4 effector cell from the cyclophosphamide-induced immune-LTS was CD4- CD8+; however, approximately 50% of those from combination-treated immune-LTS appeared to be CD4+CD8+. On day 520 immune-LTS were randomized into 2 groups. One group was re-implanted with EL4 tumor; all mice survived. The other group was killed and, even though their splenocytes developed considerable anti-EL4 activity, their allogeneic responsiveness was as reduced as that of age-matched controls. Phenotypic analysis, compared with splenocytes from young and age-matched controls, revealed changes in the makeup of each T-cell subset, except the CD4+CD8+, and all subsets, except the CD4-CD8-, had increases in CD44 positivity. On day 625, the age of these mice was equivalent to the median life-span of C57BL/6 mice; nevertheless, their splenocytes developed high anti-EL4 activity. Phenotypic analysis indicated that, compared to day 520, there was a major decrease in CD4-CD8+ splenocytes; we suggest that these cells had migrated to the site of tumor eradication.

Protective specific immunity induced by cyclophosphamide plus tumor necrosis factor alpha combination treatment of EL4-lymphoma-bearing C57BL/6 mice.

A combination treatment protocol initiated 12 days after tumor injection, when the tumor was large, by administering cyclophosphamide (CY, 150 or 250 mg/kg) intraperitoneally followed by intravenous tumor necrosis factor alpha (TNF alpha, 1000 units injection) on days 13, 16, 18, 21, and 23, resulted in about 60% long-term survival (i.e., survival for at least 60 days) in the syngeneic C57BL/6 mouse/EL4 lymphoma model system. The establishment of a specific antitumor immune memory and its possible therapeutic relevance was verified by reinjecting 60-day survivors with EL4 cells; all 60-day survivors that had received the combination treatments rejected the implants and survived for a further 60 days. Thymic cellularity was reduced during treatment and its recovery appeared to correlate with long-term survival and immunity. Thymocytes from mice treated with the combination were found to express significant levels of specific anti-EL4 cytolytic activity following a 4-day stimulation culture with X-irradiated EL4 cells and low concentrations of interleukin-2. This response could not be generated with thymocytes from naive animals. In each case the effect seen with the combination of a moderate CY dose (150 mg/kg) with TNF alpha was better than that seen with either dose of CY alone and equal to or better than that seen with the higher dose of CY combined with TNF alpha. These results indicate that treatment with a single moderate dose of CY in combination with TNF alpha is effective against a large, established tumor in this murine model. Furthermore, all the long-term survivors induced by this treatment developed protective immunity against reimplanted tumor and demonstrated a long-term specific immune memory in the thymus.