Effects of Cancer Immunotherapy with Indomethacin and Interleukin-2 on Murine Hemopoietic Stem Cells 1

We examined: (a) whether in vitro-generated lymphocyte-activated killer (LAK) cells from normal mice and splenic killer cells from tumorbearing mice subjected to interleukin-2 (IL-2) therapy alone or in combination with chronic indomethacin therapy have any detrimental effects on the spleen colony-forming units (CFU-S) of the normal bone marrow (BM); and (b) the effects of these immunotherapy protocols on CFU-S numbers in host hemopoietic organs. Effects of in vitro-generated LAK cells (normal C3H/HeN mouse splenocytes cultured with 1000 units IL-2/106 cells for 72 h) on BM CFU-S were examined by incubating macrophage-depleted BM cells with LAK cells at 1:2.5 and 1:5 BM:LAK cell ratios or with LAK cell supernatant for 4 h. The cells were washed and subsequently injected into irradiated mice. Irradiated mice were also reconstituted with BM cells or LAK cells incubated alone. Spleen colonies were scored macroscopically and microscopically on day 7 after reconstitution of lethally irradiated mice with the various cell combinations. A comparison of colony numbers produced by LAK and BM cell mixture revealed that LAK cells at either dose had no suppressive effect on the colony-forming ability of BM at the macroscopic and microscopic levels of analysis. The supernatant of cultured LAK cells had a minor suppressive effect on colony formation at the macroscopic but not the microscopic level of analysis, indicating the presence of one or more suppressive factors capable of mediating a short-term inhibitory effect. In the immunotherapy experiment, C3H/ HeN mice transplanted s.c. with 5 x 105 C3L5 mammary adenocarcinoma cells received either vehicle alone (controls), IL-2 (1.5 x 104 Cetus units i.p. every 8 h on days 10-14 and days 20-25), or chronic indomethacin therapy (10 ~tg/ml in drinking water from day 5 onwards) plus IL-2 as above. Animals were killed 24-25 days after tumor transplantation to examine: (a) the number of metastatic lung nodules; (b) the effects of co-incubating therapy-generated splenic effector cells with normal BM cells for 4 h on BM CFU-S, and (c) the CFU-S content of host BM and spleen. Results revealed a drop in spontaneous lung metastases from a mean of 50 in control mice to 18 with IL-2 therapy alone, and to 5 with chronic indomethacin therapy plus IL-2 therapy. Splenocytes from normal and tumor-bearing control or treated mice, when incubated with normal BM, had no effect on spleen colony formation at the macroscopic level. However, a small CFU-S suppression was seen at the microscopic level following incubation of BM with splenocytes from control as well as treated tumor-bearing mice, indicating the presence of tumor-induced suppressor cells acting on the less rapidly dividing CFU-S. Tumor burden alone caused a decline in femoral CFU-S content to 50% (macroscopic) and 75% (microscopic) of the controls, and a simultaneous rise in the splenic CFU-S content to 370% and 250%, respectively, indicating a CFU-S redistribution from the BM to the spleen and then expansion in the spleen. The computed total CFU-S number in the body rose by 30%. Immunotherapy augmented these changes further. The computed total CFU-S number in the body was unaffected by IL-2 therapy alone but increased by another 60% following the combination therapy. Thus, IL-2-based immunotherapies had no detrimental effect on the hemopoietic stem cells in vivo. An overall amplification of the stem cell numbers was seen, despite a minor induction of CFU-S suppressor cells in the spleen of tumor-bearing hosts, which persisted during the immunotherapy.