Proc. Natl. Acad. Sci. USA

Vol. 76, No. 7, pp. 3495-3498, July 1979 Medical Sciences

Attempt to immunize guinea pigs against L2C leukemia with leukemic cells inactivated by oy irradiation (cancer immunity/ionizing radiation)

LUDWIK GROSS AND YOLANDE DREYFUSS Cancer Research Unit, Veterans Administration Medical Center, Bronx, New York 10468

Contributed by Ludwik Gross, April 9, 1979

ABSTRACT In previous studies, intradermal inoculation of small doses of L2C leukemic cell suspensions into strain 2 guinea pigs induced immunity against challenging reinoculation with leukemic cells; however, 50% of the guinea pigs developed leukemia in the course of immunization. We have now attempted to induce immunity by inoculation of L2C leukemic cells inactivated by in vitro -y irradiation ranging from 750 to 8000 rads (1 rad = 0.01 gray). In preliminary experiments, irradiation with 750 to 2750 rads had no significant effect on leukemogenic potency of leukemic cells; however, doses exceeding 3000 rads inactivated the leukemogenic potency of L2C cells. Eighty-nine guinea pigs that survived intradermal, subcutaneous, or intraperitoneal inoculations with irradiated (10008000 rads) L2C cells were subsequently challenged by reinoculation with nonirradiated leukemic cells, and 83 of them (93%) developed leukemia. L2C leukemic cells contain spherical particles, about 103 nm in diameter; it is reasonable to assume that these particles represent the causative virus responsible for the develo ment of L2C leukemia. Inactivation of leukemogenic potency ofL2C leukemic cells by y irradiation in vitro does not necessarily imply that the virus particles consistently present in these cells were also inactivated. In previous experiments carried out on mouse leukemia, doses exceeding 1,000,000 rads were needed in order to inactivate the mouse leukemia virus in vitro.

We have reported previously that intradermal inoculations of small doses of L2C leukemic cell suspensions into strain 2 guinea pigs induced intracutaneous tumors that regressed spontaneously in about 50% of the inoculated animals (1, 2). The majority of animals in which the intradermal tumors regressed were resistant to challenging reinoculations of large doses of L2C leukemic cell suspensions. However, intradermal immunization against leukemia, even though of considerable theoretical interest, is fraught with difficulties when live leukemic cells are used for inoculations (3). Even among those animals that recovered from intradermal tumors, some developed, several months or even a year or more later, visceral lymphosarcomas that grew slowly at first but eventually led to generalized leukemia. For that reason we have investigated the possibility of inducing immunity by inoculation of inactivated leukemic cells. There are many examples in veterinary and human medicine which suggest that it is possible to inactivate by laboratory procedures the toxic or pathogenic potential of certain bacteria, bacterial toxins, or viruses, without destroying at the same time their immunizing effect. It remains to be determined whether it would also be possible to inactivate the oncogenic potency of leukemic cells, without, at the same time, destroying their immunizing potential. In previous studies we determined that immunity could not be induced by intradermal inoculation of heat-inactivated (560 C, 1/2 hr) leukemic cell suspensions (4, 5). Similarly, no immunity could be induced by intradermal inoculation of

leukemic cell suspensions inactivated by 1:500 solution of formaldehyde (3). In studies described in this paper, we have submitted L2C leukemic cells to 7y irradiation in vitro. We have first determined the dose of in vitro y irradiation required for inactivation of the leukemogenic potency of L2C leukemic cells. L2C leukemic cell suspensions, sealed in 2-ml ampoules, were irradiated with y rays varying in dose from 750 to 8000 rads (1 rad = 0.01 gray). Immediately after irradiation, the cells were inoculated into young adult strain 2 guinea pigs by either the intradermal or the subcutaneous route. Those animals that did not develop leukemia after inoculation of the irradiated cells were then challenged by reinoculation of live, nonirradiated L2C leukemic cell suspensions. MATERIALS AND METHODS Animals. Strain 2 guinea pigs bred in our laboratory since 1968 by brother-to-sister mating were used (4). L2C Leukemia. The L2C leukemia strain, used in this study, originated some 25 years ago as a spontaneous leukemia in one of the strain 2 guinea pigs maintained at the National Cancer Institute (6). We obtained the L2C leukemic strain in 1968 (4) and have continued to passage it in our laboratory by serial cell graft in strain 2 guinea pigs. On the basis of microscopic examination, primarily of blood smears, L2C leukemia can be classified as "stem cell leukemia." Preparation of Leukemic Cell Suspensions. After a guinea pig with advanced leukemia was sacrificed by ether inhalation, small fragments of the subcutaneous leukemic tumor, of spleen, and of the mesenteric tumor, were removed aseptically, weighed, and ground in a mortar, sterile physiological saline solution being added to obtain a 10% cell suspension; further dilutions were then made from the original cell suspension. A cell count was made from the 10-3 dilution. In our current series of experiments, the average number of leukemic cells per milliliter, at that dilution level, varied from 150,000 to 1,660,000 (average 488,000). "y-Ray Irradiation. Leukemic cell suspensions of either 1%, 5%, or 10% dilution, prepared in the manner described above, were sealed in 2-ml ampoules and irradiated with y rays. Control ampoules contained leukemic cell suspensions of similar dilutions, but were not irradiated. The ampoules were placed in a container filled with ice cubes; they remained on ice during irradiation. The irradiation was carried out by Cyprian Reid, Chief Clinical Physicist in the Radiotherapy Department at this hospital, in a cobalt-60 teletherapy unit; the treatment distance was 56 cm, the field size was 15 X 15 cm, and the dose rate was 160 rads per minute. The irradiated and the control cell suspensions were then inoculated without delay into 11/2 to 21/2-month-old guinea pigs. 3495


Medical Sciences: Gross and Dreyfuss

Proc. Natl. Acad. Sci. USA 76 (1979)

Table 1. Results of subcutaneous inoculations of L2C leukemic cell suspensions into young adult strain 2 guinea pigs Average latency Average Leukemic Guinea pigs Leukemia Tumors Died from no. of cells cell Developing incidence, developed, leukemia, per 0.5 ml dilution Inoculated* leukemia % days of inoculum days 10-2 241 240

Attempt to immunize guinea pigs against L2C leukemia with leukemia cells inactivated by gamma irradiation.

Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3495-3498, July 1979 Medical Sciences Attempt to immunize guinea pigs against L2C leukemia with leuke...
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