Protection of immune irradiated rats from tumor regrowth by allogenic sensitized splenic cells

PROTECTION

OF

IMMUNE IRRADIATED RATS TUMOR REGROWTH BY ALLOGENIC SENSITIZED SPLENIC CELLS

STEPHEN

M. ALAN

AUSTIN,

STARK, AND

M.D., M.D.,

BERNARD

GARDNER,

PROTECTION

OF ANIMALS from tumor growth by syngenic sensitized splenic cells has been repeatedly demonstrated [7]. The effects of humoral antibody in tumor systems are more ambiguous, however, with both inhibition and enhancement demonstrated, depending upon qualitative and quantitative differences in experimental method. The experiments reported below were designed to demonstrate the protection of a sensitized animal from a reimplantation of tumor by allogenic sensitized splenic cells. This is a technique that may be applicable to treatment of human tumors. An additional benefit would be derived if this model could be used to evaluate the relationship between antiserum and sensitized lymphocytes in influencing tumor growth. The experiments evolved by steps, and an From the Department of Surgery, State of New York Downstate Medical Center, N.Y. *Summer research fellow supported by U.S. Health Service Grant CA-08078. tFellow, American Cancer Society. This research was supported in part by U.S. Health Service Grants CA-08299 and CA-10084. Presented at the First Annual Meeting Association for Academic Surgery, Lexington, November 10-11, 1967. Address reprint requests to Dr. Gardner.

sity lyn,

326

LEON ALFRED

BROWN, CAVE,

A.B.,* M.D.,

M.D.f

overall model of the plan is shown in Figure 1. Total body radiation and establishment of chimeras have been extensively studied in experimental animals 191. The role of the hematopoietic system in recovery from the effects of radiation is amply documented, and animals exposed to from 500 to 1500 R of total body radiation die from failure of hematopoiesis. Injections of allogenic bone marrow effectively reduce mortality in guinea pigs and mice to between 0 and 300/c, while xenogenie marrow infusions are less effective with a mortality rate of about 60% [4]. In the early experiments we investigated the effects of allogenic splenic cell transfer on acute mortality after varying doses of total body radiation. Histological examinations of the

UniverBrookPublic

Public of

FRORI

the Ky.,

Fig. 1.

Plan

of

experiment.

AUSTIS

ET

AL.:

PROTECTION

OF

bone marrow, liver, spleen, kidney, and lung were also made. From our previous studies it was known that between 90 and 100% of rats sensitized against Walker sarcoma would reject a reinjection of 500,000 cells. Failure to reject this reimplantation was considered an adequate end point by which to judge the development of chimerism. The final experiments were designed to demonstrate protection from regrowth of tumor in the rat radiation chimeras by immune allogenic splenic cells.

MATERIALS

AND

METHODS

Experiment 1: 80 male Wistar albino rats weighing from 150 to 300 gm. received 500 to 1150 R of total body radiation. Forty-one of these animals were injected intravenously with seven to thirty-four million splenic cells. Finely minced spleens from female donors were diluted with Hanks’ balanced salt solution and passed through a cytosieve. The cells were washed several times, centrifuged, and the sediment resuspended. Leukocyte counts were performed on these suspensions using standard techniques and an eosin diluting fluid to check for viability. Radiation was accomplished in clear plastic containers in groups of four, utilizing a General Electric 250-kv. roentgen ray unit set at 215 kv., 15 ma. with a 1 mm. copper (Al) filter giving a beam having a half value layer of 1.95 mm. copper. Target-to-surface distance was 53 cm. and calculated output was 37.5 R per minute. Thirty-four of the irradiated rats, half of which received splenic cell transfers, were investigated with particular attention to splenic histology. The animals either died or were sacrificed between 2 and 29 days after radiation. Peripheral leukocyte, erythrocyte, and differential blood cell counts were followed and specimens of liver, kidney, marrow, and lung were examined in addition to the spleens. Experiment 2: Walker sarcoma was implanted subcutaneously in male rats and resected when the tumors were approximately 1 to 1.5 cm. in size. If after seven days no

Ihll\lONE

IRRADIATED

RATS

FROM

TUMOR

REGROWTH

regrowth of tumor was demonstrated, one half the animals received 650 R total body radiation. Reimplantation of 500,000 Walker sarcoma cells subcutaneously was then accomplished in 33 animals, 17 of which had been radiated. Experiment 3: The third experiment followed the model as depicted in Figure 1. Walker sarcoma was resected from male rats (Group A) which then received 650 R of total body irradiation. They were then divided into two groups-one receiving two million splenic cells from female rats (Group B) which had previously had Walker sarcoma resected, and a second group receiving two million splenic cells from female rats (Group C) not immune to Walker sarcoma, All splenic cell infusions were made within 24 hours after radiation. Six days later each Group A rat received 500,000 Walker sarcoma cells subcutaneously. All animals were followed for tumor growth by measurements of two diameters. Peripheral circulating blood cell counts were followed, and at autopsy specimens of liver, spleen, kidney, lung, and bone marrow were taken for histological evaluations.

RESULTS Experiment 1: The acute survival of rats after 500 to 1150 R total body radiation is shown in Table 1. The animals in the experimental group received splenic cells from female donors. Mortality was close to 100% above 700 R. At this level and below only 30% of the animals died in 15 days. No significant protection could be attributed to splenic cell infusion except perhaps in animals receiving 800 R at ten days postradiation. The most striking histological differences were noted in the spleens of these animals. Radiation in these doses uniformly produced lymphocyte depletion, loss of germinal follicles and red pulp, and fragmented, pynknotic nuclei with absence of young forms (Figs. 2, 3). With splenic cell infusions, even with seven million cells, the spleens were remarkably different. Although dark and fragmented 327

JOURNAL

OF

SURGICAL

RESEARCH

VOL.

8

NO.

7,

lymphocytes were visible, there was preservation ‘of splenic architecture with large numTabb

1.

The Eflects

of Total

Total Number of Rats

JULY

1968

bers of erythrocytes in the sinusoids. Nodules of young plump normal-appearing lympho-

Body Radiation and Splenic on Bat Survival Number (day

Dead 5)

1150R 1150R

(C) (E)

2 2

2 2

-

1050R 1050R

(C) (E)

6 10

6 10

-

-

-

900R 900R

(C) (E)

6 6

1 1

2 3

4 5

6 6

800R (C) BOOR (E)

6 6

0 1

2 1

4 1

5 4

700R 700R

6 6

0 0

0 0

0 0

2 1

7 5 6 6

0 0 0 0

1 1 1 1

1 1 1 1

3 1 2 2

600 600 500 500

(C) (E) (C) (E) (C) (E)

(day

Dead 7)

Dead 10)

Group)

Number (day -

Group

Number

Cell Infusion (Experimental

Number Dead (day 15) -

C = control animal. animal.

E = experimental

Fig. 2. Spleen of rat 2 weeks after receiving 600 R total body radiation (X 100, before 38% reduction). 328

Fig. 3. Spleen of rat 2 weeks after receiving 600 R total body radiation (x600, before 38% reduction).

AUSTIN

ET

AL.:

PROTECTION

OF

IMMUNE

IRRADIATED

RATS

FROhi

TUMOR

REGROWTH

Spleen of rat 2 weeks after 600 R total body radiation and infusion of 10 million splenic cells (x400, before 38% reduction).

Fig. 4.

Spleen of rat 2 weeks after 600 R total body radiation and infusion of 10 million splenic cells (x100, before 38% reduction).

Fig. 5.

cytes were noted with the characteristics of germinal follicles (Figs. 4, 5). It was easily possible to tell by examination of the spleens which animals had received splenic cell infusisons.Bone marrow changes followed a similar pattern but were not as marked (Figs. 6, 7) or as constant, showing preservation of architecture in some but not all animals receiving splenic cells. Vacuolization of hepatic parenchymal cells was uniformly found in both groups with minimal histological changes in the kidneys or lungs. Peripheral total leukocytes fell rapidly by the second postradiation day and remained low throughout the 15-day period (Fig. 8). Depressions were equal in both control and experimental groups, indicating that splenic histological changes were most likely due to homing of the infused cells rather than repopulation by peripheral circulating cells.

ment. We have noted in other work that animals showing regrowth of tumor after resection do not reject a second implantation. Seventeen animals received 650 R total body radiation and were reimplanted with 506,000 tumor cells as compared to 16 nonradiated controls. Ten of the 17 animals formed new tumors with the reimplantation as compared to 2 of 16 controls (p = < 0.01) (Table 2).

2: Thirty-three animals failed to show recurrence of Walker sarcoma after initial resection and were used for the experi-

3: The expected mortality was exceeded considerably. There was a marked depression in circulating leukocytes with levels averaging 2600 cells by day 6, falling from a preradiation average of 16,000 cells. Both lymphocytes and myelocytes were deExperiment

Table

2.

Experiment

Radiated Nonradiated

Takes of Walker Sarcoma Sensitized Rats

in

Tumor Growth

Tumor Rejection

10

7 14

2

329

JOURNAL

OF

SURGICAL

RESEARCH

VOL.

8

NO.

Fig. 6. Rat bone marrow 10 days after 850 R total body radiation (x400, before 38y0 reduction ) pressed at this time. Erythrocyte counts showed a similar decrease. Histological studies showed findings similar to those noted in the earlier experiments. Splenic architecture was maintained with as few as 2,000,OOO infused cells (Figs. 9, 10). Fourteen determinate animals survived this large dose of radiation (650 R), with 7 animals receiving normal female splenic cells and 7 receiving cells from females from which Walker sarcoma had been resected (sensitized). Four tumors regrew in those animals receiving normal cells (Group Al) and three tumors regrew in animals receiving cells from sensitized rats (Group A2). The growth pattern of the two groups was remarkably different, with tumors appearing an average of two weeks later in Group A2 and failing to show a significant change in size while in Group Al the tumors grew rapidly, reaching a large size at the time of death (Fig. 11). 330

7,

JULY

1968

Fig. 7. Rat bone marrow 10 days after 850 R total body radiation and infusion of 10 million splenic cells (x400, before 38% reduction).

0

2

4

6

8

IO

DAYS

Fig. 8. Demonstrating fall in peripheral leukocyte count after 650 R total body radiation with and without splenic cell infusion.

AUSTIN

ET

AL.:

PROTECTION

OF

Fig. 9. Spleen of rat 1 month after 650 R total body radiation and infusion of 2 million splenic cells (X 100, before 38y0 reduction).

DISCUSSION Homing of splenic and marrow cells in irradiated animals has been amply demonstrated previously. Main and Prehn [5] found that irradiated DBA mice injected with (DBA x Balb/c) hybrid bone marrow failed to reject skin grafts from Balb/c mice. These grafts were rejected when irradiated DBA mice were treated with isologous DBA marrow infusion. Lindsley et al. [3] demonstrated the survival of antigenically distinct donor erythrocytes in irradiated rats. Urso and Gengozian [8] demonstrated similar findings in a heterologous mouse-rat system. Using the Walker sarcoma instead of skin grafts gave us an easily measurable end point (tumor growth) by which to evaluate the effects of infused splenic cells. The treatment of human cancer patients by infusions of leukocytes from other patients

IMMUNE

IRRADIATED

RATS

FROM

TUMOR

REGROWTH

Fig. IO. Spleen of rat 1 month after 650 R total body radiation and infusion of 2 million splenic cells (x400, before 38% reduction).

previously sensitized against the tumor has been reported [6]. This implies that for some reason the patient’s own cells cannot reject

7:

6-

P

z

0

5

IO DAYS

15 POST

20

25

30

35

IRRADIATION

Fig. 11. A comparison of growth of reimplanted Walker sarcoma in radiated rats after normal or sensitized splenic cell infusion. 331

JOURNAL

OF

SURGICAL

RESEARCH

VOL.

8

NO.

the tumor. A possible mechanism for this is coating of the tumor by circulating antibody which may protect it from immune lymphocytes. It is possible that development of the tumor from the host’s tissues implies the existence of a self-marker, preventing recognition of the tumor as foreign by the lymphocytes. There is little reason to believe, however, that infused allogenic lymphocytes would not themselves be rejected by the host. A possible means of overcoming this would be to use host lymphocyte depression prior to immune cell infusion. This of course opens the possibility of reaction of these immune cells against other host antigens. In the model described in these experiments we tried to approximate some of these conditions: using an animal already sensitized against the tumor, depression of the animal’s immune response, reimplantation of viable tumor, and infusion of immune allogenic lymphocytes. The work herein reported presents background information necessary for the successful use of this model. We have shown acute survival data for total body radiation with the optimum dose for these experiments being between 550 and 650 R. Peripheral leukocyte depletion is uniformly achieved as is destruction of the hema-

Fig. 12. 332

Karyolytic

analysis

7,

JULY

1968

topoietic cells in the spleen and bone marrow. Repopulation of the spleen can then be achieved by infusion of as few as two million cells, although optimally from ten to onehundred million cells would be preferable. The histological studies and acute mortality following total body radiation confirm previously well-established data [ 11. Of particular interest is the restoration of splenic architecture by relatively low doses of splenic cells. Nodules of repopulating lymphocytes were well documented (Figs. 9, 10). Maintenance of red pulp may be due to several possible mechanisms (2)) including contamination of the infusates with erythrocyte precursors, stimulation of the spleen to erythropoiesis, or maintenance of the animal’s erythropoietic potential by the infusate. These experiments do not distinguish the possible mechanisms involved. Chimerism is attained by demonstration of failure of the irradiated animals to reject a reimplantation of 500,000 Walker sarcoma cells. As an additional step, which we have unsuccessfully attempted, karyolytic identification of the infused cells in the spleen can be made if adequate smears of chromosomal spreads can be achieved. Figures 12 and 13 show male and female chromosomal analysis

of the male Sprague-Dawley

albino

rat.

AUSTIN

Fig.

13.

ET

AL.:

Karyolytic

PROTECTlOX

OF

IMMUNE

IRRADIATED

RATS

analysis of the female Sprague-Dawley

in Sprague-Dawley rats indicating the ease with which the sexes may be differentiated. We hope to employ this method of identifying the infused cells in follow-up experiments.

TL’XIOR

REGROWTH

albino rat.

REFERENCES 1. Blair, ation.

2. Curry, topoietic

SUMMARY A model has been developed for the evaluation of the effects of sensitized allogenic lymphocytes in protecting irradiated rats from regrowth of Walker sarcoma. Doses of radiation above 700 R were rapidly lethal while doses of 700 R and less were better tolerated in 60% of the animals. Peripheral leukocyte depression was not relieved by splenic cell infusion but maintenance of splenic red pulp and development of nodules of plump young lymphocytes were uniformly demonstrated. Interference with immune response by radiation was demonstrated by failure of the animals to reject a second challenge of 500,000 Walker sarcoma cells. Significant protection against tumor growth was demonstrated after infusion of two million splenic cells from sensitized animals, as compared to cells from nonsensitized donors. We believe this model can be useful in investigations of cell-fixed antibody.

FHOM

I. Exp. 3.

H. A. Biological Effects New York: McGraw-Hill,

Radi-

1954.

J. L., Trentin, J. J., and spleen colony studies: Med. 125:703, 1967.

Wolf, M. HemaII. Erythropoiesis.

Lindsley, D. L., Odell, T. T., Jr., and Tausche, G. Implantation of functional erythropoietic ments following total body irradiation. Proc.

Exp. B&k Med. 90512, 4.

of External

F. ele-

Sot.

1955.

Lorenz, E., Congdon, C., and Uphoff, D. Modification of acute irradiation injury in mice and guinea pigs by bone marrow injections. Rndiology 58: 863, 1952. I

,

5.

Main, J. M., and Prehn, Ft. T. homografts after the administration x-radiation and homologous bone Cancer Inst. 15:1023, 1955.

6.

Nadler, S. H., and Moore, G. E. Chnical immunologic study of malignant disease: Response to tumor transplants and transfer of leukocytes. Ann. Surg. 164:482, 1966.

7.

Old, L. J., and Boyse, E. A. perimental tumors. Ann. Rec.

8.

9.

Successful skin of high dosage marrow. J. Nat.

Immunology

of ex-

Med. 15:167, 1964.

Ursa, P., and Gengozian, N. Host-graft agglutinin activity by spleen cells of heterologous radiation chimeras cultivated in diffusion chambers. Transplantation 3:762, 1965. VanBekkum,

D. W.,

tion Chimeras. London:

and

DeVries, M. Logos, 1967.

T.

Racliu-

333