-Acta Radiologica Oncology 2/ (/982) Fasc. 5

FROM THE SERVICE DE RADIOTHÉRAPIE(DIRECTOR:PROF. J. CLOSON), HÔPITAL UNIVERSITAIREDEBAVIÈRE,B-4020LIÈGE, BELGlUM.

ENHANCEMENT OF CONCOMITANT IMMUNITY AFTER RADIATIONTHERAPY AND IMMUNOTHERAPY lN A SYNGENEIC

MURINE TUMOUR SYSTEM

J.-M. DENEUFBOURG

The efficacy of radiation therapy of tumeurs de-pends on lethal effects on malignant cells but also onmodifications of vessels and connective tissue(STENSTROMet coll. 1955, PUCK & MARCUS1956,HEWITT& WILSON 1959). Tumour irradiation in-duces an immune response of the host cooperatingin the destruction of allogeneic malignant cells(DENEUFBOURG 1972, 1975). These preliminaryfindings were tested in an experiment system con-sisting of a chernically induced epidermoid carcino-ma transplanted in a syngeneic situation. The influ-ence of irradiation and active non-specifie immuno-therapy was assessed in vivo on the level of con-comitant immunity. The results are now reported.

Materials and Methods

lnbred female CBA mice 2 to 3 months old wereused. They were fed with standard pellet diet andwater ad libitum. The experiment groups contained30 to 40 mice for each data point.

An epidermoid carcinoma was induced by repeat-ed painting of the skin with 20-methylcholantrene,and a syngeneic tumour was maintained by seriaisubcutaneous transplantations. Tumour piecesaseptically prepared were inoculated intradermallyin the median dorsal region of the animais. Thetumour growth followed a linear progression andremained purely local without deep infiltration orvisible metastases. Host mean survival time wasabout 30 days.

Irradiation was performed on day 8 of tumourgrowth. An original set-up of holding and shieldingallowed a good exposure of the target volume with-out anesthesia (DENEUFBOURG1972). The irradia-tion was delivered through two parallel opposedportaIs and conferred single doses of 25 or 5 Gy atmid-depth. The radiation was generated with a Phil-ips apparatus at 50 kV and 2 mA with a 1 mm AIfilter and a focus-skin distance of 4 cm. The whole-body dose did not exceed one per cent of the tumourdose.

Active non-specifie immunotherapy. On day 8 oftumour growth 0.5 mg of a suspension of Coryne-bacterium parvum (2 mg/ml; kindly supplied by In-stitut Mérieux, Lyon, France) was injected intraper-itoneally or subcutaneously in each animal.

Assessment of concomitant immunity. Threegroups of mice (non-irradiated, irradiated, or afterimmunotherapy) bearing a dorsal tumour received asecond intradermal tumour implant on their ventralside. This challenge graft contained about 106 tu-mour cells; it was observed 3 times weekly andrejections were recorded.

Results

Tumour growth. After a latent period of at least 3days anti-tumour immunization rose to 31 per centon day 6 and to 73 per cent on day 8. Then it

Accepted for publication 7 June 1982.

345

346 J.-M. DENEUFBOURG

Rejection rate of challenge graft100908070

60~

5040 ] r-r-

i-

30 ::::

20 ~'; 1;>'-<.- f,~:10~.,-

DO 15~3 6 8 10days of tumour growth

Fig. 1. Rejection rate of a challenge graft (in per cent) implantedon days 3, 6, 8,10 and 15 ofprimary tumour growth and influenceof radiation therapy (25 and 5 Gy tumour dose on day 8). Notreatment (0). 25 Gy (0). 5 Gy (6]).

Rejection rate of challenge graft100

90

8070

60

50

4030

20

10

8

."".:~i·

15days of tumour growth

Fig. 2. Rejection rate of a challenge graft (in per cent) implantedon days 8, 10 and 15 of primary tumour growth and influence ofradiation therapy and immunotherapy (5 Gy tumour dose, intra-peritoneal or subcutaneous injection of Corynebacterium parvumon day 8). No treatment (0). Corynebacterium parvum, intraperi-toneally (E3). Corynebacterium parvum, subcutaneously (0). 5Gy and Corynebacterium parvum, intraperitoneally (0). 5 Gyand Corynebacterium parvum, subcutaneously (m).

Table

Concomitant immunity during the growth of a syngeneic epidermoid carcinoma and influence of tumour irradia-tion and immunotherapy . Rejection rate of a challenge graft implanted on days 3, 6, 8, 10 and 15 of primarytumo.ur progression. Treatment on day 8: 25 Gy and 5 Gy tumour dose, intraperitoneal or subcutaneous injection

of Corynebacterium paruum

Rejection rate of challenge graft

Day 3 Day 6 Day 8 Day 10 Day 15

No treatment 0% 31% 73% 55% 43%(0/34) (11/35) (22/30) (21/38)" (l5/35)b

25 Gy 73% 82% 95%(22/30) (31/38)" c (38/40)b. d

5 Gy 73% 45% 36%(22/30) (17/38)' (14/39)d

Corynebacterium parvum, 73% 65% 75%intraperitoneally (22/30) \26/4O)f (24/32)'

Corynebacterium parvum, 73% 54% 53%subcutaneously (22/30) (20/37) (16/30)"

5 Gy and Corynebacterium parvum, 73% 88% 72%intraperitoneally (22/30) (13/35)f· g (26/36)"

5 Gy and Corynebacterium parvum, 73% 59% 44%subcutaneously (22/30) (22/37)g (15/34)"

Statistical analysis of results:a p<O.OI. b p<O.OOI. c p<O.OI. d p<O.OOI. e p<0.05. r p<0.02. g p<O.OI. h: p<0.02.

=

ENHANCEMENTOF CONCOMITANTIMMUNITYAFTERRADIATIONTHERAPY

gradually declined and the rejection rate fell to 55per cent on day 10 and 43 per cent on day 15 (Table,Figs 1, 2).

Irradiation. The level of concomitant immunityincreased after 25 Gy to 82 per cent 2 days later andto 95 per cent 7 days later, after 5 Gy the rejectionrate of the challenge graft amounted to 45 per centand 36 per cent, respectively.

Immunotherapy. After intraperitoneal administra-tion of Corynebacterium parvum on day 8 of tumourgrowth concomitant immunity reached 65 per centon day 10 and 75 per cent on day 15. Using thesubcutaneous route the corresponding rejectionrates were 54 per cent and 53 per cent, respectively.

Immunotherapy and irradiation. A 5 Gy tumourdose associated with intraperitoneal injection ofCorynebacterium parvum induced an 88 per centrejection rate of the challenge graft on day 10. Anti-tumour resistance evaluated on day 15 was presentin 72 per cent of the mice. Results with adjuvantimmunotherapy via the subcutaneous route weresimilar to the controls: 59 and 44 per cent, respec-tively.

Discussion

The efficacy of radiation therapy depends to alarge extent on the lethal effects on tumour cells(PUCK& MARCUS,HEWITT& WILSON).Alterationsof connective tissue and vascular modifications ofthe tumour bed are al~o known to play a part (STEN-STROMet coll.).Previously it was suggested that tumour irradia-

tion induces an immune response of the host coop-erating in the destruction of allogeneic malignantcells (DENEUFBOURG1972, 1975). These prelimi-nary results were confirmed in the present experi-ments using a chemically induced epidermoid carci-noma in a mou se syngeneic system.An antigenic tumour while progressively growing

induces an immunization of the host against addi-tional tumour isografts (BASHFORDet coll. 1908).The level of this concomitant immunity reaches amaximum and then gradually declines as the prima-ry tumour becomes larger (BARSKI& YOUN 1969,DECKERSet coll. 1973). This phenomenon is relatedto several interacting factors: release of free antigenin excess, blockade by antigen-antibody complexesand production of suppressor lymphocytes and mac-rophages (BALDWINet coll. 1972, THOMSON1975,POUPONet coll. 1976).According to the present results, tumour irradia-

347

tion reverses the vanishing phase of concomitantimmunity. A 25 Gy single dose was effective within48 hours and conferred anti-tumour resistance to 95per cent of the animais 8 days later. This reactionwas dose related.Data are still scarce in the literature about such

immunologie effects. Fractionated tumour irradia-tion with a total dose of 90 to 120 Gy inducedrecovery of concomitant immunity in mice bearing atransplantable mammary tumour (BARSKI et coll.1974). Reappearance of immune resistance against afibrosarcoma was observed after a tumour dose of60 Gy but was delayed as long as vascular connec-tions between host and tumour persisted (VAAGE1973). ln both reports it was noted that a compara-ble level of immunity was achieved after surgeryand that an incomplete tumour resection preventedfull recovery. ln the present system, irradiation didnot lead to tumour elimination and yet reversed thedecline of concomitant immunity without delay.Mechanisms of radiation induced immune re-

sponse are presently under investigation. Cell mem-brane destruction by irradiation is known to releaseantigenic material (MOROSON1978). Irradiation mayinduce antigenic modifications or unmasking andenhance host immunization (MARUYAMA1968). Un-trapping of cytotoxic antibodies might also occur.Destruction of suppressor cells by whole-body ex-posure as a side-effect of tumour treatment is un-likely, due to the irradiation procedure.Adjuvant active non-specifie immunotherapy with

Corynebacterium parvum counteracted the declineof concomitant immunity associated with tumourprogression. Immunostimulation was effective with-in 48 hours and lasted 8 days at least. The discrep-ancy of activity between intraperitoneal and subcu-taneous injections (CASTRO 1977) was confirmed.Irradiation at low dosage and Corynebacterium par-vum acted synergistically. An irradiation with 5 Gyis unable to modify the tumour host balance butpotentiates non-specifie immunotherapy. Antigenavailability may be a prerequisite for full range re-sponse to immunostimulation.Detrimental immunosuppressive effects of radi-

ation therapy have been pointed out especially inbreast carcinoma treatment (STJERNSwARD1974).However, the relations are perhaps more complicat-ed than are presumed. The present results ernpha-size the fact that appropriate tumour irradiation andimmunostimulation may also exert a beneficial influ-ence on host resistance.

348 J.-M. DENEUFBOURG

SUMMARYConcomitant immunity was evaluated in vivo towards a

chemically induced epidermoid carcinoma transplanted ina syngeneic situation. Radiation therapy reversed the de-cIining phase of concomitant immunity associated withtumour progression. The rejection rate of a challenge graftamounted to 82 and 95 per cent, respectively, 2 and 8 daysafter 25 Gy as compared with 55 and 43 per cent inunirradiated controls. Radiation induced immune recov-ery was dose related and proved to be different fromrestoration of immunity following surgical removal of thetumour. Immunotherapy with intraperitoneal injection ofCorynebacterium parvum significantly improved con-comitant immunity. Immunostimulation and irradiation atlow dosage act synergistically on host anti-tumour resis-tance.

ACKNOWLEDGEMENTSThe author wishes to thank Mrs M. Evrard and Mr E.

Pierard for their skilful technical assistance. This workwas supported by a grant from the Fondation de Monsieuret Madame Braconier-Lamarche.

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