Immunoprophylaxis and immunotherapy of EL4 lymphoma

Europ. d. Cancer Vol. 13, pp. 925-935. Pergamon Press 1977. Printed in Great Britain

Immunoprophylaxis and Immunotherapy of EL4 Lymphoma* T. GHOSE,J" A. GUCLU,'~ J. TAI,++ MOLLY MAMMEN~" and S. T. NORVELL§ Departments of "~Pathology, +Microbiology and §Surgery, Dalhousie University, Halifax, Nova Scotia, Canada Abstract--Injections of appropriate numbers of irradiated, but not iodoacetate treated, EL4 lymphoma cells into C57BL/6J mice were effective in immunoprophylaxis against 106 EL4 cells and in immunotherapy against 102 EL4 cells per mouse. The addition of BCG injections made immunotherapy with irradiated EL4 cells effective against a load of 104 EL4 cells/mouse. Though inoculation of BCG into BALB/c mice inhibited the growth of the allogeneic Ehrlich carcinoma, immunoprophylaxis with BCG only was ineffective against EL4 in C57BL/6J mice. Coating irradiated EL4 cells with concanavalin A did not increase their immunogenicity. Immunotherapy with vibrio cholera neuraminidase and mitomyein treated EL4 cells or with sensitized syngeneic lymphoid cells did not protect EL4 inoculated C57BL/6J mice. However, injections of several rabbit anti-EL4 globulins completely suppressed EL4 lymphoma in a proportion of mice even when treatment was started 120 hr after i.p. inoculation of 10" EL4 cells per mouse. When administered within 72 hr of tumour inoculation 1311 or chlorambucil noncovalently bound to nontumour inhibitory anti-EL4 globulins were at least as effective tumour inhibitors as the inhibitory anti-EL4 globulins.

[2, 4]. Further, the usefulness of injections of BCG and/or irradiated EL4 cells for immunoprophylaxis was investigated and compared with the results of similar immunomanipulation in an allogeneic tumour model, i.e. Ehrlich ascites carcinoma (EAC) in BALB/c mice, in which the effectiveness of immunoprophylaxis with BCG was initially reported [11]. Finally, the therapeutic response of this tumour to active and adoptive immunotherapy has been compared with the results of immunotherapy with xenogeneic antibody bound to 131I or chlorambucil. Serotherapy with appropriate xenogeneic antitumour globulins (ATG) or A T G bound to cytotoxic agents [5, 7] appears to be more effective in suppressing EL4 lymphoma than the methods of active immunotherapy tested.

INTRODUCTION

INHIBITION of the chemically induced EL4 lymphoma in allogeneic BALB/c and A mice after passive immunotherapy with xenogeneic and allogeneic antisera, enabled Gofer [1] to unequivocally demonstrate specific antigens (i.e. X antigens) on tumour cells. Since then, this tumour has been widely used as a model to assess the effectiveness of various methods of immunoprophylaxis and immunotherapy[2-7]. In an attempt to evaluate the effectiveness of different methods of immunotherapy in this model we have first investigated the lethality of intraperitoneal (i.p.), intravenous (i.v.) or subcutaneous (s.c.) inoculation of varying numbers of viable EL4 cells in the syngeneic C57BL/6J mice and have then re-examined the efficacy of such reported methods of immunoprophylaxis against EL4 lymphoma as peritoneal tapping of ascites tumour bearing mice [3], intradermal inoculation of viable turnout cell [8] and preimmunization with attenuated [9, 10] and antigenically altered EL4 cells

MATERIAL AND METHODS

THmoHTo

The EL4 lymphoma, which originated in 1945 in a C57BL/6J mouse treated by Gorer [1] with dimethylbenz[a]anthracene, has been maintained since 1946 at The Chester Beatty Research Institute, London, from where we obtained the tumour in 1969 and have main-

Accepted 19January 1977. *This work was supported by grants from the M.R.C., the National Cancer Institute of Canada and Cancer Research Fund, Faculty of Medicine, Dalhousie University.

925

926

T. Ghose, A. Guclu, J. Tai, Molly Mammen and S. T. NorveU

tained it by serial i.p. passage in C57BL/6J mice. Despite the presence of at least three welldefined tumour associated antigens (X, L and E antigens) [6], this tumour is reported to be unhibited by immunoprophylaxis with 'unmodified' or X-irradiated EL4 cells, though allogeneic antibody against the X antigen could protect C57BL/6J mice against inocula of EL4 cells [1]. The Ehrlich Lettre hyperdiploid ascites carcinoma was maintained by serial passage in 12-16 week-old BALB/c mice. Intraperitoneal inoculation of even about 25 viable EAC cells led to solid intraperitoneal tumour formation and subsequent death of tumour inoculated mice. However, 2.5 x 10 5 viable EAC cells constitute the 'critical inoculum' for the production of ascites in 100% of the mice: the presence of less than this number of viable EAC cells in the peritoneal cavity of mice tends to give rise to solid intraperitoneal tumours [12]. The survival of EAC inoculated BALB/c mice varied inversely with the size of the tumour inoculum. After transplantation into BALB/e mice, EAC grows progressively, invades various organs, forms metastases and kills all tumour inoculated mice in spite of the probable antigeneic differences between EAC cells and the allogeneic host BALB/c mice [12, 13].

Tumour ceil and BCG preparationsfor immunization Freshly obtained tumour cell suspensions (108 cells/ml), after being washed in phosphate buffered saline (PBS, 0.01 M, pH 7.1), were exposed under sterile conditions to 15,000 rad of Y rays from a G a m m a Cell 220 60Co Irradiation Unit (Atomic Energy of Canada Ltd., Ottawa, Ontario, Canada) [7, 12]. Viable BCG organisms (Connaught Medical Research Laboratories, Toronto, Ontario, Canada) were inoculated (10v BCG cells/mouse) by various routes as stated in the different experiments. Neuraminidase treated tumour cells were prepared for immunotherapy following the method of Rios and Simmons [14]. Aliquots of 2 x 106 EL4 cells were incubated with 50 units/ ml of vibrio cholera neuraminidase (VCN, General Biochemicals, Chargin Falls, OH, U.S.A.) for 1 hr at 37°C, were washed 3 times with M199 and then reincubated for 1 hr with 50 /~g/ml of mitomycin C (Sigma Chemical Co., St. Louis, MO, U.S.A.). The cells were again washed 3 times with M199 and the viability of the cells was confirmed by trypan blue exclusion [5] before injection. Comparison groups of mice were vaccinated with EL4 cells exposed to VCN only, mitomycin C only and M199 only.

The binding of Con A to EL4 ceils was verified by autoradiography of EL4 cells [7] incubated with Con A containing tracer doses of 3H Con A (New England Nuclear, Dorval, Quebec, Canada). To coat tumour cells with Con A (Calbiochem, Los Angeles, CA, U.S.A.), 10 7 tumour cells/ml were reacted with 25 /~g Con A/ml for 30 min, at 37°C.

Spleen andperitoneal cellsfor adoptive transfer Peritoneal cells were collected by washing the peritoneal cavity with Hank's balanced solution (HBSS). Splenic cells were dissociated with 0.2% ethylenediaminotetraacetic acid in PBS and erythrocytes were lysed with 0.83% NH4C1. The nucleated spleen cells and peritoneal cells were washed 3 times with sterile HBSS, thei r viability was assessed by trypan blue exclusion and the cells were then suspended in HBSS at the desired concentration.

Anti-EL4 sera The details of the methods of production and purification of rabbit anti-EL4 sera, the tests to establish their specificity and the method of fractionation to obtain the A T G have been described recently [7, 15]. In brief, groups of female New Zealand white rabbits were intramuscularly injected twice with 4 x 108 EL4 ceils mixed with Freund's complete adjuvant (Difco Laboratories, Detroit, MI, U.S.A.) followed by repeated intramuscular injections of 108 EL4 cells/rabbit without any adjuvant. The immune sera were rendered tumour specific by repeated absorptions with homogenates of organs from adult C57BL/6J mice until the sera reacted as assayed by immunofluorescence, only with EL4 cells and neither with cryostat sections of normal C57BL/6J mouse tissues nor with suspensions or smears of C57BL/6J lymphoid cells from lymph nodes, spleen and thymus, B16 melanoma cells from C57BL/6J mice and EAC cells. The three batches of EL4 inhibitory rabbit scra were obtained after a total of 12, 22 and 35 injections of EL4 cells. Normal rabbit sera and the anti-EL4 sera after being rendered tumour specific after absorptions with homogcnates of normal C57BL/6J mouse tissues were fractionated with 33% saturated ammonium sulphate. The methods of binding chlorambucil [16] and 13'I [7, 15] to rabbit globulins have been recently described in detail.

Detection of tumour antibodies Cytoplasmic and membrane immunofluorescence of mouse tumour cells and other control preparations were carried out by the sand-

Immunoprophylaxis and Immunotherapy of EL4 Lymphoma wich method using as appropriate fluoresceihated goat antirabbit or rabbit antimouse 7S globulin (Hyland Laboratories, Los Angeles, CA, U.S.A.) [5, 7, 15]. For detection of agglutinating and complement dependent cytotoxic antibodies tumour cells were incubated in vitro with immune rabbit or other test sera with and without the the addition of eight times diluted fresh rabbit serum, guinea-pig serum or C57BL/6J mouse serum which were not cytotoxic by themselves and which were assessed for their hemolytic complement activity by the method described by Cinader et al. [17]. At the end of the incubation period, the cells were examined for agglutination, morphological changes and permeability to trypan blue [5] and were then injected into syngeneic mice. To detect tumour suppression or enhancement by these antibodies the mean survival of these test mice was compared with that of control groups inoculated with aliquots containing identical numbers of tumour cells incubated with normal rabbit serum or HBBS.

927

Table 1. Survival of C57BL/J mice inoculated i.p. with varying numbers of EL4 lymphoma cells Number of EL4 cells inoculated

Survival (days + S.D.") After i.p. inoculation

1

b

10

26.6 + 6.84

10z

23.4 + 2.60

10a

~20.0 _+ 1.0 -~ 21.3 + 2.91" [.20.7 + 1.2J

104

18.4 + 0.55 18.8 _+ 1.64" 19.8 + 1.48t

105

15.6 + 1.81

106

12.8 + +0.83

107

('12.4 + 0.89 -~ 9.0 _+ 0.71" L 12.8 _+ 1.I?

IOs

10.0 + 0.0

Mice C57BL/Icrf mice were donated by the Imperial Cancer Research Fund Laboratories, London, U.K. All other inbred mice were purchased from the Jackson Laboratory, Bar Harbor, MN. When not stated otherwise, experimental groups consisted of at least ten 12-16-week-old female mice.

Statistical evaluation The significance of the difference in mean survival between groups was assessed by Student's t-test and the significance of the difference in the numbers of tumour free survivors between groups was evaluated by the fourfold table method of X z test.

RESULTS

Survival of C57BL/6J mice inoculated with varying numbers of EL4 lymphoma cells Table 1 shows that i.p. inoculation of even about 10 EL4 cells leads to progressive tumour formation and subsequent death o f t u m o u r inoculated C57BL/6J mice. All the C57BL/6J mice inoculated i.v. or s.c. with 103 EL4 cells died approximately at the same time as the mice inoculated i.p. with comparable numbers of EL4 cells. With inocula of 10 7 EL4 cells/mouse, the mice injected i.v. died earlier (survival 9.0 -+ 0.7 days) than the mice inoculated i.p. (survival

"Each group contained at least 15 adult female mice. bOnly 4 of 42 C57BL/6J mice inoculated i.p. with a single EL4 cell developed turnout. *Figures represent survival of groups inoculated i.v. with EL4 cells. "~Figures represent survival of groups inoculated s.c. with EL4 cells. 12.4_+0.89 days) or s.c. (survival 12.8+1.1 days). None of the mice inoculated i.v. with EL4 cells had ascites. In the mice inoculated with EL4 cells s.c., ascites was seen only in those mice in which the s.c. turnout had penetrated into the peritoneal cavity. A few of the mice inoculated i.v. with 103 or 104 EL4 cells had bilateral pleural effusion containing EL4 c At autopsy, the C57BL/6J mice inoculated i.v. with EL4 cells revealed enlargement of spleen and lymph nodes. With inocula of 10 7 EL4 cells/mouse, the range of splenic weight in the mice inoculated i.v. was 402-508 mg (median, 442 rag), whereas in the groups inoculated with 10 7 cells i.p. or s.c. the range of spleen weights was 65-140 mg (median, 68 mg). The mean spleen weights of 13 normal female C57BL/6J mice of a comparable age group was 70.72-+6.25 mg. Immunofluorescence assay using xenogeneic anti-EL4 serum [9] revealed dense accumulation of EL4 ceils in the enlarged spleen and lymph nodes from the C57BL/6J mice inoculated i.v. with EL4 cells. In the C57BL/6J mice inoculated i.p. with 108,

928

T. Ghose, A. Guclu, 'J. 'Tai, 'Molly Mammen and S. T. Norvell

10 7 or 106 EL4 cells, the weight of the spleens varied between 60 to 140 mg (median, 71 mg) in contrast to the groups inoculated with 10 s cells (range of spleen weight 90-230 mg, median 145 mg); 10 4 cells (range of spleen weight 150-240 mg, median 190 rag) ; l03 cells (range of spleen weight 165-190 mg, median 182 rag) ; 10 2 ceils (range of spleen weight 140310 mg, median 185 nag). It is interesting to note that the volume of ascites fluid at the time of death in the mice inoculated i.p. with > 106 EL4 cells was >- 7 ml per mouse, wheras the volume of ascites fluid per mouse in the mice inoculated i.p. with < 1 0 s EL4 cells/mouse, did not exceed 4 ml per mouse. The splenic weight of the mice with subcutaneous tumours varied widely and was influenced by the ulceration and infection of the tumours. Irrespective of the route of tumour inoculation, survival of the tumour inoculated C57BL/6J mice was inversely related to the size of the tumour inocula and histological examination of tissues obtained at autopsy revealed widespread infiltration of EL4 cells into all internal organs. Effect of peritoneal tapping on the growth of i.p. inoculated EL4 ceils in C57BL/6J mice. There was no difference in the survival between groups of C57BL/6J mice which had their peritoneal cavity aspirated daily for 4 days beginning 24 hr (survival 11.80_+ 1.30 days), 72 hr (survival 12.4_+0.54 days) or 10 days (survival 14.6 +_4.92 days) after i.p. inoculation of 2 x 106 cells/mouse. The survival of comparison groups inoculated i.p. with 2 x 106 EL4 cells/mouse without any treatment or sham aspirated daily for 4 days beginning 24 hr after i.p. inoculation of 2 x l06 cells were 13.00_+ 1.80 and 14.0 +4.47 days respectively.

Effect of intradermal inoculation of EL4 lymphoma cells in C57BL/6J mice The survival of groups of C57BL/6J mice intradermally inoculated with 10 5 (survival of 16.20 _+ 1.64) or 103 (survival 22.6 +__0.89 days) EL4 cells/mouse was not different from the survival of groups of mice inoculated with 10 5 or 103 EL4 cells i,p. (survival 15.6 -+ 1.81, 20.0 ± 1.0 days respectively). The experiments were repeated several times, with comparable results. 'Immunogenicity' and growth of EL,I cells pretreated in vitro with iodoacetate in PBS for 1 hr at 37 °C Table 2 shows the results of inoculation of 10 7 EL4 cells pretreated with various concentrations of iodoacetate in vitro. Pretreatment of EL4 ceils even with 0.01 M iodoacetate did not

Table 2. Survival of C57BL/6J mice inoculated i.p. with 107 EL4 cells exposed to various concentrations of oidoacetate in PBS for 1 hr at 37°C Inoculation of 107 EL4 ceils pretreated with: Nil 0.001 0.001 0.005 0.01

M M M M

Iodoacetate Iodoacetate Iodoacetate Iodoacetatc

Survival (days _+ S.D.) 12.4 17.0 32.8 39.2 94.4

± ± ± ± ±

0.89 0 1.30 2.05 2.97

prevent turnout development in the tumour inoculated mice, though the appearance of tumour in these mice was considerably delayed. There was no tumour in comparison groups of mice inoculated with normal syngeneic spleen cells pretreated in vitro with 0.005 M or 0.01 M iodoacetate.

ImmunoprophyIaxis with Con A-modified turnout cells Every member of groups of fifteen mice was injected i.p. with 5 x 10 s of either X-irradiated tumour cells or X-irradiated tmnour cells coated with Con A, following the method of Martin and Wunderlich [4]. After 4 weeks each mouse received i.p. 5 x 10 v tumour, ceils prepared as in the first injection. However, consistant with our previous report [7] and that of Johnson et al. [10], i.p. inoculation of 106 EL4 cells exposed to 1500 rad in C57BL/6J or C57BL/Icrf mice, i.e. a substrain derived from Gorer's colony of C57BL/6 mice in which the EL4 lymphoma originated [1], did not prevent the development o f t u m o u r in all tumour inoculated mice irrespective of whether the tumour cells were coated with Con A. Complete tumour suppression could be obtained after exposure of EL4 cells to 2000 rad which was, therefore, the irradiation dose used in this experiment. None of the C57BL mice challenged with 104 EL4 cells 10 days after the completion of immunization survived longer than the control group of C57BL mice immunized with irradiated EL4 cells not coated with Con A (survival 18.4_+3.5 and 19.2_+2.1 days respectively). Since Martin and Wunderlich, in their experiments with induction of immunity with Con A coated EL4 cells used C57BL/6N mice, i.e. another substrain of C57BL/6 mice maintained for long in the National Institute of Health, U.S.A. [8, 18], the emergence ofhistoincompatibility between the long transplanted EL4 lymphoma and C57BL/6N mice was thought to be a possibility explaining the complete suppression in this substrain of EL4 cells exposed to 1500 rad and the induction of im

Immunoprophylaxis and Immunotherapy o f EL4 Lymphoma

munity by injections of Con A coated EL4 cells [4, 18]. We, therefore, investigated the effectiveness of Con A coated tumour cells in inducing tumour resistance in an allogeneic model, i.e. the EAC in BALB/c mice. BALB/c mice immunized with Con A modified EAC cells died earlier (i.e. within 10-16 days of challenge) compared to the survival of BALB/c mice immunized with irradiated tumour cells alone (survival 23-31 days). Prior sensitization of groups of C57BL and BALB/c mice to Con A by a method described by Wong et al. [19] did not prolong the survival of these mice when challenged as appropriate with Con A coated 104 viable EL4 or 2.5 x 106 viable EAC cells. Immunoprophylaxis against irradiated tumour cells

EL4

lymphoma

with

Immunoprophylaxis could be consistently induced in C57BL/6J mice using irradiated EL4 cells provided that the number of irradiated EL4 cells per injection and the number of immunizing injections were adequate. Injections containing 106, 10 7 or 108 irradiated EL4 cells (but not comparable numbers of irradiated syngeneic nucleated spleen cells) given i.p. once a week at least for 3 consecutive weeks, consistently protected varying proportions of mice when they were challenged i.p. with either 103 or 104 viable EL4 cells/mouse 1 week after the completion of immunization. No such protection could be observed when the number of irradiated EL4 cells per injection was less than 3. The protection increased with increasing numbers of injections and increasing numbers of irradiated EL4 cells per injection. However, immunization with 108 irradiated tumour cells (or 108 irradiated nucleated spleen cells) i.p. caused ascites and an occasional death due to peritonitis. EL4 specific antibody could be detected in the serum and ascites fluid of the C57BL mice immunized with 108 irradiated EL4 cells/mouse but not in mice immunized with comparable numbers of irradiated spleen cells. These anti-EL4 sera were not cytotoxic to EL4 cells in vitro and they neither inhibited nor enhanced EL4 lymphoma when injected into turnout-bearing mice [9]. Figure 1 shows that i.p. immunization with 10 7 irradiated EL4 cells/mouse offered better protection against challenge inocula containing 10 3 EL4 cells/mouse than inocula containing 10 4 EL4 cells/mouse, and, also that four i.p. injections of 10 7 irradiated EL4 cells were more protective than four s.c. injections or three i.p. injections followed by one intrasplenic injection of 10 7 irradiated EL4 cells.

929

I¢ i.p.

i¢ i.p.

I

li.s.

0'

' 40' ;0 20

' I00 80

PER CENT

;

' i0 20

6;8'0

I00

PER CENT

Fig. I. Survival of C57BL mice immunized with injections of 107 X-irradiated EL4 cells~mouse once a week for 4 weeks and challenged 1 week after the last injection with either 103 or 104 viable EL4 cells i.p. Empty space represents per cent of mice dead of tumour within 30 days of challenge. Diagonally hatched space represents per cent of mice dead of tumour from days 31 to 200 post tumour challenge. Crosshatched space representsper cent of mice surviving tumour-free for 200 days.

The immune C57BL/6J mice that survived a challenge inoculum of 10 4 EL4 cells/mouse were resistant 60 days after the first challenge to further i.p. challenges with 10 5 and 106 EL4 cells, but not to a challenge i.p. with 10 7 EL4 cells. However, these immune mice (i.e. those surviving tumour free 60 days after being challenged with 104 EL4 cells) had a mean survival of 22.2 +2.7 days when rechallenged with 104 syngeneic B16 melanoma cells compared to the survival of 22.4_+ 1.9 days of previously untreated C57BL/6J mice inoculated with 104 B16 melanoma cells. T u m o u r resistance in immunized C57BL/6J mice (not challenged with viable EL4 cells) declined rapidly and was undetectable 16 weeks after immunization as assessed by their survival after i.p. challenge with 103 EL4 cells (Table 3). Immunoprophylaxis against EL4 lymphoma with irradiated tumour cells and/or BCG. Figure 2 shows the survival of C57BL/6J mice challenged i.p. with 104 tumour ceils after immunoprophylaxis with irradiated EL4 cells and/or BCG. In another experiment the results of different methods of immunoprophylaxis in C57BL/6J mice against the syngeneic EL4 lymphoma was compared with the results of identical methods of immunoprophylaxis against the allogeneic EAC in BALB/ mice (Fig. 3). In immunization protocols 3 and 8, BCG and irradiated tumour cells were incubated together for 10 rain at 37°C immediately before injection. The s.c. injections during immunization protocols 5-10 were given at separate sites along the dorsal midline starting

930

IT. Ghose, A. Guclu, J. Tai, Molly Mammen and S. T. NorveU

Table 3. Survival of C57BL/6J mice challenged i.p. with 103 viable EL4 cells 1, 4, 8 and 12 weeks after completion of immunization with irradiated EL4 cells Interval between immunization* challenge'~ (week)

Survivor > 200 days t u m o u r free

Died of t u m o u r between 30 and 200 days post challenge:~

4

17/21 6/16

8

2/14

16

0/14

4/16 5/14 5/13 0/14

I

Died of tumour within 30 days (postchallenge) 4/21 6/16 14/14§ (All died of ascites tumour between 14 and 18 days after challenge.)

* I m m u n i z e d with 10 ~ irradiated E L 4 cells i.p. once a week for 4 weeks. t C h a l l e n g e d with 103 viable EL4 cells per mouse. ~ T h e majority of the mice in this group did not develop ascites. Autopsy of these mice revealed large spleen and widespread infiltration of internal organs with EL4 l y m p h o m a cells. §A c o m p a r i s o n group of previously u n t r e a t e d C57BL mice challenged with 103 EL4 cell died between 17 and 22 days after challenge.

from the caudal end on week 1 and finishing over the neck on week 4. Immunoprophylaxis with BCG alone administered either i.p. (protocol 1) or s.c. (protocol 6) did not protect C57BL mice against challenge with EL4 cells, but both these protocols of immunization offered varying degrees of protection to BALB/c mice against the allogeneic EAC. However, in terms of tumour free survival for more than 200 days in C57BL/6J mice, the protection resulting from i.p. immunization with irradiated tumour cells alone (protocol 2) was increased (P< 0.01) when the mice were simultaneously immunized with BCG s.c. (protocol 5). As immunization with i.p. injections of only irradiated EAC cells offered complete protection to the BALB/c mice (protocol 2) any additive effect of BCG

injections in protocols 3, 4 and 5 could not be elucidated. BCG given i.p. lowered the protection (i.e. tumour free survival > 200 days) produced by s.c. injection of irradiated EL4 cells alone (protocols 7 and 10) in C57BL mice (P<0.01), but did not significantly alter the protection offered by s.c. injections of irradiated EAC cells in BALB/c mice. Injections of BCG s.c. at the opposite side (protocol 9) did not increase the protection (i.e. tumour free survival > 200 days) resulting from s.c. injections of irradiated EL4 cells or EAC ceils alone (protocol 7). Incubation of irradiated EL4 or EAC cells with BCG prior to subcutaneous injection appears to lower the protection provided by immunization with irradiated EL4 or EAC cells alone (protocol 8).

cells IP immed BCG S.C.

I00

""i"" ............................... I

i ~40-

J~--

g

.

~. I I

.

.

~'J~i1

.

.

BCG IP I

I

.

.

.

.

[[

O

' " i "'I ' 10 20

I

30

~ ' ~

cells IP 3hrsBCG IP

~ . . . .

"celIs+BCGIP"

I . ~cells S,C. + BCG S.C. opp, side L ----'-//'- . . . . . . " cells + BCG S.C. " "" // cells S.C. + BCG IP

104 EL4 only

'

~ n

~,

.

I I

0 I

-"//

irradcellslP .

cells S.C.

--/z---

~

O'-,

i

0

II ......................................

'

l

40

l

50

I / /

60

I

180

'

l

190

1

200

'

]

210

DAYS AFTERCHALLENGE,WITH104 EL4 CELLS I IP )

Fig. 2. Survival of C57BL/6J mice challenged with 104 EL4 lymphoma cells/mouse 1 week after immunization with injections of 10 7 irradiated tumour cells and/or 10 7 viable BCG once weekly for 4 weeks. 'Cells + BCG' i.p. representsgroups of mice inoculated with irradiated EL4 cells incubated with BCG before injection.

Immuno prophylaxis and Immunotherapy of EL4 Lymphoma EL4 LYMPHO/VIA

,cc,.p. I

EAC

I '

ToMou,ce,s,.,.

I

I

2

931

(15.20 _+2.28 and 11.40 _+2.60 days, respectively). However, immunotherapy with BCG and irradiated tumour cells resulted in the survival of two out of twenty mice given 104 EL4 cells i.p.

TUMOUR cells + TUMOUR cells Lp. 3

hrs.-BCC i.p. ~ ~ - . x . ~

Immunotherapy of EL4 lymphorna with 'adoptive' transfer of syngeneic immune splenic and peritoneal exudate cells

4

'TUNtOUR cells i.p.

,c~,.c. ~

s

"c°'-°.l

I

I"UMOURcells +

s.c.

N

I

8

J

9

I

I

TUMOUR cells s.c. BCG s.c. opposite side

~x.~.~x"~

TUMOUR cells s.c. ,p

~

l

I .;o ; z; 4; 6; 8'o 1~0

; 10 4b ~; ~0 1®

PER CENT

PER CENT

Fig. 3. Comparisonof the survival of C57BL and BALB/c mice challenged respectively with 104 EL4 lymphoma and 2.5 x 106 EAC cells per mouse 1 week after immunization with injections of 10v irradiated tumour cells and/or 107 viable BCG once a weekfor 4 weeks as explained in the left hand column. For convenience of reference each protocol of immunization has been assigned a number presented in the third column. Irnrnunotherapy of EL4 lymphoma with irradiated tumour cells only or irradiated turnout cells and BCG

After i.p. inoculation of 10z-106 viable EL4 cells/mouse, three i.p. injections of 108 irradiated EL4 cells were given 2, 48 and 168 hr later. Nine of 10 mice inoculated with 102 cells and 1 of 10 mice from groups inoculated with 103 and 104 EL4 cells/mouse survived tumourfree for 300 days. None of the mice given 10 5 or 106 cells before immunotherapy or treated with 3 i.p. injections of irradiated syngeneic nucleated spleen cells survived longer than did the untreated controls. Injection of irradiated tumour cells i.p. followed by BCG s.c., i.e. protocol 5 (Fig. 3), the most potent protocol for immunoprophylaxis, was also used in a modified form for immunotherapy. Two hours after i.p. inoculation of 10 ~, l0 s or 106 EL4 cells, each C57BL/6J mouse received injections of 108 irradiated EL4 cells i.p. and 106 BCG cells s.c. The injections of irradiated EL4 cells and BCG were repeated on days 3 and 7 after tumour inoculation. The survival times of the treated mice receiving 10 5 and 106 EL4 cells (14.20-t3.19 and 11.25-+0.5 days, respectively) were not different from those of untreated controls

Spleen and peritoneal cells from tumour-free immune C57BL/6J mice 60 days after challenge with 103 viable EL4 cells inhibited EL4 cells. This was established by the longer survival i.e. (24.4 -+ 3.64 days for splenic cells and 27.6 -+4.51 days for peritoneal cells) of mice inoculated i.p. with 106 EL4 cells mixed with either 10 7 immune nucleated splenic or peritoneal cells compared to the survival of groups of C57BL/6J mice inoculated with 106 EL4 cells mixed with splenic or peritoneal cells from previously untreated mice (survival 14.4_+1.05 days and 14.4_+1.34 days respectively). For immunotherapy, C57BL/6J mice were first given 104 viable EL4 cells i.p. and were then treated 1 or 2 hr later with 10 5 immune spleen or peritoneal cells. Survival was slightly prolonged (22 days) in 1 of 10 mice inoculated i.p. with immune spleen cells and 2 of 10 mice (23 and 24 days) given immune peritoneal cells 1 hr after tumour inoculation. The survival times of all other mice in these groups did not differ from the survival of 10 mice inoculated i.p. with 104 EL4 cells only (16-19 days). Active Immunotherapy of EL4 neuraminidase treated tumour cells

lyrnphoma with

Groups of C57BL mice were treated 2 hr after receiving 104 EL4 cells/mouse with i.p. injections of either 10 7 EL4 cells exposed in vitro to both VCN and mitomycin, mitomycin only or M199 only. The mice injected i.p. with VCN and mitomycin treated EL4 cells (survival 15.2_+0.44 days) or only mitomycin treated (survival 17.0 __ 1.87 days) or only VCN treated tumour cells (survival 17.2 + 1.92 days) did not survive longer than the control group of mice which after receiving 104 EL4 cells/mouse were injected i.p. with PBS only (survival 17.2 + 1.92 days). The group of mice, which after receiving 104 EL4 cells i.p. were injected with 10 7 EL4 cells exposed to M199 only died earlier (survival 11.0 +0 days). Passive immunotherapy of EL4 lymphoma

While examing the biological properties of more than fifty batches rabbit and goat anti-

932

T. Ghose, A. Guclu, J. Tai, Molly Mammen and S. T. NorveIl

E L 4 sera, 3 batches of r a b b i t a n t i - E L 4 sera were found to consistently inhibit t u m o u r growth in C57BL mice preinoculated with E L 4 cells even t h o u g h these anti-sera did not r e n d e r E L 4 cells p e r m e a b l e to t r y p a n blue after exposure in vitro to these A T G preparations and complement. T a b l e 4 shows the survival of groups o f C57BL/6J mice which were tested with 5 injections of 2 m g A T G / m o u s e / d a y , either 2, 24 or 120 hr after i.p. inoculation of 10 3, 10 4, 10 5

A T G preparations ( T a b l e 4). H o w e v e r , w h e n t r e a t m e n t was started at 120 hr after i.p. inoculation of 103 or 10 4 cells/mouse, daily injections of 2 m g A T G ( T I ) / m o u s e for 5 days completely suppressed the E L 4 l y m p h o m a in a p r o p o r t i o n of these mice. C o m p a r i s o n groups of E L 4 inoculated C57BL mice treated with 0.1 m g of c h l o r a m b u c i l or 2 m g A T G ( N T I ) per mouse daily for 5 or 9 days died between 18 and 22 days after t u m o u r inoculation [5, 15].

Table 4. Survival of C57BL/6J mice treated either with tumour inhibitory antitumour globulin (ATG) (A) or non-tumour inhibitory A TG bound to chlorambucil (B) or a31I (C) 2, 24, 72 and 120 hr after i.p. inoculation of various numbers of EL4 cells. Each group contained at least 10 mice

No. of EL4 cells inoculated (per mouse) i.p.

Interval--(hr) between tumour inoculation and beginning of treatment and per cent survival after the 3 modalities of treatment, i.e. A, B and C* 2 24 72 120

10 a

A--80 B--100 C--100 (400BCi)

A--80 B--100 C--100 (400~Ci)

A-- 80 B--80

A--80 B--0 (P.S.)'~

104

A--40 B--100 C--100 (540pCi)

A--60

A--50

A--50

B--90

B--60

B--0 (P.S.)'~

105

A--20 B--100

106

A--0 B--60

107

A--0 B--0

C--80 (500pCi)

*A--Treated with tumour inhibitory anti-EL4 globulin (2 mg daily for 5 days). B--Treated with nontumour-inhibitory anti-EL4 globulin bound to chlorambucil (2 mg globulin + 0.1 mg chlorambucil daily for 5 days). C--Treated with nontumour inhibitory anti-EL4 globulin bound to 1311 (approximate activity of 1311 in parenthesis). All injections were intraperitoneal. J'Prolonged survival only, no cure. or 10 6 E L 4 cells/mouse. E i g h t y per cent of the mice inoculated with 10 a E L 4 cells/mouse and a p p r o x 5 0 % of the mice inoculated with 10 4 E L 4 cells/mouse survived t u m o u r - f r e e irrespective of the interval between t u m o u r inoculation and t r e a t m e n t .

Immunochemotherapy of EL4 lymphoma with A TG noncovalently bound to chlorambucil W h e n administered within 72 h r of t u m o u r inoculation, chlorambucil noncovalently b o u n d to a xenogeneic A T G (non t u m o u r inhibitory, N T I ) was a better or at least equally effective t u m o u r inhibitor as the t u m o u r inhibitory (TI)

Immuno-radiotherapy of EL4 lymphoma X e n o g e n e i c A T G (not t u m o u r inhibitory, N T I ) w h e n b o u n d to 1311 could suppress the growth of E L 4 l y m p h o m a in mice preinoculated with 10 4 cells p r o v i d e d that a d e q u a t e a m o u n t s of radio-iodinated A T G was administered within 72 hr of t u m o u r inoculation ( T a b l e 4). For t u m o u r inhibition in mice preinoculated with 10 4 E L 4 cells/mouse longer t h a n 72 hr it was necessary to administer more t h a n 1 m C i of 1 a lI/mouse, which causes severe bone m a r r o w depression and d i a r r h a e a in these mice. T h e details and survival of a p p r o p r i a t e comparison groups of mice have been described elsewhere [7, 14].

Immunoprophylaxis and Immunotherapyof EL4 Lymphoma DISCUSSION

The localization of large numbers of i.v. inoculated EL4 cells into the spleen and lymph nodes of C57BL/6J mice is similar to the lymphoid tissue specific localization of i.v. inoculated lymphoma cells in A K R mice [20, 21]. The decreased volume of ascites tumour and increased spleen weight in the C57BL/6J mice inoculated i.p. with < 10 5 EL4 cells compared to the mice receiving > 106 EL4 cells i.p. might indicate either localization of EL4 cells in the spleen in preference to the peritoneal cavity in these mice or the ability of these mice to offer effective resistance only against inocula containing < 10 s EL4 cells. Increased splenic cellularity and organ weight has been postulated to be associated with tumour I esistance in mice bearing syngeneic tumours [22] including lymphomas [23]. It is interesting to note that splenic enlargement, decreased volume (or absence) of ascites and disseminated tumour were also seen at autopsy in those C57BL/6J mice which after immunotherapy or immunoprophylaxis against i.p. inoculated EL4 lymphoma survived longer than tumour inoculated untreated control mice. Contrary to the report of Rubin [8] that viable EL4 cells after intradermal injection into C57BL/6J mice do not proliferate and induce immunity against further challenges with EL4 the results presented here demonstrate that inoculation of as few as 10 a EL4 cells into C57BL/6J mice leads to the death with widely disseminated tumour in all tumour inoculated mice irrespective of i.v., i.p., s.c. or intradermal routes of tumour inoculation. This discrepancy might be due to minor degrees of histoincompatibility between the EL4 lymphoma and the C57BL/6N mice, the evidence for which has already been discussed. In our laboratory, intraperitoneal injection of blood into C57BL/6J mice from mice preinoculated i.p. 24 hr ago with as few as 10 5 EL4 cells/mouse led to the development of EL4 lymphoma in the recipient mice, thus demonstrating the presence of EL4 tumour cells in the circulation of C57BL/6J mice 24 hr after i.p. inoculation of l0 s EL4 cells/mouse. Further, histological examination of internal organs of C57BL/6J mice inoculated i.p. with 106 EL4 cells/mouse had revealed small loci of tumour cells in the lungs 72 hr after tumour inoculation and also definite invasion of the liver and kidney 120 hr after tumour inoculation [7, 15]. Using the 'Pondville subline of C57BL mice', Apffel and Arnason [2] have reported the induction of immunity against EL4 lymphoma

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after injections of EL4 cells 'inactivated' with 10 -2 , 10-3 and 1 0 - 4 M iodoacetate. T h e y also reported in this subline of C57BL mice the complete suppression of EL4 lymphoma followed by the development of tumour immunity after serial peritoneal tapping beginning 7-14 days after i.p. inoculation of 2.5 x 106 EL4 cells

[3]. However, the suppression of EL4 lymphoma after serial peritoneal tapping [3] and after immunoprophylaxis with iodoacetate treated cells can be explained on the basis of genetic heterogeneity of the C57BL mice used, i.e. a tumour indigenous in the 'C57BL]6 Pondville mice' was rejected by another substrain of C57BL/6 mice used by these investigators [2]. Consistent with our observation, iodoacetate treated tumour cells have been reported to be not very effective in tumour immunotherapy [24] and VCN and mitomyein treated tumour cells are therapeutically effective mostly in tumour models with relatively long survival of turnout inoculated mice [14] compared to the survival of EL4 inoculated C57BL/6J mice. Studies on immunoprophylaxis and immunotherapy using tumours of long transplantation history like the EL4 lymphoma are complicated by the possibility of viral contamination and the emergence of histoincompatibility between the various tumour sublines and the substrains of mice evolving from the strain of origin of the primary tumour. Our results confirm the effectiveness of immunoprophylaxis with BCG and/or irradiated tumour cells in allogeneic tumours like the EAC [11] or the $91 melanoma in BALB/c mice [25]. O n the other hand, immunoprophylaxis with BCG and/or irradiated tumour cells failed in our laboratory to inhibit in A K R mice the syngeneic L2 lymphoma which has a much shorter transplantation history [9]. It is, therefore, not certain that the immunity induced in C57BL/6J mice with injections of irradiated EL4 cells by us and others [9, 10] is truly tumour specific. The superiority o f t u m o u r inhibition by ATG linked cytotoxic agents over tumour inhibition by A T G or cytotoxic agents by themselves is now well established [7, 15, 26-30]. However, although the therapy of malignant tumours with xenogeneic and allogeneic antisera has been generally disappointing [31], more recently there have been reports on the prolongation of survival by a few days, but not 'cell cure' of EL4 bearing C57BL mice [32] and rats bearing a syngeneic lymphoma [33] after injections of appropriate rabbit antitumour sera. The A T G preparations obtained by us appear to be more potent as cell cure could be obtained

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, T. Ghose, A. Guclu, J. Tai, Molly Mammen and S. T. Norvell

in a p r o p o r t i o n of mice p r e i n o c u l a t e d with 10 s E L 4 cells (i.e. 104 times m o r e t h a n the minim u m n u m b e r o f E L 4 cells necessary for 100% t u m o u r take). T h e complete t u m o u r suppression in a p r o p o r t i o n of mice, even w h e n passive i m m u n o t h e r a p y was instituted 120 hr after i.p. inoculation with E L 4 cells, is especially interesting because there is definite evidence o f t u m o u r dissemination 120 hr after i.p. inoculation of E L 4 cells and even chlorambucil b o u n d A T G only p r o l o n g e d the survival and failed to pro-

duce any 'cell cure' w h e n t r e a t m e n t was initiated 120 hr after t u m o u r inoculation. W e have so far failed to detect t u m o u r inhibition b y several goat anti-mouse l y m p h o m a s including goat a n t i - E L 4 sera. Further, m o r e t h a n 50 different batches of r a b b i t and goat antisera against the L2 l y m p h o m a have consistently failed to cause a n y t u m o u r inhibition. This lack of universality of t u m o u r inhibition by xenogeneic antisera at present limits the usefulness of passive i m m u n o t h e r a p y of cancer.

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