Macrophage migration inhibition with mouse tumor antigens: Properties of serum and peritoneal cells during tumor growth and after tumor loss

CELLULAR

3, 113-122

IMMUNOLOGY

(1972)

Macrophage Migration Inhibition Properties of Serum and Tumor

Growth

and

W. J. Departrkkekzt

of

with Mouse Tumor Antigens: Peritoneal Ceils During After

Tumor

HALLIDAY

Pathology, Uuiversitp Seattle, Waslzirtgton

of

Loss 1

’

bvashiqtokk 98105

Mcdiral

Sclkool,

Sera and peritoneal cells (PC) were obtained from mice bearing- primary Moloney virus-induced tumors or transplanted chemically induced tumors (“progressor” mice) and from mice in which these tumors had spontaneously regressed or had been surgically removed (“regressor” mice). No stimulus was given to produce peritoneal exudates. PC from the two types of tumor-treated animals were distinguishable. “Regressor” cells had their migration in culture inhibited by the corresponding soluble tumor antigen, whereas “progressor” cells did not. Mixtures of the two kinds of PC were not inhibited. “Progressor” PC produced a soluble substance in vitro which could block the normal inhibition of “regressor” cells. ,Cera from the two types of mice were also different. “Progressor” serum blocked the migration inhibition usually found with “regressor” PC. “Regressor” serum not only lacked this property but was able to unblock “progressor” PC, so that mixtures of the latter cells with this serum were inhibited by tumor antigen. Rlacrophage migration inhibition thus revealed cellular immunity and humoral factors analogous to those found by other techniques in animals and human subjects exposed to tumor antigens.

INTRODUCTION

Many recent advances in cancer immunology have resulted from the observations that tumors have specific antigens (1-3) and that these antigens can stimulate a cellular (lymphocyte-mediated) immunity (4). The anomalous ability of tumors to persist in the face of the host’s specific cellular immunity has been at least partly explained by the demonstration of soluble “blocking factors” (5). These factors, 1 The work reported in this paper was undertaken during the tenure of an American Cancer Society-Eleanor Roosevelt-International Cancer Fellowship awarded by the International Union against Cancer. It was supported by grants CA-10188 and CA-10189 from the National Institutes of Health to Dr. K. E. Hellstrb;m and Dr. I. HellstGm, respectively, and by contract NIH-69-2061 from the National Institutes of Health to Dr. Charles McKhann, University of Minnesota, subcontracted to Dr. I. Hellstriim and Dr. K. E. HellstrGm. 2 On hustralia;

leave from the Department requests for reprints to this

of Microbiology, address. 113

0

1972

by

Academic

Press,

Inc.

University

of

Queensland,

Brisbane,

114

HALLIDAY

found in the serum of human patients and animals with progressing tumors, block or inhibit the anti-tumor cytotoxicity of immune lymphocytes in vitro. The precise molecular nature of blocking factors is unknown but there is suggestive evidence that they are complexes of tumor antigen with specific antibody (6). Confirmation of the above findings by an independent technique would greatly improve their credibility. Macrophage migration inhibition (MMI) offers such a possibility since it is a method of measuring cellular immunity which does not necessarily involve living target cells. Previous work (7-13) has shown that tumor cells and extracts specifically inhibit ilz vitro the migration of suitable cells taken from individuals sensitized to tumor antigens. MM1 is known to correlate in some systems ( 14-16) with delayed hypersensitivity reactions, which in turn resemble the cellular phenomena taking place in graft and tumor rejection. The basic premise of the present study was that blocking factors should be active in MM1 if they are of general importance in modifying cellular immunity to tumors. This paper describes the use of MM1 with soluble tumor antigens and mouse peritoneal cells, and the finding of blocking factors in the sera of mice with progressing tumors and in peritoneal cell cultures prepared from these mice. “Self-blocking” of peritoneal cells could explain their apparent inertness under some circumstances, since they could be shown to be inherently active by a suitable technique. A preliminary report of a portion of this work has appeared elsewhere ( 17). MATERIALS

AND

METHODS

Mice and tu~tool-s. Sarcomas were induced in adult BALB/c and A/.% mice by the intramuscular injection of 0.05 ml of Moloney sarcoma virus (MSV : a 1 : 7 dilution of lot #206-R from Dr. J. B. Moloney, National Institutes of Health, Bethesda, MD) ; these tumors grow progressively for about 10 days then spontaneously regress over a l-2 week period (lS, 19). Tumor 969 was the 7th-10th transplant generation of a fibrosarcoma induced in A/Sri mice by methylcholanthrene (MCA). Tumors 1013 and 1030 were the 4th-7th generation of fibrosarcomas induced in BAT>B/c mice by MCI. MCA tumors were removed surgically after 2-4 weeks growth. A transplantable Moloney tumor was maintained by serial passage in -q/Sri mice and was used only to prepare tumor extract (antigen). Peritonea,l cells. PC were obtained from groups of living mice without the production of exudates. The animals were anesthetized with ether and injected intraperitoneally with S ml of warmed Eagle’s minimal essential medium with heparin (5 units/ml). through a multiperforate l&gauge needle (20). The fluid w:ts withdrawn and re-injected several times and collected in tubes in an ice-bath. These tubes were centrifuged (all centrifugations of cells at 5” and 200s for 5 min), and the cells of each type were pooled in 3 ml of cold Eagle’s medium (with penicillin, streptomycin and 12% newborn calf serum). After counting and further centrifugation, the cells were resuspended m~iformly in the same medium (with 12% serum) to a density of al)out S X lO’/ml, and drawn into capillaries (20 ,~l Drummond Microcaps, Helena Laboratories, Beaumont, TX) which were then sealed at one end in a flame. The capillaries were centrifuged, cut at the cell-fluid interface, and the stubs containing the packed cells were placed horizontally in 35 X 10 mm tissue culture dishes (#3001, Falcon Plastics, Oxnard, CA) with no coverslips or silicone treatment, and were secured with a drop of silicone grease

MACROPEIAGE

hZ IGRATIOP;

INH

TRITION

115

(Dow Corning Corp., Midland, MI). The above medium ( 1.2 ml) and appropriate tumor extract (0.2 ml) were then added to each dish; where mouse serum or cell culture fluid were to be used, they were added (0.2 ml/dish) before the tumor estract. Incubation was at 37” in 5% CC&/air for 48 hr. Every effort was made to handle all capilIaries similarly. A group of 6 mice usually gave about 30 X IO6 PC, which yielded 20 capillaries; the samemice could be used repeatedly. PC consisted of about 40% small lymphocytes and 60% larger cells, which were not tested for phagocytic activity but were assumedto be macrophages; there was variable contamination with erythrocytes and suspensionsnoticeably red in color were discarded. “Progressor” PC were obtainecl from mice with ~~relI-deveIol~edgrowing tumors (7-10 days after MSV injection or 2-3 weeks after MCA tumor transplantation). “Regressor” PC were obtained about 2-5 weeks after Moloney tumor regression or about 4-8 weeks after surgical removal of transplanted MCA tumors. Molise Scrtllz-1 . Serum was from A/Sri mice bearing transplanted tumor 969 (969P serum) ; A/S n and BALB/c mice with progressing primary Moloney sarcomas (MP serum) ; and A/% and B&&LB/c mice whose primary Moloney tumors had regressed (MIX serum). Different pools of serum were used in separate experiments. -111sera were stored at -20’ ; they were filtered to sterilize and inactivated at 56” for 30 min before use. Tzt~zor extract, Antigen was prepared from fresh tumor tissue by homogenizing in 4 volumes of pllosphate-b~iffered saline, and centrifuging at 1OOOgfor 30 min then 100,000~ for 30 min (10, 11). The resulting clear supernatants were stored at -70” until required, then thawed and centrifuged at IOOOgfor 10 min before use. They were tested for toxicity against normal PC; usually it was found that 1 part of iextract in 9 parts of medium was not illh~l~ito~~ to nlig~atio~l, but larger amounts were toxic. CatEtivation of PC. Cells harvested and washed as described above were suspended in Eagle’s medium with 12% newborn calf serum, to a concentration of 4 X lo6 cells/ml. One lnilliliter volumes were placed in plastic dishes (Falcon #3001) and inculcated without agitation at 37” for 48 In-, with one addition of fresh medium (0.5 ml/dish) at 24 hr. After harvesting, the culture fluids were pooled and concentrated about tenfold by membrane ultr:~filtratiotl (Amicon Corp., Lexington. &I?r >1then sterilized by filtration. JJeasarr~~zcnfsand calculations. Areas of cell migration were traced with the aid of a camera lucida at 20X magnification, and were measured by planimetry. Tracings were made without knowledge of the numbering system for the different mixtures. The measnred migration areas (in cm*), usually in quadruplicate (4 capillaries/dish), were subjected to analysis of variance and t-test, comparisons being made only between dishes containing the same PC and different antigens, sera or culture fluids. The u~igratiotl of PC from different groups of mice, or from the samemice at different times, was variable. RESULTS MMI

~~t~ Afottse PC and ~~~~,~~~?~e Tumor

~~t~~f?~~s

Mouse PC prepared as described usually migrated well and gave easily measured zones, Successful demonstration of MM1 depended on the proper preparation of

116

IlALLIDAY

the PC donors. It was eventually realized that only mice which had lost their tumors some time previously (“regressors”) gave PC which were directly and consistently reactive. Reactivity gradually appeared 24 weeks after transplanted MCA tumors were removed surgically, but a shorter time sufficed for MSVinduced primary sarcomas (spontaneously regressing). Animals bearing progressing tumors (“progressors”) had PC which appeared to be inactive (that is, not inhibited by antigen), as shown in Table 1 which is representative of many similar experiments. The experiment shown is a double one, done in a “criss-cross” pattern which provides constancy of serum and extract concentrations and adequate controls for specificity and toxicity. The PC of “regressor” mice (but not of “progressors”) had their migratio~l inhibited by the corresponding tumor antigen extract, but not by an unrelated antigen.

Since mice have circulating anti-tumor antibodies and possibly other factors both during tumor progession and after loss of tumors (5, 21)) it was of interest to examine the effect of serum from such mice on MMI. Accordingly, pooled serum from animals exposed to either of two tumors was adder1 to the migration dishes. This was not autochthonous serum, but was obtained from other groups of mice appropriately treated (both “progressors” and “regressors”). “Regressor” PC were used as the test cells, since these were known to be reactive. Table 2 shows the experimental design employed and the results obtained in three consecutive experiments. Each extract and each serum were tested against each type of PC to exclude possible non-specific toxicity and stilnlllation. For the MCA tumor 969, serum TABLE EFFECT

OF

SPECIFIC

TINOK

,tNTIGEN

1

ON nillCKATION PERITONEAL Cmrs

Migration Area (mean & SE)

PCU

OF “P1~0GR1?SS0l~”

Change in IVligration

969P 969P

M 969

16.5 17.8

f f

2.2 1.0

+

969R 969R

M 969

24.4 9.2

f f

0.7 1.9

-620/,

MP MP

$1 969

15.7 18.5

zt 1.3 zk 0.8

-15%

MR MR

M 969

12.4 26.7

* i

-54%

1.5 0.4

3%

AND

“~
P Not significant < 0.00 1 Not significant
t’ Peritoneal cells obtained from mice exposed to tumors as follows: 969P-Bearing MCA 969 tumors transplanted 2-3 weeks previously (A/!% mice). 969R-MCA 969 tumors remoxved 5-6 weeks previously (A/Sri mice). MP--Injected with MSV 7 days prexriously and tumors progressing (BALB/c mice). 1~lR-Injected with MS\: 5 uTeeks previously and tumors regressed (BAf.B/c mice). b Tumor extracts prepared from >‘loloney (21) aud MCA (969) tumors in A/.% mice.

&fACROPHAGE

hIIGRA’I’JOK

117

INHIBITIOiX

from syngeneic mice bearing this tumor neutralized the inhibition of 969R PC caused by 969 extract (compare mixture 6 with 5 or 4), but this serum had no stimulator,y effect on MR cells. Conversely, MP serum from syngeneic or allogeneic mice reversed the migration inhibition inflicted on MR cells by allogeneic Moloney tumor extract (compare mixture 8 with 9). In the latter situation, migration was consistently augmented above the controls (compare mixture 8 with 10, 11, 1.2). In some experiments (I and II), MR serum neutralized partially the inhibition of MR cells (compare mixture 7 with 9 and 10, 11, 12). Are

‘~Progressor”

PC Inert?

The apparent inactivity of PC from animals with growing tumors was noted above. Since this observation seemedto be at variance with some findings reported to the literature (22-25) although consistent with some others (20) (see Discussion), it u’as decided to investigate the effect of “progressor” PC or their products on the MM1 of “regressor” cells. The first procedure used was to mix “progressor” (not inhibited) and “regressor” (inhibited) PC in equal numbers, and to see whether or not the cell mixture was inhibited by the appropriate antigen. Continued inhibition would indicate inertness of the “progressor” PC; abrogation of inhibition would show that these cells were releasing or actively producing a blockin g substanceand might themselves be potentially active in MM1 under the proper conditions. As seen in Table 3, “regressor” and “progressor” PC, active and inactive respectively when tested alone

JIisture No.

PC

.\ntigen

Serum

Experiment I

1

969 I<

2 .3 4 5 6

969K 969K 969K 969R 969R

7

and and

11 11

n1 I< XlP

M 969 969 969

iv I<

8 9 10 11

n1 M M M

12

iv1 Ii

969P 1Ll Ii VI I’ 969P

XI

K Ii I< Ii

10.6 14.3 14.5 5.9 7.0 14.3

M I<

II

=!I 0.7

12.3

i

0.5

f i It i

0.9 1.4 0.4 0.6

1.1.2 10.7 5.6 3 9

f f f f

2.2 0.4 0.2 0. 3

+

2.1

10.8

AZ 1.3

p

2 and
‘1

~____~~~ 10.0

III

~~~

+

0.4

9.0 10.9 4.0 4.3

f f i i

1.2 1.9 0.9 0.6

9.7

f

0.4

8.7

f

0.7

8.6

f

0.2

8.7

f

1.3

11 11 969 969

41 I’ 969P >I Ii MP

17.5 3.7 10.4 8. 5

f f

1.5 0.7

13.8 4.3

i f

0.7 0.7

18.1 7.3

f *

1.3 1.1

969

969P

9.0

f f +

0.6 0.3 0.3

IO.6 12.2 12.5

i 1.3 ztz 1.1 i 1.0

11.4 13.3 14.2

+ 0.8 3~ 0.6 f 0.9

n Conditions identical in each experiment, except I< and P sera from from ,4/S mice in II. PC were from mice which had lost their Maloney sarcoma in BALB/c) 4-7 weeks previously. * The statistical significance of some of the differences is as follows

ment III): Mixtures mixtures 8 and 9,

No.

5, p
BALB/c tumors (t-test

mice (WICA applied

”

in I and I I I, 969 in A/Sri to

Experi-

; mixtures 6 and 5, p O.OS; mixtures 8 and 11, p
;

118

Iv1 igrxtion Area (mean i SE) _..~__ 10.9 i 1.0 14.3 i 0.6

969R 969R 969P 969P

969 M

19.9 21.0

f 1.1 zk 0.5

969P 969P

and 969R and 969R

969 M

14.8 13.4

i f

969K 969R

and normal and uormal

969 M

10.2 15.6

969P 969P

aud normal and normal

969 M

17.7 16.6

zr,fj PC and antigen

as for Table

Change Migration

iu P

-22%

<0.05

Not significant

0.7 1.1

-i-10%

Not significant

+ 1.3 zk 1.2

-33%


* i

-I- 7%

2.6 2.8

Not significant

1.

for LIxMI, were inactive when mixed together. That is, the “progressors” had imposed their properties on the other cells and inhibition was abrogated. Truly inert cells (from normal control animals) did not have this ability. Similar experiments, with comparable results, have been done with other tumors also (MCA and Moloney). Further evidence for the procluction of blocking factors by “progressor” PC was sought by cultivating these cells in vitro for 2 days. The culture media were then concentrated and tested for their effect on the MMI of “regressor” cells as described above for serum. Once again two different tumors were used and a double experiment was set LIP to provide controls as in Table 4. MM1 was specifically and

-.--

PCs

Antigeu

h

.__~-

PC furuishing culture fluid

Migration area (: (mean f SE) ~ ___33.8 f 1.6 37.8 xk 1.4

Change in migration

1013R 1013R

1013 1013

10 I 3 t’ 103UP

101311 1013R

1030 1030

1013P 103OP

49.6 50.6

f 1.8 zk 2.3

-

1030R 103OR

1013 1013

1013P 10301’

35.3 41.2

It 2.5 f 1.2

+16%

f030R 103OK

1030 1030

lOI3P 103OP

22.9 41.3

f 2.5 rk 4.3

+8Q%

-___.-__

+16% 2%

P <0.05 Not significant Not SignifiCilllt


a Peritoneal cells obtained from BALB/c mice exposed to tumors as follows: 1013R-MC.S. 1013 tumors removed 4-5 weeks previously; 1030R-MCA 1030 tumors removed 4-5 weeks previously. h Tumor extracts prepared from MC.4 tumors 1013 and 1030 in BALB/c mice. c Concentrated fluid from cultures of PC from BALB/c mice bearing progressing tumors 1013. and 1030.

MACROPHAGE

MIGRATION

119

INIIIRITION

significantly reversed by the two culture fluids. The results shown here suggest that “progressor” PC do indeed produce or release soluble factors which can specifically abrogate MM1 in appropriate systems.

If PC from tumor-hearing animals were “self-blocked,” it seemed possible that their potential activity might be revealed by antagonizing the blocking factors. Serum from “regressors” which have recovered from Moloney tumors has an “unblocking” activity, i.e. it is capable of reversing the Hocking caused by “progresso? serum in cell inhibition studies (21 j. “Regressor” serum was therefore examPC. Syngeneic mouse serum from 3 ined for it:5 ability to unblock “progressor” sources (i\lR, MP and normal BAT,B/c) was added separately to the migration dishes, together with either Jlolonev antigen or control antigen. “Regressor” serum appeared to have an effect which could be ascribed to unblocking : it permitted MM1 to take place with “progressor” PC, whereas “progressor” and normal sera did not (Table 5). DISCUSSION The application of two simple technical advances has made the use of MM1 in mouse tumor systems a more convenient and reliable test than it was in the past (8). Firstly, soluble tumor extracts as described for guinea-pig (10) and human tumors (11.) were found to be effective antigens, as well as being easily handled, stable when stored, and free from optical interference in the observation of migration patterns. Secondly, the PC collection technique of Bat-ski and Youn (20) permitted the obtaining of adequate numbers of cells from living unstimulated mice, and this enables serial studies to he done on the same groups of animals. Previous successful attetnpts to demonstrate MM1 in experimental oncology employed animals whose tumors had heen removed or had regressed after a period of

Experiment PC

Antigen”

Seruni’

.\ligration (mean + SE)

I

Experiment -____~

Change in Migration

hligratim (meatl i

P

SE)

2

Change in Jligration

p

MP

M 969

N N

21.2 22.4

f f

2.1 1.1

-

6%

NS

36.7 31.6

f f

0.9 1.4

+160/o

NS

511’

?iI 969

I’ I’

21 .a f 19.6 f

0.7 0.7

+

9%

NS

32.6 29.7

f f

1.3 1.4

+lo%

NS

11 I’

M 969

R R

16.7 29.5

< 0 00 1

Lh..Z 38.2

f f

0.6 1.0

-31%


r!z 1.1 zt 0.7

-43yo

I’ Peritoneal cells from female BALB/c mice injected ing large growing tumors. h Antigen as for Table 1. c Serum from normal BALB/c mice (IN). “progressor” and “regressor” mice (MS\’ 1 weeks previously).

10 da>,s previously

B.-\I.B/c

mire

with

(MS\’

MSV,

7 days

and

bear-

previously)

120

ITAILIUAY

growth (8, 9) or animals which were hyperimmunized with tumor preparations (7, 8, 10). Studies with human patients are usually done while the tumors are still present or only recently removed, and inhibition of migration of blood leukocytes has been observed in many cases (11-13). In addition to showing that PC taken from mice a certain time uftev tumor loss were uniformly active (Table l), the present work defines more clearly the status of mice with progressing tumors. These animals’ PC were generally not inhibited by specific antigen (Tables 1 and 3), and furthermore, this apparent inactivity persisted for several weeks after tumor removal in the NCX-induced sarcoma systems used. It is of great interest to note that a similar “inertia” of PC was described in connection with the colony inhibition test for cellular immunity, and activity against tumor target cells was apparent only some weeks after removal of the tumors (20). PC may be a special cell population not strictly comparable to the cells used by others in studies of cellular immunity to tumors; blood leukocytes and lymph node cells from tumor-bearing individuals have been found to be reactive against tumor cells (4)) as discussed below. The blocking or abrogation of MM1 by the serum of specific tumor-bearers was shown for two different tumors (Table 2). An MCA tumor system showed simple restoration of migration to the level of controls incubated under comparable conditions. In the Moloney tumor system, not only was inhibition abrogated by serum from “progressors” but the cells consistently seemed to have gained increased migratory activity. “Progressor” sera thus contain tumor-specific blocking factors, possibly but not necessarily identical with those described in cytotoxic systems using lymphocytes and live tumor cells (22). Whatever the mode of action of the blocking factor or factors (attachment to tumor antigen or to immune lymphocyte or to macrophagej, the relative proportions of the reactants are undoubtedly important in the in vitro test; at present the quantities of these are arrived at empirically and may not always be optimal. The fact that MM1 of “regressor” PC could be blocked by extrinsic factors from serum, and the knowledge that lymph node cells of “progressor” animals were highly active in other tests of anti-tumor immunity, suggested that the “progresso? PC might be potentially active but “self-blocked.” That this may be true is supported by the findings reported in Tables 3 and 4. “Progressor” PC have a blocking capacity, as shown by their behavior in cell mixtures with “regressor” PC and by their elaboration of soluble blocking factors when cultivated separately in vitro. This is of course not a proof that “progressor” PC are inherently sensitive to MM1 but this possibility is supported by the data of Table 5. Here “progressor” PC were persuaded to undergo MM1 when “regressor” serum was added to the medium. Although these particular experiments are preliminary and some desirable controls are lacking at present (for example, controls for tumor specificity), the results are of possible importance in several ways. They confirm, by a different technique, another phenomenon discovered first by colony inhibition studies (21) ; they permit an analysis of the mechanisms of blocking and unblocking ; and they indicate that “progressor” PC contain a sub-population, sensitive to MM1 and therefore immunologically reactive against tumor antigens. The relationships between so-caIIed unblocking and other properties of “regressor” sera (such as com-

MACROPHAGE

MIGKATION

INHIBITION

121

as also are the conditions for ohplement-dependent cytotoxicity) are unknown, taining highest unblocking activity. The figures given in Table 2 indicate that sera map sometimes have some l~locking activity. A further enigma is “regressor” the nature of the blocking factor(s) from PC and whether or not they are the same as those detected in serum by MM1 and colony inhibition tests. Is the same material also produced by other cells or organs, for example the spleen, in viva and in vitro ?

“Progressor” PC may owe their distinctive properties to a population of antibody-forming or antibody-bearing cells or to release of antigen by macrophages, or to a combination of these and possibly other factors. It may be of interest to point out some resemblancesand differences in comparison with other findings in this field. As noted above, Barski and Youn (20) found that PC were active against tumor cells in colony inhibition only when obtained from “regressor” animals, and suggested that an immunological inertia existed under the influence of tumor growth in “progressors.” Mikulska, Smith and Alexander (26) arrived at a similar conclusion by the use of spleen cells and an assay in viva. On the other hand, Hellstrijm and Hellstrtim (22) found lymph node cells and peripheral leukocytes from both “progressors” and “regressors” to be active; activity could be blocked by “progressor” serum but this humoral property was lost after tumors regressed or were removed. The present results with MM1 of PC resemble thclse of Barski and Youn (20) but can be reconciled with the opposing findings by the observation that “progressor” PC produce their own blocking factors. The appearance of detectable immunological activity only some days or weeks after loss of tumors is known for some other experimental systems (19, 27, 28) in addition to those already mentioned, It is difficult to compare these techniques with other immunization methods, such as those utilizing killed cell vaccines, since tumor progression is not part of the procedure in these cases. Further work is necessary to determine the correlation of MM1 with specific delayed hypersensitivity and tumor growth in viva and with other tests in vitro. It would be interesting to know the detailed kinetics of these phenomena: their rise and fall during carcinogenesis, tumor progression, tumor removal or regression, recovery and subsequent life of the individual. Preliminary experiments by the author (unpublished ; also see 29) indicated that delayed hypersensitivity to tumor antigens was correlated with MMI, both being minimal in “progressor” animals and gradually becoming more pronounced after tumor loss. The lack of hypersensitivity in tumor-bearers was not a general anergy, since delayed reactivity to sheep erythrocytes could be developed (29). Another important correlation is that between tumor growth and presence of serum blocking factors, recently studied with polyoma tumors in rats (30). As these and other studies progress, a picture of the total immune responseto tumors is gradually emerging. ACKNOWLEDGMENTS The hospitality and advice of Dr. assistance of Mrs. Linda Katzenberger, .gratefully acknowledged.

K.

E. Hellstri;m Mrs. Sherrie

and Dr. I. Hcllstriim and the skilled Wilke and Miss Char Crossman are

122

IIALLIDAY

REFERENCES I. Old, L. J., and Boyse, E. .4., /Iwz. Rev. Med. 15, 167, 1964. 2. Sjogren, H. O., Prog. EXp. 7‘tbrrzor RES. 6, 289, 1965. 3. Klein, G., .-1,z~. Rrrj. Microbial. 20, 223, 1966. 4. Hellstriim, K. E., and Hellstrom, I., .i2dva11,. Cawcv Res. 12, 167, 1969. 5. Hellstrom, K. E., and Hellstrom, I., Ann. Reu. Micl-obiol. 24, 373, 1970. 6. Sjogren, H. O., Hellstrom, I., Bansal. S. C., and Hellstriim, K. E., Proc. Nat. Acud. Sci., in press, 1971. 7. Pekarek, J., Svejcar, J., Vonka, \-., and Zavadova, H., %. I~lwr~tJorsch. 134, 449, 1968. 8. Halliday, W. J., and Webb, M., J. Il’nt. I‘a~zcev Inst. 43, 141, 1969. 9. Kronman, B. S., Wepsic, H. T., Churchill, W. H., Zbar, B., Borsos, T., and Rapp, H. J., Science 165, 296, 1969. 10. Bloom, B. R., Bennett, B., Oettgen, H. F., McLean, E. P., and Old, L. J., Proc. Nat. Acad. Sri. 64, 1176, 1969. 11. Andersen, V., Bendixen, G., and Sch$dt, T., Acta Med. Scazd. 186, 101, 1969. T., and Dissing, I., Id. J. Cawcr 5, 12. Andersen, V., Bjerrum, O., Bendixen, G., Schigdt, 357, 1970. 13. Steiner, T., and Watne, -4. L., Carlcer Kes. 30, 2265, 1970. 14. George, M., and Vaughan, J. H., Proc. Sot. Exp. Biol. Med. 111, 514, 1962. 15. David, J. R., Al-Askari, S., Lawrence, H. S., and Thomas, L., J. 1wwz~noZ 93, 264, 1964. 16. Rosenberg, S. A., and David, J. R., J. I~zmunol. 105, 1447, 1970. 17. Halliday, W. J., J. Inzwwzol. 106, 855, 1971. 18. Perk, K., and Moloney, J. B., I. Nat. Cafrccr Iwt. 37, 581, 1966. 19. Fefer, .4., McCoy, J. L., Perk, K., and Glynn, J. P., Calzcrr lies. 23, 1577, 1968. 20. Barski, G., and Youn, J. K., J. i$‘ut. Cnnccr Iwt. 43, 111, 1969. 21. Hellstrom, I., and Hellstrom, K. E., Irlt. J. Cancer 5, 195, 1970. 22. Hellstrom, I., and Hellstrom, K. E., lilt. J. Cawcr 4, 587, 1969. 23. Hellstrom, I., Hellstrom, K. E., and Sjogren, H. 0.. Cell. Ir~rr~ur~ol. 1, 18, 1970. 24. Datta, S. K., and Vandeputte, hi., C’trncev RES., in press, 1971. 25. Sjijgren, H. O., and Borum, K., Cwcer Res., in press, 1971. 26. Mikulska, 2. B., Smith, C., and Alexander, P., J. ATat. Cancrr Inst. 36, 29, 1966. 27. Pilch, Y. H., and Riggins, R. S.. Ca~irrr Res. 26, 871, 1966. 28. Harder, F. H., and McKhann, C. F.. J. Nat. Cwcer Iwf. 40, 231, 1968. 29. Hoy, W. E., and Nelson, D. S., Sutwr 222, 1001, 1969. 30. Bansal, S. C., and Sjiigren, H. O., Progress ire Irrwrutzolog~ (Procecdi~r~s of the First