Relationship of cellular and humoral immunity in virus carcinogenesis

Lite Sciences Vol . 3, pp., 1415-1488, 1984. Pergamon Press, Inc. Printed in the United Bt~Ees .

REIJITION$HIP OF C~:r .Tltr.sR AND HUMORAL IMF~IUNITY IN VIRUS CARCINO(3ENESIB A .C . Allison National Institute for Medical Research Dill Hill, London, England

(Rec:eived 17 Itovember 1984) Virus carcinogenesis is much more easily achieved in newborn than is adult animals .

However, adult animals inoculated with an oncogenic

virus are subsequently resistant to challenge with transplantable Lsologous tumours induced by the virus .

The accepted explanation of

these fladings has been summarised by Hebel (1) : the virus transforms twraal cello to tumour cells in the infected animals, both newborn and adult ; the transformed cells contain a aer "foreign" cellular antigen ; sad the newborn, "being immunologically incompetent tolerates the new antigen and allows tumour growth, whereas the immuaologically mature adult rejects the antigen and thus becomes hypersensitive or immune to later challenge with the tumour" . With the first and second of Habel'e postulates no dieagreeme.nt !.a poeaible .

There is now very substantial evidence that tumour cells

have asr antigens which are shared by all tumours induced by a particular virus .

These antigens as a rule do not cross-react with those of tumours .

induced with other virueea .

Specific aatigena are also demonstrable in

tumours induced by some viruses by complement-fixation teats with sera of tumour-bearing animals (2) . Certain problems are not yet solved, e .g . the relationship between tumour antigens sad viral antigens ; but these need not concern us here . ühat does concern us is Habel'a third postulate, that the newborn, being imsunolugically incompetent, becomes tolerant to the new antigen and this Rllow tumour growth .

We believe that thin view is incorrect, and that

as alternative explanation for the efficiency of carciaogenesia in the nerbora gnat be found . The Brat paint to bs recognized is that is all the systems studied hnmoral antibody appears after injection of virus into the newborn . Indeed, in some cases such a. BV-40 and adenovirus type 12, on] .y animals anbsequeatly developing tumours have demonstrable antibody .

Hence the

nerbosap rhich are going to produce tumours are not tolerant as regards eatibo~ production .

However, the point is not decisive because it is

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clear that cellular rather than humoral immune reactions are responsible for tumour rejection (3) and it is conceivable that an animal might become tolerant as far as cell sensitization is concerned but not as regards antibody production .

It might also be argued that the antigen

against which humoral antibody is directed has no relevance to transplantation, and that there is some kind of dissociated tolerance . The second point is more telling .

This is the fact that animals

injected with virus as newborns and transplanted with isologous tumours containing the corresponding tumour antigen do not, in fact, show pro longed tolerance .

In the case of mice injected with polyoma virus,

Babel's own experiments show a decreased resistance against tumour transplants for about

3

weeks followed by a much increased resistance .

Moreover, when rats are injected neonatsl'.y with polyoma virus, animals developing tumours show increased resistance against transplants of tumour cells containing the polyoma-specific antigen (4) .

Thus tolerance,

if it existe at all, is partial and transient . An alternative explanation for the temporarily decreased resistance against tumour transplants during the first three weeks is a particular relationship between cellular and humoral immune responses, comparable with enhancement .

The sdenovirus type 12 system is convenient for analysis

of the situation because antibody can be measured by complement fixation with tumour homogenates, and effects of cellular and humoral immunity can be dissociated .

In this paper preliminary results of such experiments

will be presented . Material and Methods Virus .

Adenovirus type 12, strain 1131, isolated and shown to be

oncogenio by M .S . Pereira and MacCallum (5)~was kindly provided by Lr . H .G . Pereira .

This strain of virus has never been passed in monkey cells, and

wss propagated and titrated by cytopathic effects in Hela cells and human embryo kidney (H .E .K .) cells .

Infected H .E .K . cell cultures contained

approximately 106 TCD~ per ml ., infected HeLa cell cultures approximately 2 x 105 TCD50 per ml . ; 0 .2 ml . of frozen and thawed infected cell suspension was injected aubcutaneouely into the interacapular region of golden hamsters ( Cricetus auratus ) about 24 hours old . Tumou~celle .

Two transplant lines (H-75 and H-118) of tumour cells

induced by adenovirus type 12 were maintained by cheek-pouch transfer in young adult animals ; both of these gave complement fixation with sera of tumour-bearing animals .

Suspensions of cells were obtained

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by cutting tumours into small pieces and incubating for~15-20 minutes at 37 ° in 0 .25îô trypsin or pronase (California Biochemicals) in (iey's saline solution without calcium and magnesium .

Celts were centrifuged,

resuspended in complete saline solution, lightly centrifuged to remove clumps and counted in a haemocytometer .

Cells were considered viable

if they resisted staining with 0 .0Nî eosin in saline for 2 minutes . The appropriate number of viable cells was injected is a volume of 0 .1 ml . into the hamster cheek pouch, and the pouch inspected for at intervals usually of

3

days .

The size of tumours was measured,

and tumours were weighed when animals were sacrificed after

3

weeks .

The transplant was taken as rejected if there was no sign of a tumour

3

after

weeks .

Preparation of lymph node cell suspensions .

Axillary lymph nodes

were pooled, cut into small pieces with a razor, suspended in Parker's solution 199 + 10A6 calf serum inactivated at 56° for

30

minutes, and

gently pressed through a stainless steel gauze to obtain suspensions of lymph node cells . above .

Total and viable cell counts were made as described

Tumour cell suspensions were incubated with ten times their

number of sensitized or control lymph node cells before injection into hamster cheek pouches . Observations Resistance of animals bearinK tumours to tumour transplants . The majority of hamsters inoculated subcutaneously with H .E .K . stocks of adenovirus type 12 as newborns developed tumours (table 1) .

When

tumours first appeared, injections of tumour cells from transplant lines were made into the cheek pouch .

As shown in table 1, tumour-

bearing animals were resistant to transplantation with cells containing the corresponding antigen .

This is similar to the findings of

Vandeputte and de Somer (4) in rats with polyoma-induced tumours . In interpreting these experiments, the curious immunological behaviour of the hamster cheek pouch must be borne in mind .

The

cheek pouch provides a barrier on the afferent, but not the efferent side of the immune reaction . Billingham et al . (6) have shown that an incompatible skin graft established in the cheek pouch is promptly rejected when the recipient is sensitized by a second graft from the same donor on the skin .

Similarly, in our experiments, animals

sensitized by prior exposure to virus subcutaneously reject grafts containing the . corresponding antigen made into the cheek pouch .

141 7

118

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CELLULAR AND SUMORAL ~I1âUNITY TABLE I Number of Hamsters Developing Cheek-Pouch Tumours Compared with Total Number of Animals Inoculated with Adenovirua type 12 Tumour cells

No . of cells inoculated

10 5

106

Normal hamsters

9/10

Tumour-bearing hamsters

1/8

10/10

Tumour cells + lymph node cells of tumour-bearing animals

6/8

2/8

6/8

Tumour cells + normal lymph node cells

4/5

5/5

In other experiments, lymph node cells from axillae of animals bearing tumours were shown to be sensitized because when mixed with tumour cells from the transplant line they reduced the number of takes in transplants (table 1) . Role of humoral antibody in oncogenesis .

The results just described,

as well as the findings of others previously mentioned, sugf;est that newborn animals do not become tolerant to the tumour antigens .

Never

theless, the immune cellular sensitization occurs more promptly in the adult, and reasons for the delay in the newborns can now be considered . one point that emerges from Habel's experiments (1) is that in the newborn mouse injected with polyoma virus, humoral antibody formation ~s delayed only slightly as compared with adults, whereas the appearance of resistance against transplants is much delayed in the newborn .

If

this proves to be generally true, it would seem that the limited number of lymphoid cells in the newborn exposed to this type of antigenic stimulus respond earlier and more efficiently by antibody formation than by cell sensitization .

Tndeed, the antibody formed early might

inhibit or delay the immune cellular reaction in a manner analogous to enhancement in adult animals .

The phenomenon of enhancement has been

studied in detail in a variety of systemB (see Kaliss, ~ and Brent, 8) . It is most simply demonstrated by passive administration to the recipient of antiserum directed against the antigenic components distinguishing the graft from the host ; the graft will then be accepted even though under comparable conditions it would be rejected in the absence of humoral antibody .

Experiments were carried out to ascertain wt;ether

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passive administration of antiserum would have air effect on the appearance of tumours is hamsters given adenovirus type 12 subcutaneously z4 hours after birth .

The serum was a pool from hamsters bearing

adenovirus type 12 tumours, mostly primary but a few with transplants shown to fiz complement with tumour homogenates .

Twenty four hours

.after administration of virus, one half of each hamster litter ras givea~0 .2 ml . serum subcutaneously at the site of injection of virus, and this wan repeated

7

days later .

The other animals remained as

littermate controls receiving virus only . TABLE II Peroentage of Tumours and Kean 7,atenay (Days) before Appearance of Tumours in Hamsters Inoculated with Adenovirua Type 12 on the Second Day .

One hall of each litter received Injections

of Antibody Subcutaneously on Days

Initial Viru~ Inoculum

Subsequent treatment

3

and 10

No . of Animals

H .E .K .

None

12

H .E .K .

Antibody

14

HeLn

None

14

HeLa

Antibody

16

~ tumours

Latency

75 "0 85 .5 7 .1 31 .3

37 .3

32 .1

56 48 .5

As shown in table 2, administration of antiserum certainly did not retard tumour formation ; instead, there were atroag indications of enhancement .

Other experiments in which whole litters had been treated

with antiserum also showed earlier appearances of tumours than controls . The differences as shown in table 2 are not statistically significant, but at certain stages (e .g . 4 de~ys after the appearance of the first tumour in the H .E .K . series) the differences between members of tumours and théir size attained significance at the 5A6 level . Decreased number of tumours is animals given repeated injections of virus .

Having established that antibody plays no part in preventing the

appearance of tumours in newborn animals, we were looking for methods of boosting the cellular immune response .

At this point we were informed

of the observations of Dr . Bernice Eddy that hamsters inoculated with SV-40 virus as newborns and again later developed fewer tumours than animals receiving ealy the first injection . were published by Deichman and Kluchareva (9) .

Similar observations

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We carried out similar experiments with adenovirus type 12, of which representative results are shown in table 3 . TABLE III Percentage of Tumours and Mean Latency (Days) Before Appearance of Tumours in Hamsters Inoculated on the Second Day of Life with H .E .K . Adenovirus Type 12 .

One Half of Each Litter was then (liven

Further Subcutaneous Injections of Virus on Days 8 and 15, or a Further Subcutaneous injection of Yirus in Adjuvant on Day 8, Followed by Virus Alone on Day 15 .

Second treatment

No . of animals

Sir Tumours

Latency

None

12

83 .3

36 .4

Virus

14

14 .3

42 .5

None

10

80 .0

38 .5

Virus + adjuvant

12

83 "3

33 " 7

These observations, together with other comparable results recently obtained by Eddy et al . (10), show unambiguously that subsequent injectia~na of virus delay the appearance and reduce the number of tumours in animals injected neoaatally with oncogenic viruses .

This shows that the immune

cellular mechanism is capable of responding to later antigenic stimuli, In animals

which is îtu~ther evideaee against the concept of tolerance .

that are tolerant, subsequent antigenic stimuli prolong tolerance and do not break it .

On the other hand, subcutaneous injection of

virus seems to be a relatively inefficient way of inducing humoral antibody responses .

We have ao far been unable to detect circulating

antibody in hamsters given repeated subcutaneous Injections of adenovirus type 12 and failing to develop tumours . Failure of subsequent injections of virus in adjuvant to reduce the incidence of tumours .

The simplest interpretation of the results

just described ie that repeated injections of virus boost the immune cellular response but not the humoral antibody response .

We thought

that it might be interesting to give the second injection of virus in Freund's complete adjuvant, which should boost both immune responses . Experiments on adult hamsters had previously shown that a single subcutaneous injection of adenovirus type 12 with adjuvant produces quite high levels of circulating antibody as well as sensitizing

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lymph node cells eo that they prevent establishment of homologous tumour cell grafts . Ae shown in table

3,

hamsters injected with adenovirus type 12

as newborns and subsequently given the same virus in complete Freund's adjuvant do not show retarded tumour development ; if anyDr . J . Samaille has recently

thing, tumour development is enhanced .

informed us privately that he has had very similar results in experiments carried out for other reasons .

Hamsters developing tumours

after exposure to virus and then virus with adjuvant were found to have relatively high titres of antibody, which was mainly directed against the virus rather than the tumour, as shown by reaction with the group-specific A antigen which is usually absent from tumours (11) . 'these results support the view that humoral antibody can exert an enhancing effect in virus carcinogenesis . Discussion A number of observations reported by other workers support the general view put forward in this paper .

Thus, Qoldner et _al . (12)

found that repeated immunization of hamsters with extracts of SV-40 or polyoma tumours in adjuvant enhanced rather than depressed the ßppearaace of tumours in animals injected with the corresponding viruses as newborns or transplanted with corresponding tumour cells es adulte .

Rauscher et al .

(13)

observed that by using Freund's

adjuvant tumours cold be obtained in chickens with doses of Rous sarcoma virus (RSV) too low to produce tumours in the absence of adjuvant ; much better antibody responses were obtained in the presence of adjuvant .

Conversely, Rubin (14) found that in chickens made

tolerant to RSV by embryonic exposure to RIF,]D00 times as much RSV was required for tumour induction than in chickens not tolerant to RSV .

Although interpretatiôn is complicated by the fact that RIF

in the tolerant birds could have interfered with RSV, the results can hardly be construed as supporting the concept that tolerance is required for oncogenesis . In general, the results suggest that tolerancq cannot be accepted as the explanation for the eusceptibiZity of newborn animals to virus oncogenesis .

It seems that a delicate balance between cellular

8~nd humoral immune responses takes place . experimentally+ .

This balance can be altered

Repeated administration of virus favours the cellular

immune response and inhibits tumour forme~tioa .

Passive administration .

~f antibody, or generation of antibody by the use of virus with

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adjuvants, diminishes the effectiveness of the cellular immune response and enhances tumour formation . Acknowledgements I am indebted to Dr . Bernice Eddy for making available her t~esults which are still in press, and to Dr . H .G . Pereira for allowing pe to quote serological findings which will be published jointly is fore detail later .

The technical assistance of Miss J . Wilson and

!'Ir . C . Gilchrist ie also gratefully acknowledged . References

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