The Abrogation of Immunological Tolerance by Means of Allogeneic Confrontation

Transplant. Rev. (1972), Vol. 12 180-197

Published by Munksgaard, Copenhagen, DenmarkNo part may be reproduced by any process without written permission from the author(s)

The Abrogation of Immunological

Tolerance by Means

of Allogeneic Confrontation

PETER MCCULLAGH

This article is a resume of a series of investigations on, the abrogation, bymeans of the injection of allogeneic lymphocytes, of the state of immuno-logical tolerance of rats for sheep erythrocytes. A preliminary attempt willbe made to correlate the interpretations of the various groups of experiments.However, speculation on the nature of 'allogeneic abrogation' will be con-fined to sheep erythrocyte tolerance, and the possibility that similar phe-nomena may be demonstrable in other examples of immunological unre-sponsiveness (for example, homograft tolerance and tolerance of self) willnot be discussed. Reports of experiments to which reference is made in thetext but which are not specifically identified in the reference list have beensubmitted to the Australian Journal of Experimental Biology and MedicalScience.

1. CHARACTERISTICS OF SHEEP ERYTHROCYTE TOLERANCEIN RATS

To assess the significance of 'allogeneic abrogation' of erythrocyte tolerance,it is necessary to outline the features of this form of unresponsiveness.

a. The induction and maintenance of tolerance. Immunological tolerance ofsheep erythrocytes was induced in the rats used in these experiments bymeans of a series of injections of erythrocytes. This regime entailed thebi-weekly intraperitoneal injection of 0.25 ml of a 50 per cent suspension of

Department of Immunology, TTie John Curtin School of Medical Research, TheAustralian National University, Canberra, Australia.

ABROGATION OF TOLERANCE 181

sheep erythrocytes. The first injection was given on the day of birth, halfof the inoculum being administered intravenously. The intraperitoneal in-jections were continued for approximately 10 weeks after birth, with thecourse of injections being terminated 8 or 9 days before a rat was to be usedin an experiment. Rats were bled several days after the last injection so thatserum haemolysin titres could be measured. Immunological tolerance, assessedby the absence of haemolysis at the highest serum concentration examined (1in 4), was achieved in excess of % of the animals prepared in this manner.

b. Stability of tolerance. In any investigation concerned with the abrogationof tolerance, it is necessary to take account of the possibility that spon-taneous cessation of the tolerant state has intervened. If rats which havebeen rendered tolerant as described in the preceding paragraph are leftwithout any further erythrocyte injections, a gradual reversion towardsnormal reactivity to sheep erythrocytes occurs. To observe this spontaneousreturn of reactivity, a group of 20 rats in which tolerance had been inducedwas foilowed by means of routine, weekly examinations of sera for haemo-lysin after cessation of the bi-weekly injections of erythrocytes. This wassupplemented by occasional challenges with sheep erythrocytes. Of these20 rats

- Four remained unresponsive to challenge with sheep erythrocytes as lateas 120 days after the cessation of bi-weekly erythrocyte injections.

- One rat failed to respond to challenge at 17 days, but spontaneously pro-duced haemolysin after 59 days.

- Tn 7 other rats, haemolysin appeared spontaneously after 52 days, althoughno response had been detected following earlier antigenic challenges.

- In a further 3 rats, haemolysin, although detected for the first time between20 and 30 days, at a titre of 1 in 4, did not increase above this level untilthe fifty-second day.

- In the case of the remaining 5 rats in this experiment, a serum haemolysintitre of 1 in 4 was recorded in response to challenge between 30 and 50days, but higher titres were not observed until after 50 days.

From these results it can be seen that immunological tolerance of the typeunder review f>ersists for at least a month, and often for periods in excessof 7 weeks, after cessation of bi-weekly sheep erythrocyte injections.

182 PETER McCULLAGH

c. The consequences oj transferring normal syngeneic thoracic duct lympho-cytes to erythrocyte-tolerant rats. To place the sequelae of allogeneic lympho-cyte transfer to tolerant rats in perspective, some features of the transferof syngeneic cells should be noted. If erythrocyte-tolerant rats of an inbredstrain are injected intravenously with thoracic duct lymphocytes from nor-mally reactive (non-tolerant), syngeneic rats and then challenged with sheeperythrocytes, no haemolysin response is detected (McGregor, McCullagh &Gowans 1967), regardless of the number of lymphocytes transferred. Notonly are serum haemolysin and splenic plaque-forming cell responses absentin the recipients, but the transferred, nonnal cells appear to become immuno-logically unreactive themselves after a period of 3 days residence in thetolerant host (McCullagh 1970b). If lymphocytes from normal donors whichhave been immunologically stimulated by incubation in vitro with erythro-cyte-primed peritoneal macrophages are transferred to a tolerant host, ahaemolysin response is observed, but such tolerant recipients fail to respondto rechallenge. That is, although an immune response cannot be initiatedin a tolerant host, a response already initiated outside the tolerant rat canbe completed in it. In contrast to this situation in the intact, tolerant rat inreceipt of unstimulated normal syngeneic lymphocytes, a haemolysin re-sponse is observed if similar cells are transferred to a sublethally irradiatedtolerant rat prior to its challenge with erythrocytes (McGregor et al. 1967).However, this response is much weaker than that observed in an irradiated,non-tolerant recipient, unless the lymphocytes to be transferred have beenstimulated by incubation in vitro with antigen-primed macrophages (MeCullagh 1970a). This indicates that, even after irradiation, the tolerant ratis less suitable than its non-tolerant equivalent as a host in which trans-ferred, normal lymphocytes can be stimulated.

d. Repression of immune responsiveness exerted by the cells of tolerantrats. The failure of normal, syngeneic lymphocytes to mount an immunerespt>nse against sheep erythrocytes when transferred to a tolerant hostsuggests that there is some factor in the tolerant rat that is inimical to theinitiation of an immune response against this antigen. This has been con-firmed with the demonstration that thoracic duct lymphocytes transferredfrom erythrocyte-tolerant donors are able to completely suppress the specificresponsiveness of normal recipient rats (to be published). From the natureof the cells transferred in these experiments, it is highly improbable that thetransfer of tolerance is antigen dependent. Furthermore, the prolongedduration of the tolerant state that is observed to follow its transfer to normalrats in this manner is inconsistent with a requirement for the persistence ofantigen in the recipient if tolerance is to be maintained. It is surmised that


TABLE IThe effect of transferring syngeneic or allogeneic lymphocytes

to erythrocyte-tolerant rats at the time of challenge with sheep erythrocytes

Strain oferythrocyte-tolerant rat

(Lewis X DA) Fi

"

""

Lewisn

n

n

DA"

"

Strain oflymphocyte

donor

Lewis""

"

DAH

tt

II

Lewis X DA) F"

DA

"

"•1

"

Number oflymphocytestransferred

(K 10")

38

47.26.2763.62.56

69

"II

II

"

Total splenicpi aque-form ing

ceil content(X I0-")

170720434340107

993102(X)25

450240

n.d.n.d.n.d.n.d.n.d.n.d.n.d.

Interval betvv-eenimmunization and

plaque-formingcell a.ssay (hrs)

89

90929496868992

9395

110666911

92116122165

n.d. — none detected.

Each tolerant rat was injected intravenously with the number of lymphocytes indicatedin the third column, together with IC sheep erythrocytes. The results presented havebeen selected from larger groups previously published. Reproduced with permissionfrom J. exp. Med. (1970) 132, 916 and Aust. J. exp. Biol. med. Sci. (1970) 48, 369.

transferred tolerance (and, presumably, the tolerant state produced by therepeated injection of antigen from birth) can be maintained by an activerepressive response on the part of lymphocytes in a tolerant animal. Morerecently, it has been found that the ability of transferred, normal, syngeneiclymphocytes to mount a response in. sublethally irradiated, tolerant hosts isattributable to an interference with the activity of repressor cells in the host,by means of the irradiation (to be published).

2. THE EFFECTS OF CONFRONTING ERYTHROCYTE-TOLERANTRATS WITH NORMAL, ALLOGENEIC LYMPHOCYTES

In sharp contrast with the consequences of transferring normal, syngeneiclymphocytes to erythrocyte-tolerant rats, the transfer of allogeneic lympho-

184 PETER McCULLAGH

cytes results in a strong immune response on the part of the tolerant recipientto challenge with sheep erythrocytes (Table I). By the use of iso-antisera,the plaque-forming cells differentiating in the tolerant host in this situationhave been shown to be predominantly of host origin (McCullagh 1970c).There is appreciable variation between individual animals in the magnitudeof the response when tolerance is abrogated in this manner. This variationmay be attributable to the measurement at a single time of a response, thetiming of which varies slightly from one rat to another. The strongest re-sponses attained during abrogation may exceed the peak response of normalrats immunized with sheep erythrocytes alone. The weakest may be less than1/lOth of the response of normal rats.

The time of occurrence of the peak response differs from that observedfollowing the challenge of normal rats with sheep erythrocytes. Whereas thepeak splenic plaque-forming cell response of normal rats is usually observedduring the 5th day, the peak of the response, if tolerance is abrogated byconfrontation with allogeneic lymphocytes, occurs some 90 to 92 hoursafter challenge. Apart from this difference in its timing, the peak responseof rats in which tolerance is undergoing abrogation extends over a muchshorter period than that observed in nonnal rats that have been challengedwith sheep erythrocytes. Thus, the splenic plaque-forming cell responsedeclines to low levels within hours of attaining its peak. Abrogation oftolerance by means of confrontation with allogeneic lymphocytes has beenobserved to occur in a similar manner in horse erythrocyte-tolerant rats(unpublished).

3. FACTORS INELUENCING THE CAPACITY OE ALLOGENEIC CELLSTO ABROGATE ERYTHROCYTE TOLERANCE

a. The type of allogeneic cell transferred. The type of cell transferred froman allogeneic donor to an erythrocyte-tolerant host influences the efficiencyof abrogation. Thus, allogeneic thoracic duct lymphocytes and spleen cellshave a comparable, high efficiency. Thymus cells are much less effective,while bone marrow cells have very little capacity to abrogate tolerance.Such a distribution of activity reflects the general immunological activityof cells from these sources and suggests that the ability to abrogate toleranceresides in mature, immuno-competent cells rather than in their precursors.

b. Tfze genetic relationship of the donor to the erythrocyte-tolerant recipient.Lymphoid cells from a variety of types of donor are capable of abrogatingerythrocyte tolerance. Thus, lymphocytes transferred from a rat of unrelatedstrain are effective, as also are cells from a parental strain rat that have

ABROGATION OF TOLERANCE ]85

been transferred to a tolerant Fi hybrid recipient and cells from Fi hybriddonors transferred to tolerant parental recipients (McCullagh 1970c). Intwo closed, but non-inbred, rat colonies, lymphocyte transfer between themembers of one colony was not effective as a stimulus to the abrogationof tolerance (unpublished).

It is evident that allogeneic or semi-aJlogeneic lymphocytes are effectivein abrogating tolerance. However, it is not necessary that the transferredcells be able to mount a graft-versus-host reaction against the host, nor isthe tolerant host required to mount a homograft response against the trans-ferred cells. Genetic dissimilarity between the cell donor and the tolerantrecipient suffices for abrogation. The failure of lymphocytes transferred be-tween outbred rats lo initiate abrogation of tolerance indicates that a majorgenetic difference between cell donor and erythrocyte-tolerant recipient isrequired.

c. Tfte immunological relationship of tfw cell donor to the erythrocyte-tolerant recipient. Although a major genetic difference between the lympho-cyte donor and the erythrocyte-toierant recipient is required, this differencealone is not sufficient to ensure that tolerance is abrogated. Lymphocytesfrom a donor which differs markedly from the erythrocyte--tolerant recipientare ineffective if the donor has been rendered imm.unologically tolerant ofthe recipient's tissues. This is so, regardless of whether the erythrocyte-tolerant recipient is allogeneic or semi-allogeneic with respect to the lympho-cyte donor that is tolerant of its tissues. Thus, cells from a parental strainrat that is tolerant of Fi hybrid tissues are ineffective in an erythrocyte-tolerant Fi hybrid, while cells from a rat tolerant of the tissues of anunrelated strain are ineffective in an erythrocyte-tolerant recipient of thatstrain. Whereas both graft-versus-host and homograft responses are pre-cluded in the former situation, a homograft reaction against the transferredcells remains possible in the latter.

d. The immunological capacity of the lympfiocyte donor itself to respondto sheep erythrocytes. The capacity to mount an immune response againstthe sheep erythrocyte antigen of which the recipient is tolerant is not aprerequisite for a lymphocyte donor if its cells are to abrogate tolerance tothis antigen in an allogeneic recipient. Thus, lymphocytes from a rat whichis itself tolerant of sheep erythrocytes are able to abrogate tolerance of thisantigen in a recipient provided that the recipient is allogeneic or semi-allo-geneic with respect to the cell donor.

e. The immunological maturity of the lymphocyte donor. TTie capacity of

186 PETER McCULLAGH

allogeneic lymphoid cells to effect abrogation of tolerance is dependent uponthe immunological maturity of the cell donor. Thus, spleen cells fromparental strain rats do not acquire this capacity (as assessed after transferto erythrocyte-tolerant Fi hybrids) to any marked extent until the third weekof age. Although some reduction in ability to abrogate tolerance might beattributable to a predominance of haemopoietic over lymphoid cells in theneonatal spleen, this is unlikely to account for inefficiency of the extent ob-served with spleen cells from these young rats. In contrast to this inefficiencyis the facility with which four-day-old F, hybrid strain rats effect theabrogation of tolerance in lymphocytes transferred from adult, erythrocyte-tolerant donors of parental strain (to be published). It is apparent that theacquisition of the ability to evoke abrogation of tolerance on the part ofthe cells of immature rats does not necessarily parallel the developmentof other immunological activities.

f. The numbers of allogeneic lymphocytes transferred. The magnitude of theimmune response when tolerance is abrogated is not closely related to thenumber of allogeneic lymphocytes transferred to effect abrogation. Increasebeyond 3 x 10^ in the number of lymphocytes transferred does not increasethe extent of the response generated in the erythrocyte-tolerant recipient.The transfer of as few as 2.5 x 10' lymphocytes is still capable of evokingan appreciable immune response (McCullagh 1972a). This lack of a firmcorrelation between the number of allogeneic cells transferred and thenumber of plaque-forming cells penerated is not surprising in a situationwhere the transferred lymphocytes are acting indirectly.

g. The temporal relationship of allogeneic lymphocyte transfer to antigenicchallenge. The transfer of allogeneic lymphocytes to an erythrocyte-tolerantrat is not sufficient, on its own, to abrogate tolerance. Antigenic challengeof the erythrocyte-tolerant recipient with sheep erythrocytes is also required.Furthermore, this antigenic challenge should be administered simultaneouslywith, or soon after allogeneic cell transfer. There is a marked decrease inthe size of the immune response of the tolerant recipient if antigenic challengeis deferred for 2 hours or longer after allogeneic lymphocyte injection(McCullagh 1972a). This suggests that the stimulus for abrogation generatedby the confrontation with the transferred allogeneic cells is short lived.

h. The viability of the allogeneic lymphocytes transferred. Thoracic ductlymphocytes from an allogeneic donor which have been exposed in vitro to6,000 r of gamma irradiation retain little capacity to initiate abrogation(McCullagh 1972a). It is unlikely that irradiation of the cells has interfered


with their abrogating ability by altering their pattern of migration in thetolerant recipient (Goodman 1971). Furthermore, it is unlikely that thisdose of irradiation would have modified histocompatibility antigens on thetransferred lymphocytes (McKhann 1964). Consequently, it is inferred thatthe presentation of foreign histocompatibility antigens to an erythrocyte-tolerant rat is not sufficient, on its own, to abrogate tolerance. The ineffec-tiveness of allogeneic bone marrow cells in abrogating tolerance (section3 (a)) may be interpreted similarly.

i. The ability of transferred lymphocytes to undergo mitosis. Neither DNAsynthesis nor mitosis is required on the part of transferred allogeneic lympho-cytes if they are to abrogate tolerance. Allogeneic lymphocytes which havebeen incubated with mitomycin C (25 //g/ml for 30 min) retain their capacityto abrogate tolerance (McCullagh 1972a). This result is obtained regardlessof whether the mitomycin C-treated lymphocytes are completely allogeneicwith respect to the erythrocyte-tolerant recipient or are semi-allogeneicparental cells which are being transferred to a tolerant Fi hybrid recipient.In the former situation, it remains possible for the erythrocyte-tolerantrecipient to mount a homograft reaction against the transferred cells. How-ever, in the latter, the capacity of the parental strain lymphocytes toinitiate graft-versus-host disease has been blocked. In reactions of mixedlymphocyte type, mitomycin-treated lymphocytes from a rat of one strainremain capable of stimulating lymphocytes from an unrelated donor, butare unable to respond to stimulation themselves. Furthermore, parentalstrain cells fail to stimulate Fi hybrid strain lymphocytes in this situation(Wilson & Nowell 1970). Thus, allogeneic interactions capable of abrogatingtolerance can take place with combinations of lymphocytes which wouldnot produce a positive mixed lymphocyte reaction.

j . The ability of transferred lymphocytes to synthesize RNA. If allogeneiclymphocytes which are to be transferred to an erythrocyte-tolerant recipientare first incubated with actinomycin D at a concentration sufficient toprevent synthesis of RNA, they lose their capacity to abrogate tolerance.The concentration of actinomycin D to which lymphocytes must be exposedin order to block their abrogative capacity (1-2 /ig/ml for 3 hours) correlatesclosely with that which interferes with their ability to incorporate tritiateduridine. The observation that retention of the ability to synthesize RNA ismandatory if the transferred lymphocytes are to abrogate tolerance doesnol clearly define the metabolic activities which are required for the abro-gating effect. Many cellular activities are indirectly dependent on RNAsynthesis.

188 PETER McCULLAGH

4. THE CAPACITY OE XENOGENEIC LYMPHOCYTES TO ABROGATEERYTHROCYTE-TOLERANCE IN RATS

Lymphocytes obtained from the efferent popliteal lymphatic duct of normalsheep are able to abrogate tolerance of recipient rats for sheep erythrocytes(to be published). Similar lymphocytes, from normal sheep, are ineffectivein abrogating tolerance of fiorse erythrocytes in rats. However, lymphocytesthat have been collected from the efferent popliteal duct in the course ofthe cellular response mounted by the popliteal lymph node to the sub-cutaneous injection of rat lymphocytes in its area of drainage (Hall &Morris 1963, Lafferty, Walker, ScoUay & Killby 1972) have been observedto abrogate tolerance of rats for horse erythrocytes. That sheep lymphocytescan survive in a rat for a sufficient period to interact with host lymphocytesmight be anticipated from the experiments of Alexander, Delorme & Hall(1966). The ability of sheep lymphocytes to abrogate tolerance of sheeperythrocytes in rats is consistent with two earlier conclusions, namely thatunresponsiveness of the transferred, foreign lymphocytes to the toleratedantigen does not impair their abrogative capacity (3 (d)) and that theabrogative stimulus acts over a short period of time (3 (g)).

5. THE ABROGATION OE ERYTHROCYTE TOLERANCEWITH SUBCELLULAR FRACTIONS

Sub-cellular fractions can be substituted for intact lymphoid cells as a meansof abrogating tolerance (to be published). Active fractions were preparedas follows. Normal rats were injected intravenously with allogeneic thoracicduct lymphocytes and, 2 to 3 hours later, their spleens were removed (Fig-ure 1). Cold phenol/ethanol extracts of these spleen cells were prepared bythe method of Harris & Watts (1962). Each extract was incubated withthoracic duct lymphocytes from an erythrocyte-tolerant donor of differentstrain from the spleen cell donor. After incubation, the mixture was injected,together with sheep erythrocytes, into the erythrocyte-tolerant lymphocytedonor. In experiments of this type, abrogation of tolerance frequently oc-curred as indicated by the appearance of splenic plaque-forming cells.Extracts prepared from spleens which were removed between 90 and 210minutes after allogeneic lymphocyte transfer to the spleen donor were mosteffective. In 25 experiments of this type, a mean total splenic plaque-formingceil response of 83,000 was obtained. In contrast, extracts prepared fromthe spleen cells of syngeneic or untreated allogeneic rats were ineffective.Ribonuclease treatment of an active extract obtained from the spleens ofrats injected with allogeneic lymphocytes reduced its abrogative capacity.

ABROGATION OF TOLERANCE 18Q

Thus, incubation of the extract for 60 min with 10 //g of ribonuclease perml reduced this activity by 55 per cent, while 25 /^g/ml reduced it by 85per cent. However, higher concentrations of ribonuclease (up to 200 //g/'ml)consistently failed to reduce the activity further. This result suggests, butdoes not definitely establish, that allogeneic RNA may be effective in abro-gating erythrocyte tolerance. It is possible that other molecules in the cellularextract may be responsible for this effect but, if this is so, it appears thatthe presence of intact RNA is necessary for the maintenance of their activity.Regardless of the chemical nature of the active material in the phenol/ethanolextract, it cannot automatically be assumed that this material mediates theabrogative effect when intact, allogeneic lymphocytes are transferred to atolerant host. For example, if abrogative activity was to reside in RNA,it is conceivable that this molecule might itself be transferred between theinjected, allogeneic cells and the lymphocytes of the host. However, it isequally feasible that a protein, the production of which was directed bythe RNA in the injected lymphocytes, might be transferred to stimulateabrogation.

Allogeneic lymphocytes which have been exposed to ribonuclease retaina nonnal capacity to abrogate tolerance. Fifteen erythrocyte-tolerant ratswere each injected with 2.5-3.0 x 10^ allogeneic thoracic duct lymphocytes

NORMAL ( A X B ) F, HYBRID 5TRAIN

NORMAL B

SRBC TOLERANT 1 A X B ) R HYBRIDSPLEEN CELLS

EXTRACTION WITH PHENOLAND ETHANOL

INCUBATION OF T.D 1. i ,WITH EXTRACT + SRBC ^

REINJECTION TO TOLERANT DONOR

Figure 1. The use of a subcellular fraction prepared from the spleens of rats injectedintravenously with allogeneic lymphocytes to abrogate tolerance.

190 PETER McCULLAGH

which had been incubated in medium 199 containing 1 mg of ribonuclease/ml for one hour. At this concentration, the enzyme failed to impair thecapacity of the allogeneic lymphocytes to abrogate tolerance, lymphocyterecipients having an average of 2.5 x 10^ plaque-forming cells per spleen92 hours later (unpublished observation). It does not appear that carbo-hydrate-containing structures on the surface of the allogeneic lymphocytesare required to mediate interaction with host cells. Thus, lymphocytes whichhave been treated with neuraminidase in such a way as to effect the maxi-mum release of sialic acid retain a nonnal capacity to stimulate abrogationof tolerance (A. S. Kemp, unpublished observations).

6. THE NATURE OE THE CELLS WHICH INTERACTTO INITIATE ABROGATION OF TOLERANCE

The cell type of the erythrocyte-tolerant host which interacts with the in-jected, allogeneic lymphocytes to initiate the abrogation of tolerance, occursin the circulating lymphocyte population. The nature of this interacting hostcell was resolved by observing the abrogation of tolerance in populations of

SRBC TOLERANT A STRAIN SRBC TOLERANT B STRAIN

NON TOLERANT A OR B STRAIN

Figure 2. 'CultLire' of erythrocyte-tolerant cells in irradiated third party hosts (seetext). Results are indicated in Figure 3.


cells 'cultured' together in third party hosts which had been rendered un-

reactive by irradiation as outlined in Figure 2 (McCullagh i972b). Lympho-

cytes from a normal, non tolerant, allogeneic donor would be able to mount

an immune response themselves if transferred to an irradiated host which

was challenged with antigen. Such a response might mask any response

mounted as a result of abrogation in the tolerant population. To obviate this

too.ooo

50,000

25,000

O 10.000uL

2 5000UJ

^ 2500

O 1000

500

250

• D

• D O

B6 89 P2 95 9fl 101 lOi 107 110 :13

TIME OF P.F.C. DETERMINATION (HOURS)

116

Figure 3. The abrogation of immunological tolerance in populations of cells transferredto lethally irradiated (910 rad) recipients and then confronted with allogeneic thoracicduct lymphocytes and sheep erythrocytes. In each experiment, the first cell inoculumwas injected intravenously on the day of irradiation and was followed two days laterby lymphocytes plus 10'' sheep erythrocytes. Plaque-forming cell content of the recip-ients' spleens were measured at the times indicated. All cell donors were tolerant ofsheep erythrocytes.* 2.5x10* thoracic duct lymphocytes followed by 10* allogeneic thoracic ductlymphocytes.O 2.5 X IO'* thoracic duct lymphocytes followed by 10* syngeneic thoracic ductlymphocytes.• 2.5 X 10* thymus cells followed by IO" allogeneic thoracic duct lymphocytes.• 1.5 X 10̂ bone marrow cells followed by 10* allogeneic thoracic duct lymphocytes.(Reproduced with permission from Aust. J. exp. Biol. med. Sci. (1972), 50, 49, 'Thenature of the response of the cells of the sheep erythrocyte-toierant rat to transferredallogeneic lymphocytes'.)

192 PETER McCULLAGH

difficulty, each of the cell populations to be mixed in the third part hostwas collected from an erythrocyte-tolerant donor. It has been indicatedpreviously (3(d)) that lymphocytes from erythrocyte-tolerant, but allogeneicdonors are as effective as lymphocytes from normal, allogeneic donors inabrogating tolerance. Consequently, in the experiments outlined in Figure2, the splenic plaque-forming cell response to challenge with sheep erythro-cytes was examined in irradiated recipients of mixtures of various typesof cells from an erythrocyte-tolerant donor of one strain together withthoracic duct lymphocytes from a second erythrocyte-tolerant donor of adifferent strain. Immune responses of large magnitude were observed onlyafter the mutual confrontation of two allogeneic batches of thoracic ductlymphocytes (Figure 3). If thymus or spleen cells were 'cultured in vivo'with allogeneic thoracic duct lymphocytes, the ensuing plaque-forming cellresponses were weak.

7. THE RELATIONSHIP OF THE INTERACTIONTHAT OCCURS BETWEEN ALLOGENEIC LYMPHOCYTES TO THE

SUBSEQUENT ABROGATION OF TOLERANCE

The experiments reviewed to this point, while indicating some of thefeatures required if the interaction of injected, allogeneic lymphocytes withhost eells is to initiate abrogation, have not been directed to the meanswhereby such an interaction triggers abrogation. As mentioned in (l(d)),the maintenance of the tolerant state is facilitated by an active repressiveeffect of lymphoid cells. Whether these repressing cells have the potentialto differentiate for antibody formation themselves has not been established.However, the faet that tolerance can be transferred by means of these cellsindicates that they are able to repress differentiation for haemolysin forma-tion in other cells which have this potential.

The direct effects of allogeneic interaction could be exerted either onrepressed cells with antibody-forming potential (to stimulate them to becomeplaque-forming cells in the face of a continuing repressive effect) or, alter-natively, on the repressing cells themselves (to suspend the repressive effect).A combination of these effects is also conceivable.

A distinction between these possibilities has been drawn experimentally.The procedure was as follows (Figure 4). Rats of 3 inbred strains (denotedas A, B, and C) were each rendered tolerant of sheep erythrocytes. Addi-tionally, tolerance of C strain tissues was induced in both A and B strainrats by means of the neo-natal injection of bone marrow cells, and the Cstrain rats were similarly rendered tolerant of both A and B strain tissues.It will be recalled that in experiments described previously (6, Figures 2


and 3), sheep erythrocyte-tolerant lymphocytes from two unrelated strainsof rats were 'cultured' together in an irradiated third party host resultingin the abrogation of erythrocyte tolerance in the cells of the mixture. In thepresent experiment a mixture of A strain and B strain lymphocytes wasinjected, together with sheep erythrocytes, into the C strain rats. As bothA and B strain lymphocyte donors were tolerant of C strain tissues, therewould be no tendency for the infused ceils to interact with host cells toabrogate erythrocyte tolerance in the latter. Furthermore, as the C strainhost was tolerant of both A and B strain tissues, the infused lymphocyteswould not be subject to rejection as a homograft. It was independentlydemonstrated that the A and the B strain lymphocytes in the mixture retainedthe capacity to interact with erythrocyte-tolerant lymphocytes of the otherstrain to abrogate tolerance. However, when the mixture of A and B strainlymphocytes was transferred to erythrocyte-tolerant, C strain rats there wereno pi aque-forming cells of any strain - A, B or C - generated (to bepublished). The failure of any pi a que-form ing cells of A or B strain to appearin this situation is likely to reflect the persistence of a repressive influenceexerted by cells of the erythrocyte-tolerant C strain host. In the situationexisting after this transfer, C strain cells would not be directly involved inan allogeneic interaction, as the host was tolerant of and tolerated by bothcell donors. Hence, it is additionally inferred that, if abrogation of erythro-cyte tolerance is to be produced by the interaction of allogeneic cells, it isnecessary to interfere with the activity of all of the repressor eells present.In contrast, if allogeneic interaction had the sole effect of augmenting the

S STRAIN I TOLERANT OF C STBAIN AND OF SKK * " R A t ^ | TOLERANT OF C STRAIN AND Of SRBC

C STRAIN ( TOLERANT OF ( A K B 1 FI HYBRD STRAIN AND OF SRBC

Figure 4. The transfer of mixtures of erythrocyte-tolerant lymphocytes from differentstrains of rats to erythrocyte-tolerant rats of a thid. unrelated strain (see text).

Transplant. Rev. {1912) 12 13

194 PETER McCULLAGH

capacity of repressed cells to differentiate for antibody formation, plaque-forming cells derived from A and B strain cells should have appeared inthis experiment. It is also clear from this experiment that allogeneic inter-action does not abrogate tolerance by releasing a material which acts non-specifically to stimulate any contiguous tolerant cells. If this were so, theappearance of plaque-forming cells of A, B and C strain would have beenpredicted.

If responsiveness to sheep erythrocytes is examined in rats in whicherythrocyte tolerance has been abrogated by means of the injection ofallogeneic lymphocytes, it is frequently found that the rat is again tolerant.That is, the response to an antigenic rechallenge administered subsequentto allogeneic abrogation is either markedly reduced or undetectable (Mc-Cullagh 1972b). However, if challenge with sheep erythrocytes is accom-panied by the transfer of a second inoculum of aJlogeneic lymphocytes,the transferred cells interact with the repressing cells of the host and so inter-fere with this repressive activity. As a result of this cessation of repression,the repressed antibody-forming cell precursors are permitted to differentiateto plaque-forming cells under the influence of the erythrocyte challengewhich has been administered with the allogeneic lymphocytes. However,the suspension of repression following allogeneic interaction is ephemeraland, accordingly, if the initial antigenic challenge is deferred for very longafter lymphocyte transfer, the resulting plaque-forming cell response isweak. For a similar reason, rechallenge, in the absence of a secondarystimulus with allogeneic cells, produces litde response in rats in whichtolerance has been abrogated previously.

8. THE EFFECT OF ALLOGENEIC CONFRONTATIONON IMMUNE RESPONSES OF NORMAL ANIMALS

If antigenic challenge is accompanied by the transfer of allogeneic lympho-cytes, augmentation of the immune response may be observed in normal,non-tolerant animals (Katz, Paul, Goidl & Benacerraf 1971, McCullagh1972b). The mechanism whereby confrontation with allogeneic lymphocytesincreases the magnitude of a concomitant immune response is not known.The simplest model for this phenomenon would be that the stimulationof an antigen reactive cell by allogeneic lymphocytes non-specific ally in-creases its responsiveness to the antigen presented simultaneously. If thisis the case, and if the previous suggestion of a mechanism whereby allo-geneic confrontation facilitates the abrogation of tolerance (namely, thatthe activity of a repressing cell is suspended) is also correct, the detailedmechanism by which allogeneic lymphocytes augment immune responsive-


ness in these two situations would be different. It is unwise to emphasizethese differences in mechanism when so much remains to be learnt aboutboth phenomena. However, there are a number of features in which augmen-tation of the response of normal rats to sheep erythrocytes by allogeneiclymphocytes differs from allogeneic abrogation of erythrocyte tolerance.These may be summarized as follows:

a. The transfer of parental strain lymphocytes to Fj hybrid recipients iseffective in abrogating erythrocyte tolerance and also in augmenting theresponse of normal Fi hybrids to this antigen. However, the transfer oflymphocytes from rats of a completely unrelated strain, while highly effectivein the abrogation of tolerance, fails to augment the immune response ofnormal rats.

b. Parental strain lymphocytes that have been treated with mitomycin Cremain capable of abrogating erythrocyte tolerance in Fi hybrids but donot augment the immune response of normal hybrids.

c. Aug;mentation of the immune response in nonnal recipients of allogeneiclymphocytes is achieved even if antigenic challenge is deferred for somehours after lymphocyte transfer. However, if tolerance is to be abrogated,antigenic challenge must be given much sooner after allogeneic confrontation.

Perhaps allogeneic confrontation stimulates antigen reactive cells in bothnormal and erythrocyte-tolerant rats. This mechanism could completelyaccount for augmentation of the immune response in the former case. How-ever, if an immune response is to be evoked in tolerant rats, a second effectof allogeneic confrontation, namely a temporary interference with the actionof repressor cells, may also be required.

9. CONCLUSIONS

Abrogation of tolerance of sheep erythrocytes following confrontation ofa tolerant rat with aiiogeneic lymphocytes has implications in two contexts.

a. The cellular nature of immunological tolerance. The rapid reappearanceof antibody-forming cells in tolerant animals is a strong indication of theexistence of tolerant cells. It is quite inconsistent with any interpretationof artificially induced tolerance as a condition resulting from the eliminationof those cells capable of responding to the appropriate antigen. The inter-pretation of tolerance as a positive state of repression is supported by ob-

196 PETER McCULLAGH

servations of the reappearance of antibody-forming cells among tolerant

populations of cells submitted to in vitro manipulation (McCullagh &

Gowans 1966, Moller & Sjoberg 1972) and of the rapid return of reactivity

in tolerant animals in other situations (Howard 1972, Biro & Arroyave

1970).

b. The manipulation of immunological responsiveness by means of allo-

geneic confrontation. The concurrent initiation of an interaction with allo-

geneic lymphocytes is one of the most potent measures available to non-

specifically augment immune responsiveness. The importance of non-specific

influences in determining the nature of the response to antigens is requently

overlooked. However, it is possible that techniques such as allogeneic con-

frontation may have some relevance in augmenting immunological reactivity

in states of general unresponsiveness and in modifying the reactivity of an

animal to neoplasms.

REFERENCES

Alexander, P.. Delorme, E. J. & Hall, J. G. (1966) The effect of lymphoid cells fromthe lymph of specifically immunized sheep on the growth of primary sarcomatain rats. Lancet i, 1186.

Biro, C E. & Arroyave, C. (1970) On the existence of a live immunologically un-responsive lymphoid cell. Immunology 18, 387.

Goodman, J.W. (1971) Cellular interaction between thymocytes and transplantedmarrow stem cells. Transplant. Proc. 3, 430.

Hall, J.G. & Morris. B. (1963) The lymph-borne cells of the immune response. Quart.J. exp. Physiol. 48, 235.

Harris, H. & Watts, J. W. (1962) The relationship between nuclear and cytoplasmicribonucieic acid. Froc. roy. Soc. B 1S6, 109.

Howard, J. (1972) Cellular events in the inducation and loss of tolerance to pneumo-coccal polysaccharide. Transplant. Rev. 8, 50.

Katz.D. H., Paul, W. E., Goidl, E. A. & Benacerraf, B. (1971) Carrier function inanti-hapten antibody responses. III. Stimulation of antibody synthesis and facili-tation of hapten specific responses by graft-versus-host reactions. J. exp. Med.133, 169.

Lafferty, K. J., Walker, K. Z., Scollay, R. G. & Killby, V. A. A. (1972) Allogeneic inter-actions provide evidence for a novel class of immunological reactivity. Transplant.Rev. (this issue).

McCullagh, P. (1970a) The transfer of immunological competence to rats tolerant ofsheep erythrocytes with lymphocytes from normal rats. Aust. J. exp. Bio!, med.Sci. 48, 35L

McCullagh, P. (1970b) The immunologlcai capacity of lymphocytes from normaldonors after their transfer to rats tolerant of sheep erythrocytes. Aust. J. exp.Biol. med. Sci. 48, 369.

McCullagh, P. (1970c) Tbe abrogation of sheep erythrocyte tolerance in rats by meansof the transfer of allogeneic lymphocytes. J. exp. Med. 132, 916.


McCullagh, P. (1972a) The contribution of transferred allogeneic lymphocytes to theabrogation of sheep erythrocyte tolerance in rats. Aust. J. exp. Biol. med. Sci.SO, 35.

McCullagh, P. (1972) Tbe nature of the response of the cells of the sheep erythrocyte-tolerant rat to transferred allogeneic lymphocytes. Aust. J. exp. Biol. med. Sci.50, 49.

McCullagh, P. & Gowans, J.L. (1966) Immunologically tolerant lymphocytes. In:The Lymphocyte in Immunology and Haemopoiesis, ed. Yoffey, J.M., p. 234.Arnold, London.

McGregor, D. D., McCullagh, P. &. Gowans, J.L. (1967) The role of lymphocytes inantibody formation. I. Restoration of tbe baemolysin response in X-irradiatedrats. Proc. roy. Soc. B 168, 229.

McKhann, C. F. (1964) Tbe effect of X-rays on tbe antigenicity of donor cells intransplantation immunity. J. Immunol. 92, 811.

Moller, E. & Sjoberg, O. (1972) Antigen-binding cells in immune and tolerant animals.Transplant. Rev. 8.

Wilson, D. B. & Nowell, P. C. (1970) Quantitative studies on tbe mixed lymphocyteinteraction in rats. IV. Immunological potentiality of the responding cells. J. exp.Med. 131, 391.

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The Abrogation of Immunological Tolerance by Means of Allogeneic Confrontation