Lymphocyte-A Disgraceful Gap in Medical Knowledge

8/14/2019 Lymphocyte-A Disgraceful Gap in Medical Knowledge

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rom time to time, I am sent col-ourcd wall charts that illustratecurrent knowledge about lym-phocyte subsets, their origin, de-velopment and interactions together with

the battery of lymphokincs that control thecellular changes underlying immune re-sponses. Whether or not tltey rire accurate,they paint a large and complex picture, andone that must be difficult for instructors toexplain and for students to grasp. I have anadditional difficulty. This is to conjure upthe time, 40 years ago, when virtually theonly property of lymphocytes about whichthere was general agreement was that they were motile.

It was Howard Florey, the Professor of Pathology at OxfordUniversity at that time, who suggested that I should work on lym-phocytes. I had returned to Oxford at the end of 1953 after a year atthe Pasteur Institute with littlc idea what to do next. The laboratoryin Oxford already had a record of work on lymphocytes that, JC-cording to Florey, had blunted the wits of a number of his col-leagues and he could see no reason why I should be spared a simi-lar fate. As is well known, Florcy led the team that transformedpenicillin from a laboratory curiosity into the most powerful agentyet available for treating human infections. Florey drew my atten-tion to the current state of the lymphocyte field and liked to quoteArnold Rich: The complctc ignorance of the function of this cell isone of the most humiliating and disgraceful gaps in all medicalknowledge. Rich was talking about small lymphocytes and thesmall round cells of the histopathologists. Literally nothing of im-portance, he concluded, is known regarding the potentialities ofthese cells. This was tltc situation in 1953,

The rec i r cu la t ion o f l ymphocytesFlorey suggested that I should work on the problem known at thattime as the mystery of the disappearing lymphocytes. The prob-lem could be stated v e r y simply: sufficient lymphocytes cnier theblood each day from the major lymphatic ducts to replace ,-ll thosein the blood many times over. For example, in the rat, all the lyil,phocptes in the blood are replaced about ten times daily by thoseentering from the thoracic duct. The fate of these disappearing lym-phocytes was accounted for by two rival theories. The first regardedlymphocytes as primitive stem cells which, according to the 1949

edition of Maximov and Blooms Textbookof Histology, were endowed with haem-atopoietic, phagocytic and fibrocytic poten-cies. This was the prevailing view at thetime 1 entered the field. The other view wasthat lymphocytes were end cells, dying afew hours after having performed their I.IG~~known functions. Various graveyards weresuggested, notably the gut and skin.

Thus, at the beginning of 1954, thesesmall cells became my obsession, andFlorey said if 1 could find out where theywent, we might know what they did. Hedrew my attention to a technique for col-

lecting lymph and lymphocytes from the thoracic duct of un-anaesthetized rats, which Bollman ef nl.? had described in 1948. Witha little practice, this became a routine procedure. However, in 1950,Mann and Higgins3 found that, although very large numbers oflymphocytes could indeed be collected initially from a thoracic ductfistula, continuous drainage for several days resulted in a precipi-tous fall in the output of cells. By 1957 we had shown that, if all thecells from the fistula were reinfused continuously into the blood,the output remained constant: the lymphocytes that normally dis-appeared from the blood were somehow responsible for maintainingthe output of cells from the thoracic duct.

The simplest explanation for this finding was a recirculation oflymphocytes from blood to lymph. This was not a new idea but onethat had not previously been taken seriously; however, it gainedplausibility from an experiment I still remember with some satis-faction. Thoracic duct lymphocytes were labelled irl z , i f r owith in-organic ?I, horoughly washed and then reinfused into the blood ofthe same rat; a substantial wave of cell-associated ?I appeared inthe lymph of these animals, overlapping the rise and fall of a waveof increased cell output. Tritiated thymidine ([H]TdR) had recentlybecome available commercially and this enabled us to show that theincreased output was no: due to the Troduction of new cells: over95% of the lymphocytes in the wave of lymph-borne cells were un-labelled when tritiated d~ymidine was given along with the cellularinfusion; the remaining 5%, consisting of large lymphocytes, wereheavily labelled. The larger lymphocytes were new cells and providedan intental control for the effectiveness of the procedure.

The paper describing these results was published in 1959 (Ref. 5)but not before one referee had recommended its rejection on thegrounds that all the experiments could be explained by an exchange


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of ??Ibetween the transfused cells and thosethat were recovered from the thoracic duct.The referee was not alone in their criticisms.At a haematology meeting in the USA In1959 (Ref. 6), there was a discussion aboutthe uptake and persistence of radioactivity in

HozonrdFlorq (1953).

small lymphocytes after giving [H]TdR iiz CZU. The labelling dataindicated a surprisingly long lifespan for blood lymphocytes which,of course, supported the idea of recirculation -no other rxplanationcould accommodate the disappearance each day of very large num-:>ers of long-lived cells. However, another interpretation was con-sidered: the DNA from old lymphocytes might be re-utilized inlymphoid tissue during the formation of new lymphocytes. For ashort while, this idea of DNA re-utilization was taken sufficientlyseriously to urge caution in accepting the interpretation of my ownexperiments in which the cells comprising the \vdve of increasedoutput from the thoracic duct had failed to label with [7H]TdR. 1 re-turned from the USA with only a small concession: if recirculationoccurred at all, it only happened on a small scale.

Recirculation still seemed to me to be the simplest explanation ofall our experiments, but it was necessary to: (I) show that it occurredon a large enough scale to account for the turnover of the bloodlymphocytes; and (2) trace the pathway of individual cells alongtheir proposed route from blood to lymph. This was accomplishedby labelling the RNA of thoracic duct cells irk &o with a tritiatedprecursor and then determining their location by autoradiographyat various times after intravenous infusion. The labelled cells be-haved as we had predicted. They migrated from the blood into thecortex of the lymph nodes, passed into the lymph sinuses and thenreappeared in large numbers in the thoracic duct lymph. High con-centrations of labelled cells were also found in the Peyers patchesbut none in the thymus and few in nonlymphoid tissues. Largenumbers migrated into the white pulp of the spleen from whichthey were known to re-enter the blood directly. The cells that recir-culated into the lymph were the small lymphocytes; the minority oflarger, dividing lymphocytes in which the DNA could be labelled,did not reappolr in the lymph but migrated into the lamina propriaof the small intestine, where they had assumed the morphology ofplasma ceils. This last observation, worked on by Julie Knight, wasthe basis of subsequent studies on local immunity in the gut.

The autoradiographic study also locatedihe precise route taken by thesmall lympho-cytes in their journey from the blood illtothe lymph nodes. At short intervals after in-travenous infusion, labelled cells were seento be migrating across the end&helium ofan unusual set of small blood vessels in thecortex of the lymph nodes and the Peyerspatches. These postcapillary venules hadthe property of mediating a selective migra-tion of lymphocytes (and, under normalconditions, no other class of leukocyte) fromthe blood. Vincent Marchisi had been work-ing with Florcy on the migration of cellsfrom blood vessels during acute inflamma-tion and had produced a series of e~,z~.tronmicrograph pictures of poly~~iorphonucle~~rleukocytes passing between the enduthelialcells of venules in inflamed tissues. Marchisidid a similar study on serial sections

through normal rat ly.nph nodes and identified lymphocytes at allstages in their passage across the endothrlium of the venules. Thisincluded an accumulation under the endothelium, a location firstoccupied by the labelled cells soon after their intravenous infusion.Ci comse, the electron micmgraphs did not reveal the direction oflymphocyte migration, but we knew this from the infusion experi-ments. A detailed examination of the serial sections led to an unex-pected conclusion: that lymphocytes passed through the endo-thelial cells of the postcapillary venules and not behveen them. Theappearance was quite different from that of polymorphouucledrleukocytes passing between the endothelial cells of venules in in-tlamed lymph nodes. Such an intracellular mlgr,ltion is certainlyunorthodox and this point has still to be satisfactorily resolved. Theselectivity of postcapillary venules, now known as high endothelialvenules, presumably means that the normal traffic through them ismediated by a repertoire of ligands on lymphocytes different fromthat on polymorphonuclear leukocytes.

Two papers in 1964 (Refs 7, 8) appear to have convinced moslpeople that small lymphocytes recirculate from blood to lymph byway of the lymph nodes and that recirculation explained the turn-over of the blood lymphocytes. The mystery of the disappearinglymphocytes was solved: they did not disappear at all but, rather,continually reappeared in the lymph. However, Florey turned outto be wrong about one aspect-that discovering the fate of the bloodlymphocytes would uncover their function. Unfortunately the dem-onstration that lymphocytes recirculated from blood to lymph gaveno clues to their function.

immuno logyA t t ] l e ime h e ecirculation story was developing, there was littlein the literature to convince anyone that small lymphocytes had im-munoIogical functions. In 1959, Burnet wrote that: An obj~cti~csurvey of the facts could well lead to the concIusion that there was

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no evidence of immunological activity in small lymphocytes.Certainly, by 1939, we knew from the work of McMasteP thatthe lymph nodes were the site of antibody formation after regionalantigenic stimulation, and Coons, using his technique of im-mmofluorescence, had shown that plasma cells make antibodyin both the primary and the secondary response. However, therewas still no place for small lymphocytes. Coons thought that theprecursor of the plasma cell might be a large undifferentiated cellhe called a primitive reticular cell. The idea that lymph nodeswere the seat of immunological responses was further strengthenedby the fact that the production of antibody, delayed hypersensitivityand the reactions against both tumour and normal tissueallografts could be transferred to suitable recipients by means oflymph node cells from immunized donors. However, even thoughsmall lymphocytes are a major component of lymph nodes, the celltypes responsible for such adoptive immunization had not beenidentified.

The view that Burnet expressed in 1959 had to be updated a fewyears later, when an immunological role for small lymphocytes wasunequivocally demonstrated in work influenced by a long-standingfriendship with Peter Medawar. Like others, Peter Medawar wasinitially rather sceptical about recirculating, long-lived lbmpho-cytes and, at his instigation, we did an experiment together to see ifa nucleoprotein fraction would substitute for living lymphocytes inmaintaining the output of cells from the thoracic duct. The resultswere negative and he later regarded recirculation as an agreeableidea, likening the blood lymphocytes to a chorus of soldiers in aprovincial production of Faust - they make a brief public appear-ance and then disappear behind the scenes only to re-enter by thesame route.

Medawar introduced me to the main players in the field of trans-plantation biology and, particularly, to the work of Simonsen, andof Billingham and Brent, on graft-versus-host (GVH) reactions. Ithad a!,ready been shown that these reactions could be induced byinjecta,ng allogeneic lymph node cells or blood leukocytes into neo-natal recipients, so suspicion was naturally falling on some kind oflymphocyte as the inducing cell type. In 1958, I discussed withMedawar the idea of coifirming this suspicion by injecting thoracicduct cells. However, the demonstration in 196041 by a number ofUS that thoracic duct cells could, indeed, induce GVH reactionsleft the precise identity of ti-- iLnducing cell type unknown. Somethought that large, dividing lymphocytes might be responsible be-cause donor cell proliferation in the lymphoid tissue of the host wasa feature of the early stages of the disease. We had a simple way ofresolving this problem by testing the ability of small lymphocytes,purified from the thoracic duct lymph of parental-strain rats, tocause GVH reactions in adult F, hybrid rats. Such inocula induceda vicious reaction in the recipients, with progressive wasting anddeath after a time that depended on the cell dose. By contrast, inoc-ula containing mainly large, dividing lymphocytes failed to inducethe disease.

At first, it was not at all clear how the small lymphocytes wereexerting their devastating effect, particuIarIy as one of the currentviews, based on infusing [jH]-TdR in zlizn, was that small lympho-

cytes were long-lived end cells. This idea was demolished whenwe showed, by injecting radioactively labelled lymphocytes into F,rats, that during the early stages of the resulting GVH reaction, thedonor small lymphocytes enlarged and began dividing. The donororigin of these dividing cells was confirmed by means of a chromo-some marker~? at last, small lymphocytes did something. Also in1961, Jacques Miller published his work on the lymphopenia andimmune deficiency of neonatally thymectomized mice; this was thefirst of his many contributions t3 knowledge about the function ofthe thymus in immunity and about what subsequently becameknown as T lymphocytes. A quite independent line of enquiry duringthis period also showed that small lymphocytes could be provokedinto growth and mitosis.

In 1960, Nowell had shown that incubation with phytohaemag-glutinin initiated mitosis in cultures of normal human leukocytes,and Marshall and Roberts established conclusively in 1963 that thedividing cells were derived from small lymphocytest7.

Our subsequent experiments on alloreactivity drew heavily onMedawars analysis of the allograft reaction and of itnmunologicaltolerance. We were able to show that the state of tolerance in an ani-mal reflected a deficiency in its lymp!locyte population. Thus, tho-racic duct cells from tolerant animals were specifically deficient intheir ability to induce GVH reactions] and long-standing skin allo-grafts on tolerant animals were destroyed following an injcsction ofsmall lymphocytes from normal donors. Bill Ford also contributedmuch to the understanding of the alloreactivity of lymphocytes,and the phenomenon of lymphocyte migration.

It seemed to us unlikely that the immunological activity of smalllymphocytes was restricted to alloreactivity so, concurrently withthe work on GVH reactions, Douglas McGregor had begun studieson their possible role in the induction of antibody responses.Support for this possibility came from studies in rats depletedof recirculating lymphocytes by prolonged drainage of cells froma thoracic duct fistula. Such animals showed a profound depressionof pritnary antibody responses to tetanus tnxoid and to sheeperythrocytes, which could bt: restored to normal by injectionsof small lymp!locytes from the thoracic duct. Similarly, the primaryresponse of X-irradiated rats to sheep ervthrocytes could be re-stored by small lymphocytes from normal, but not from specificallytolerant, donors. On the other hand, immunized rats that hadbeen drained of recirculating cells still responded normally toantigenic challenge: secondary responses, unlike the primary re-sponses we had studied, did not seem to be sensitive to lymphocytedepletion. These findings, published in 1963 (Ref. 20), were con-firmed three years later while working with Jonathan Uhr in NewYork, when we made the additional point that recirculating lym-phocytes carried the property of immunological memory. Thus,irradiated rats given thoracic duct cells from donors immunizedwith a bacteriophage gave brisk secondary responses after chal-lenge; but so did the lymphocyte-depleted donors. We never ex-plained this duel endowment of primed animals: all the cellularingredients for mounting a secondary response were present bothin the recirculating pool and in lymphocyte-depleted lymphoidtissue.


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-r&e bnction of ymThe work with Dou$~ McGregor was sufficientlyadvanced by 1962for us to suggest a general immunological function for small lym-phocytesz2. We had interpreted the experiments on GVH reactions asshowing that small lymphocytes had initiated the immune responsefollowing interaction with the foreign histocompatibility antigens ofthe host. We extended this notion to suggest that these small, recircu-lating cells, not yet in cell division, are the population that endows ananimal with its immune respomiveness. Furthermore, followingtheir stimulation with antigen, they would be triggered into divisionand differentiation to produce the various effecters of immune re-sponses, including antibody-forming cells. The weakness of the ar-gument was the absence of any firm evidence that small lymphocyteswould give rise to plasma cells, although we thought this very likelySuch a function at last gave a rflisnli fYPtre or lymphocyte recircu-lation: it would allow specifically reactive cells to be selected from thelarge recirculating pool into regional lymphoid tissue following lo-calized autigenic stimulation. A selection of this kind was subsequently demonstrated ill P~UOor histocompatibility antigens2 andfor antigens yielding conventional antibody responses2,25.Thus, thesmall Ijrmphocyte was the antigen-sensitive cell that met the require-ments of Bumets clonal selection theory.

The dispute about the cellular origin of plasma cells still neededresolving. Our obsession with this problem continued into the pe-riod \&en the focus of interest in the field had shifted to studies oncell colIabora+ 3n, but we were anxious to complete the picture. Ourpredictions were confirmed by Susan Roser (nee Ellis) and JonathanHoward in transfer experiments in which donor and recipient cellscould be distinguished in suitably chosen irradiated hosts by theuse of alloantisera: cellsmaking a primary antibody to sheep eryth-rocytes and a secondary response to tetanus toxoid were, in bothcases, derived from the donors small lymphocy&+?. So theseversatile cells not only initiated reactions against alloantigens, butthey were also triggered by antigen to generate antibody-producingplasma cells.

Of course we never did complete our picture. We could notexplain what we had termed the functional heterogeneity thatallowed small lymphocytes to engage in all classes of immuneresponse. We made no contribution to the solution of this problemand it is for this reason that I have deliberately avoided any refer-ence to T and B lymphocytes in interpreting our old work. TO mymind, the first persuasive evidence for the existence of two classesof lymphocytes came in 1968 from the work of Mitchell and Miller.In 1971, the idea that thymus-derived (T) lymphocytes helped thedevelopment of marrow lymphocytes was used by Mitchison to ex-plain the effect of prior carrier immunization in increasing the pro-duction of antihapten antibody following challenge with a hapten-carrier conjugate3. Cooperation between B and T lymphocytes hadnow become the topic of the day.

Concluding remarksSince these early studies, the lymphocyte has risen to become one ofthe most studied of all mammalian cells. We now know about the

generation of antibody diversity, the structure of the surface mol-ecules of lymphocyte subsets, and the role of these moiecules andof the major histocompatibillty complex in cell cooperation andantigen recognition. However, 1 have to admit to one small disap-pointment: to my knowledge it is still not possible to describe theevolution of any immune response in terms of the highly dynamicstructure of lymphoid tissue ill zlizl~and of the migratory pathwaysof cooperating lymphocytes within it. Maybe my next colol-red wallchart will have the answers.

James Gowans is f i3 CHI~IIZO~jili, O.+I{, J_IK 0x2 gf+X.eferences

1 Rich,A.R. (193h) .&I./I, Pnfiwl. 22, 228-2542 Bollman, J.L., Cain, J.C. and Grindley J.H. (lY#) J Ir?h C/N. MP~. 33,1349-13523 Mann. J.D and H&gin%G.M. (1950) Blw0 5, 177-1904 Gowans, j.L. (1957) Br. j. E.xp. Pni/r~)l. 38. 67-Z5 Gowans. J.L. (1959) I. Piysiol. IJb, 54-696 Stohlman, F., ed. (1959) T/w Kiwhrs of Cc hh Pdifm fi~w , GITXW an dStratton7 Gowans, J.L. and Knight, E.J. (1964) Pmt. X. Soi. Lor~dorzSu 6 159,257-2828 Marchesi, V.T. and Gowans, J.L. (1964) Pwr. R. Soi. Lom tw jr,: B 159,283-2909 Burnet, EM. (1959) in 7%~~ kwrl S&h~lrr Thy of Aq ~md Imrnuuify.p. 110, Cambridge University Press10 McMaster, l?D. and Hudack, 5.5. (1935) 1, Erp Mcci. bl, 783 +O511 Leduc, E.H., Coons, A.H. and Connolly, J.M. (1%) J, Evp !&

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Lymphocyte-A Disgraceful Gap in Medical Knowledge