Activation of lymphocytes

J. clin. Path. (1967), 20, 795

Activation of lymphocytesR. J. V. PULVERTAFT AND ISOBEL PULVERTAFT1

41 From the Department ofPathology, Makerere University College, Kampala, Uganda

SYNOPSIS The technique involved in studying the activation of lymphocytes in the resting form,and their recognition as dividing and functional cells was studied, using phase contrast and agaras well as fluid culture. Standardization of technical methods was found to be essential, and theeffect of variables was studied. Lymphocytes from human umbilical cord vein blood were found tobe spontaneously activated. Infestation by microfilaria either diminished or entirely inhibitedactivation. The significance of lymphocytic cohesion was considered and the formation of coloniesof activated lymphocytes on agar is described. The effects of non-human vertebrate lymphocytes andof human cells other than lymphocytes were studied. Spontaneous activation of abnormallymphocytes was noted.

Blood grouping has been of such importance inensuring compatibility in transfusion that it isinevitable that a search for an analogous laboratorytest applicable to organ transplantation should bemade. It has long been recognized that when a graftis rejected sections show dense infiltration withlymphocytes. When Schrek and Donnelly (1961)noted that segregated lymphocytes from twoindividuals suffered morphological changes whenmixed, the significance, at first, was not appreciated.More recently, however, many workers have studiedthe phenomenon, in some cases specifically inrelation to its possible importance in organ grafting(Bain, Vas, and Lowenstein, 1964; Gordon andMaclean, 1965; Kasakura, Shinpei, and Lowen-stein, 1965).

These changes in the morphology of lymphocyteswere studied initially on live cells, but later workershave used other methods. Staining of films is used,but obscures many significant features (e.g., motility,cytoplasmic granulation, etc.). Autoradiographyusing 'tracers' is an elegant technique, but unlikelyto be applicable to clinical pathology owing totechnical difficulties. Simple cell counts are employed,and may well prove the best test of reaction betweenindividuals. However, marked morphologicalchanges occur much earlier than cell division, whichis often sparse. Up to the present only one method ata time is used by most workers and it is not provedthat all are studying the same phenomenon.

In this communication a return is made to the'Present address: Hedges, Stour Row, Dorset.Received for publication 19 May 1967.

original method of Schrek and Donnelly and themorphological and other changes readily observedin living cells following lymphocyte admixtures arealone considered. An attempt is made to outline thetechnical details necessary and observations on thebehaviour of lymphocytes in normal and abnormalconditions are related.

TECHNIQUE

MATERIAL Blood samples were obtained by venepuncturefrom 150 healthy young blood donors, of Ugandan orAsian race; no European samples were tested except thosefrom the authors, which were regularly used as controls.

In addition, 34 samples of venous blood from theumbilical cord were examined and 23 samples fromvarious animals, including reptile, fish, and hen.

SEGREGATION OF LYMPHOCYTES Ideally samples shouldcontain only lymphocytes but in practice it is neverpossible to ensure this. Red blood cells were alwayspresent in varying numbers.Lymphocytes can be separated from heparinized blood,

but the use of heparin is to be avoided as during laterprocessing small fibrin clots often formed and vitiatedexperiments; moreover heparin for clinical use usuallycontains a preservative.

Defibrinated blood, obtained by shaking samples withglass beads, gives good results. Even a small clot in thefinal product, however, removes many lymphocytes.The actual segregation was carried out by methods

described by Coulson and Chalmers (1964). Blood wasslowly rotated in boro-silicate tubes with wisps of glasswool for one hour at 370C. A 30% solution of gelatine(May and Baker) was made up in phosphate buffersaline pH 7-2. This must be prepared afresh daily. Stale

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gelatine produces a granular precipitate. Two othercommercial samples of gelatine proved unsatisfactory.Of this solution, 5 ml. was placed in a plastic measuring

cylinder and 25 ml. of blood was added; the cylinder wasplaced in a water-bath at 37 C. and left for 30 minutes.Bubbles which form at the surface must be broken, asthese hold up cells.

Sedimentation of red cells, polymorphs, and monocytesis rapid. The supernatant fluid contains most of thelymphocytes but also some eosinophils, polymorphs, andred blood cells. Many of these are removed by centri-fugation at 500 r.p.m. for two minutes. This deposit isdiscarded and the supernatant again spun at 2,500 r.p.m.for five minutes.The deposit is suspended in culture medium. This, of

course, must not contain any cells. The serum used, at20% concentration, was obtained from Ugandans andAsians. It was centrifuged at high speed and filtered;before use it was frozen at -20°C. No cells were foundon examination.The diluent was 199 (Glaxo), containing penicillin and

streptomycin, and neomycin and nystatin were added.It should be noted that this complex medium containsfour antibiotics and many other constituents to which anindividual may be sensitized; and it is known thatlymphocytes cultured with a reagent to which the donoris sensitized may be activated. Hence until entirelysynthetic media are evolved 'false positive' reactions,though few, are inevitable.The lymphocytes were counted, and diluted until each

donor's cells equalled 2,000 per c.mm. This figure is notcritical but as in all forms of tissue culture there is anupper and a lower limit consistent with efficiency.

CULTURE CHAMBERS As in all forms of tissue culture agas-tight seal is essential. Our chambers were cylindricalvials of boro-silicate glass measuring 1 cm. X 4 5 cm.,to which 3 ml. of cell suspensions was added. The vialswere cultured statically and upright for six days. Thesedetails are very important. For example, if the vials arerotated practically no morphological changes are seen inmixed lymphocytes. Almost all leucocytes and red bloodcells sink to the bottom of culture chambers, andlymphocytes do not adhere to glass and thus do notclimb the sides. Hence, although the total number ofcells and the number per c.mm. may not be varied, ifa culture is made in a conical container the cells are toocrowded to survive and all die quickly. Conversely if thecontainer was a wide plastic ring, no activation occurredin mixed cultures; as will be shown, cell cohesion appearsto be an essential requirement; controls in both caseswere activated.

RESULTS

Tests were made of 150 human normal bloodsamples. In 64 cases lymphocytes from two donorswere mixed, and in 56 activation was found. Ineight cases there was no activation; this was due toCO2 loss, to stale gelatine, or to fibrin clots; notechnically successful culture was negative. In mostcases the cultures were duplicated.

The phenomenon of activation as seen morpho-logically will be discussed later. Here it may bestated that 'activation' implies that practically all thelymphocytes are affected by the gross and veryobvious changes. The effect is not in any way gradedwhen equal numbers of lymphocytes from any twoindividuals are added; and no distinction was seenwhen an individual's lymphocytes were grown withpreparations from two or more other individuals.

It follows that simple admixtures of equal numbersof lymphocytes from any two individuals (other thanuniovular twins) cannot be used as a test for graft orcell compatability as it is always followed bylymphocyte activation and is not graded orquantitative.

In all cases controls were set up; and in only onecase did a control give a positive result. In this case,in which several cultures were set up, all the lympho-cytes were 'activated'. The donor was clinically anormal Ugandan adult male.

THE PHENOMENON OF ACTIVATION: PHASE CONTRAST

This was studied by continuous observation ofcultures of lymphocytes on agar-coated slides at370C.When lymphocytes from one individual alone are

studied they are at first spherical and on warmingmove sluggishly with their characteristic gait. Whenthey touch each other they never cohere. Theyenlarge slightly but do not vary from their originalmorphology, showing very few cytoplasmic organoidsand a homogeneously dark nucleus without nucleoli(Fig. 1).The average diameter of the spherical cell is 6[t.

They never show mitosis.When mixed lymphocytes are studied the first

difference seen is cohesion or agglutination. Thespherical cells are kept in motion at first by gravita-tion and convection and when two cells make contactthey never separate. On warming, and becomingmotile, they move towards each other and towardsthe groups already formed. Thus within twelve hoursin the cultures of a single individual's cells these arewidely scattered; and those of two individuals mixedare almost all in three-dimensional clumps (Fig. 2).

In 48 hours small colonies of circular outline formwith mixed lymphocytes and in six days they areupwards of 3 mm. in character. If many red cells arepresent they are red; if not they are white. Lympho-cytes from one individual (other than from cordblood) never form colonies (Fig. 3).

Morphological changes in mixed lymphocytesoccur as early as 24 hours after explantation. Thefirst change is the appearance of a large nucleolus.The whole cell enlarges and the nucleus becomesmottled; granules and large filamentous mito-

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Activation of lymphocytes

FIG. 2.

All photographs except Fig. 3 weretaken at 37°C. by phase, x 1,500 Thecells illustrated arefrom unmixed culturesof umbilical cord lymphocytes butidentical changes occur in mixed cultures.

FIG. 1. Lymphocytes on inocu-lation.

FIG. 2. Lymphocytic agglutin-ation after 96 hours.

FIG. 3. Colonyformation by cordlymphocytes on agar x 3.* - > e . ..

X

... .;.i....^....

....... ^: ..

..; si ..i::.. .-.:... ^ . .

..' aR.Rw....:':* ............ ..... ... ..::: .: :..

FIG. 3.

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R. J. V. Pulvertaft and Isobel Pulvertaft

FIG. 4. FIG. 5.

chondria appear in the ctyoplasm. Eventually afterfive days the whole cell shows five times its originaldiameter. Pseudopodia, instead of being blunt andbulbous, are pointed. Mitosis occurs in most, butnot all, cases. Motility ceases (Fig. 4-8).

In fluid cultures colonies develop at the bottom ofcontainers. Attempts to subculture or to maintaincultures have failed. No effect, morphological orotherwise, is seen following the addition of phyto-haemagglutinin to activated cultures of mixedlymphocytes and the end-cell following mixedculture is indistinguishable from the cell found afterphytohaemagglutinin stimulation.

According to Gordon and Maclean and toKasakura and Lowenstein (loc. cit), the culturemedium in which lymphocytes from one individualhave been grown stimulates lymphocytes fromanother individual, causing great multiplication. We,however, have found no morphological changes insimilar circumstances.

Single source lymphocyte cultures were grown forsix days and the medium removed with a pipette withan up-turned point. This fluid was centrifugalizedand, surprisingly, showed an obvious deposit of cells.

The supernatant fluid was removed, and again spun,this time without yielding a deposit.Lymphocytes from another individual were ex-

planted in fresh medium to give a concentration of4,000 cells per c.mm. Three ml. of these cells wascentrifuged and the deposit re-suspended in themedium in which other lymphocytes had grown.Control suspensions were mixed with anotherindividual's lymphocytes and always showed charac-teristic activation in six days. In contrast the testlymphocytes never showed any morphologicalchanges.

In addition to single source lymphocyte culturemedia, medium in which cells from two individualshave been grown and shown activation, and mediumin which cord blood lymphocytes had been grownwere used. No morphological change was ever found.

In a previous communication using culturesseparated by millipore filters, activation was statedto have been found (Pulvertaft and Pulvertaft, 1966).This, however, was an erroneous result, due in allprobability to admixture of small amounts of otherlymphocytes since the lateral apertures were closetogether. When this was prevented by the use of

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FIG. 6. FIG. 7.

FIG. 4. Lymphocytes (18-hr.culture).

FIGS. 5-7. Activatedlympphocytes(three tofive days).

FIG. 8. Colony ofagglutinatedand activated lymphocytes (five days).

F.r: 8.

FIG. 8.

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chambers with widely separated apertures noactivation was found.

INACTIVITY OF CELLS OTHER THAN LYMPHOCYTESMonolayers were prepared from the kidney of a greymonkey, from two cases of human thyroid enlarge-ments, and from a malignant melanoma. Segregatedlymphocytes were added to these cultures fromseveral individuals: the cultures were examined at37°C. by phase contrast. The lymphocytes remainedalive; they did not change their appearance ormultiply.

ACTIVITY OF NON-HUMAN VERTEBRATE LYMPHOCYTESHeparinized blood was obtained from four speciesof monkey, and from rat, rabbit, and hamster. In allcases, when mixed with human lymphocytes, theirsegregated lymphocytes gave full activation. Bloodwas collected with heparin from a crocodile (shot inthe head) and from an anaesthetized python, lungfish, and hen. Small cells segregated from thesespecimens by the technique used for mammalianblood and mixtures were incubated at 37°C. in thestandard medium with human lymphocytes. Noactivation occurred.These animal experiments were performed during

the early stages of our investigations and beforetechnique was standardized.

LYMPHOCYTES FROM VENOUS UMBILICAL CORD BLOODBy venepuncture up to 25 ml. of blood can beobtained from the umbilical cord vein, if puncturedimmediately after ligature of the cord. and beforethe placenta is expelled. Delay in puncture involvesthe extraction of a much smaller volume. Althoughblood is often collected by section and drainage ofthe cord this is to be avoided owing to the danger ofmixture of maternal blood and of tissue elementsfrom the cord itself.

It was found that segregated lymphocytes fromsuch specimens all showed activation in all specimenstested without addition of any other cells. Thetechnique was identical; 4,000 lymphocytes perc.mm. were cultured for six days in a 3 ml. volume.They were, indeed, by far the most active cells

tested. It has been stated that culture of too few cellsin mixed cultures leads to failure to produceactivation. But in the case of cord blood, it was foundthat serial dilution showed complete activation downto a concentration of 150 cells per c.mm., the lowesttested; below this concentration it is difficult toobserve the cells.When first examined all the cells were normal,

highly motile, small lymphocytes. Changes were firstnoted in 48 hours and were complete in six days.

Maternal blood was collected immediately after

the cord blood. The lymphocytes showed no activa-tion after six days.Up to the present, no information is available as

to the behaviour of lymphocytes from prematureinfants, or from infants 6 days or more old.

THE INFLUENCE OF FILARIAL INFESTATION Micro-filaria perstans is exceedingly common in Uganda.It is found not only in the blood, but in pericardial,pleural, and peritoneal exudates and also in prepar-ations of neoplasms such as Burkitt's tumour and innon-malignant enlargement of thyroid. Methods ofconcentrating lymphocytes also concentrate micro-Mfiaria, which are found in the supernatant fluid whenred cells are sedimented and in the deposit when thesupernatant fluid is centrifugalized. Their vigorousmovement renders them easily visible; and theyremain alive and motile for at least a fortnight.Eosinophils have been noted to become adherent tothem, covering them almost completely.They were commonly found in blood from

volunteer blood donors, so that all serum for tissueculture had to be filtered. However, their presence intransfusion in the tropics is not important; they haveto pass through their normal vector in order tobecome viable in the human host, according toauthority.

Unfortunately, they were often present in bloodcollected for experiments with lymphocytes. It wasfound that when they were present, even in smallnumbers, activation of lymphocytes either did notoccur at all, or if it did, was very poorly demon-strable with a mixture of another donor's cells.When lymphocytes from a donor were culturedwith his own microfilaria alone, there was noactivation at all. Cord blood never showed micro-filaria, even when the mother washeavilyinfested.Eosinophils were, however, common in cord blood,sometimes in great numbers.

Non-activation of mixed lymphocytes in thepresence of microfilaria may possibly be due toutilization of growth factors by the parasite. Thelymphocytes, however, remain motile and apparentlynormal in their presence at least for 14 days. Theviolent agitation caused by their movements mayprevent lymphocytic cohesion, which, as notedelsewhere, is a marked feature in activation.The non-activation of lymphocytes from an

infested donor is anomalous. The antigen, themicrofilaria, is by definition present: lymphocytesin the presence of an antigen to which a donor hasbeen exposed commonly are activated.Another interesting possibility should be con-

sidered. Microfilaria have, by definition, come toterms with their host and, although they provokea profound eosinophilia, obviously suffer no harm

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from host antibodies. In the case of Perstansinfestation, the only one which we encountered inUganda, the host himself, so far as is known, is inno way discomfited.

It is to be noted that, when lymphocytes from asubject with Perstans microfilaria are added to thoseof a normal subject, in most cases neither group oflymphocytes is activated. It is possible that inPerstans infestation a mechanism has been developedwhich specifically inhibits activation, and thatwithout this mechanism the microfilaria could notsurvive in the blood.The expert committee on filariasis of the World

Health Organisation (Geneva, 1962) suggested thatresearch on immunochemistry of filarial infectionsshould be prosecuted and search for antibodies inold chronic human infections should also be con-ducted. It does not mention any work on immunereactions in filariasis.We have performed only one experiment to see

whether a child's red cells heavily infected withPl.falciparum would induce activation of lympho-cytes from an adult recently recovered from a similaracute infection. In fact, no activation occurred; it isobvious, however, that the experiment must berepeated many times before any valid opinion can beexpressed.

TITRATION OF INDIVIDUAL A'S CELLS AGAINST THOSEOF INDIVIDUAL B The standard technique involvedthe culture of cells in cylindrical vials, 1 cm. indiameter, the volume being kept constant at 3 ml.,and the total number of cells at 12 million.

If the two individuals' cells are mixed in equalnumbers, i.e., 1-5 ml. of each containing 6 millioncells, activation normally occurs in six days, and inthis way it is not possible to determine whether thecells of one individual induce more activation thanthose of another. In our experiments the contents ofvials are inverted, and transferred to centrifugetubes; after centrifugalization all the deposit wastaken up with a pipette, and transferred to amicroscope slide, care being taken not to disperseaggregates by raising the deposit carelessly. Normally(unless, for example, microfilaria are present or CO2has escaped from ill-stoppered vials) aggregates ofhundreds or thousands of cells are seen, the greatmajority being the large, activated lymphocytes.One method of distinguishing the activation

potency of an individual's lymphocytes is to performdaily counts on the mixed cultures, which in itselfinvolves dispersal of aggregates with possible mod-ification of the final result, as we have found whenvials are rolled during culture. A second method is tokeep the total number of cells of individual Aconstant, and to add diminishing numbers of

individual B's cells. Strictly speaking, in order to besure which individual's cells are the most active, theexperiment should then be performed by keepingB's cells constant and varying those of A.

In practice the volume of blood which canproperly be taken from volunteers, who in our caseswere nearly all blood donors offering normal con-tributions for transfusion, is an additional 20 ml.;and this volume will not permit, as a rule, of morethan six experiments on any one specimen, if eachvial is to contain 12 million lymphocytes from oneindividual alone. It is not possible therefore toconsider in any one experiment many variables.As examples of the kind of results which may be

obtained, the following experiments may be cited:

TABLEB Cells B Cells B Cells B Cells B Cells

400,000 40,000 4,000 400 40

A cells12

million

B cells12

million

Activation+ + NegativeC cells C cells400,000 40,000

Negative + + +C cells C cells4,000 400

NegativeC cells40

+ + + + Negative Negative Negative Negative

A cells A cells400,000 40,000

C cells12 Not done + + +

millionControls-No activation.

A cells A cells A cells4,000 400 40

++± +++ ±++

The results demonstrate clearly that serial dilutionof cells does not necessarily give a clear-cut result;in the case ofA cells against B cells after two negativeresults a strong positive result was given with only400 cells.

However, in two cases 400,000 cells mixed with12 million cells induced marked activation. A cellswere activated by C cells at all concentrations tested,even when a nominal number of only 40 cells wasadded. The errors of dilution and of counting are ofcourse considerable.

THE RELATIONSHIP OF ACTIVATED LYMPHOCYTES TOMALIGNANT LYMPHOMAS It has been pointed outthat the activated lymphocyte has certain points ofsimilarity to the cells of Burkitt's lymphoma(Pulvertaft, 1964). The formation of cellularaggregates in particular is a significant commonfeature.However, the process of activation is one of

continuous development; it begins with the appear-ance of a nucleolus; it continues with the develop-ment of granules and gross enlargement; it concludeswith mitosis and the development of the property ofstaining with pyronin.

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We have made a study of the cells of malignantlymphomas in London, Nigeria, and Uganda formany years, using the same methods of examination,i.e., agar culture and phase examination of livingcells. It is possible to find, in the series of changesoccurring in the activated lymphocyte, resem-blances with all the cells of malignant lymphomata,however named, in each new annual reclassification.A cytologist presented with a fully activated

culture of normal lymphocytes would find it difficultto distinguish it from a culture, for example, of a'reticulosarcoma'.

It may be that lymphocytes can be 'activated'either by an immune process, or a carcinogenicstimulus, and that morphologically there is difficultyin distinguishing them. One marked distinction isthat the malignant lymphocyte can be cultivatedcontinuously in vitro; we, at least, have failed to doso with the non-malignant cell.

'Activated' cells have, in general, a family resem-blance, differing chiefly in size from each other. Butsometimes quite different cells are seen with a muchdarker cytoplasm and nucleus. Indeed, on oneoccasion a typical small lymphocyte, from cordblood, was seen in mitosis. It appears therefore thatactivated lymphocytes can develop along more thanone channel and that the most usual type of develop-ment studied here is dependent on the environmentalconditions occasioned by the cultural techniques.

Nucleated red cells are present in small numbers incord blood, but do not multiply; myelocytes arepresent in small numbers; mitosis has been seen.

ABNORMAL LYMPHOCYTES Since this communicationdeals with the behaviour in vitro of normal lympho-cytes, consideration of the culture of cells fromdisorders of the haemopoietic system is not in generalrelevant.

There are, however, a few considerations whichshould be kept in mind. Segregated leukaemia cells,both of the myeloid and lymphoid series, survivereadily in fluid culture medium for several weeks,and a number of cell lines, both of human andanimal leukaemia, are maintained. We have studiedmany primary cultures, although to date none hasbeen established by us as a cell line. Reference willbe made only to leukaemia of the lymphoid series.

In Uganda 'lymphatic leukaemia' is regularlyencountered, and in our experience the commonestcell type, both in children and in adults, is a smalllymphocyte differing when first isolated little if at allfrom a healthy lymphocyte, when studied by phasecontrast at 37°C. When grown in fluid culture,however, the lymphocytes of normal appearancesdie out, and are seen as ghosts.

Bizarre forms, often in mitosis, appear within a

few days, and these clearly resemble activatedlymphocytes as above described.One case deserves notice in more detail. A young

man of 23 complained of weakness and no physicalsigns were found. His blood showed a gross anaemia,with 3,000 leucocytes per c.mm., 90% of which werenormal small lymphocytes. His bone marrow showed80% small lymphocytes, with 10% 'blast' cells.The blood lymphocytes were segregated, andcultured in fluid medium for six days by the standardtechnique. At the end of that time only very large'clumps' of activated lymphocytes were present, andthe original small lymphocytes were no longer to beseen. The diagnosis in this case was 'aleukaemicleukaemia'.Although a single case can be no moie than

suggestive, the study of segregated lymphocytes insuch conditions by culture may prove valuable.There are some disorders as yet not classified, inwhich gross splenomegaly is a presenting symptom;the culture of blood lymphocytes in these cases oftropical 'big spleen disease' should be considered.We have seen two cases with this diagnosis whichterminated as malignant reticulosis, diagnosedhistologically as 'stem cell sarcoma'.

DISCUSSION

In this paper the word 'activation' is used to denotethe changes found in lymphocytes instead of theconventional word 'transformation' which is used inmany senses. It appears first in pathology in the firstpaper in the first number of the Journal ofPathologyand Bacteriology where Virchow used it in relationto changes seen in malignant cells. When tissueculture was introduced, it was used to cover alldevelopments seen in explanted cells; and when'cell lines', such as Hela cells, were established theywere known as 'transformed' cells. These have allchromosome abnormalities; the other categorieshave not, or not necessarily.The word itself was first used in relation to the

drama, in the Elizabethan period, to allude to thealteration in an actor's appearance when he changeshis costume. Hence its use as a polite synonym fora wig in Victorian times. It is used, of course, inevery science as in the electrical transformer.Presumably the word implies the preservation ofessential individuality while 'mutation' implies achange of individuality. In our present state ofignorance 'activation', which implies merely that thelymphocyte acquires new properties, is preferable.The activation of lymphocytes has been achieved

in many ways since the influence of phytohaemag-glutinin was discovered, and there is still no agree-ment as to the essential nature of the phenomenon.

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The end result seems always to be the same, namely,the conversion of a dormant cell to one of greatactivity, and all the evidence points to the belief thatone of these forms of activity is the production ofantibodies.Why lymphocytes from one person should

stimulate those of another, or more probably whythey should reciprocally react, is not at present clear.There is at present no evidence that any other cellhas this property in vitro. In the living animal therejection of grafts shows that a host reaction takesplace, involving any type of cell whatsoever. But souniversally is the lymphocyte distributed in everyorgan that it is quite impossible to graft a piece oftissue without also introducing lymphocytes. If ourexperiments are confirmed, very few lymphocytesindeed can activate an enormously larger number ofalien cells.

There is massive evidence that lymphocyticactivation is harmless to the individual; indeed,everything points to its being essentially beneficial.Every transfusion involves the transfer of greatnumbers of lymphocytes. We have not concludedwork on post-transfusion lymphocyte behaviour;stored blood six days after transfusion caused noactivation of lymphocytes in the recipient. Whathappens if fresh blood is used, and the recipient'slymphocytes collected while the transfusion is stillrunning has yet to be determined; in one case thisgave a positive result.The behaviour of cord blood lymphocytes was

unexpected; the segregated cells were very activeindeed, and divided even in the smallest numberwhich could be examined. Cord serum itself did notactivate adult lymphocytes; and cord lymphocyteswere activated in the presence of human adultserum. Maternal lymphocytes simultaneously col-lected did not show activation spontaneously.

Until proof to the contrary is obtained, the mostlikely suggestion is that during parturition andseparation of the placenta maternal blood escapesinto the foetal circulation. This is in fact known tooccur. Desai and Creger (1963) collected maternalblood, and labelled the leucocytes with atebrine;just before parturition they were re-injected into themother, and when born, the infant's blood wasexamined for fluorescence. Some labelled cells werefound but in such small numbers that it was thoughtthat they could not have any effect on the infant.However, since in our experiments very few

lymphocytes indeed evoked a profound reaction,this opinion must be reconsidered. It could have noeffect greater than the transfusion of maternal blood,which is often performed with advantage. It could notcause 'runting' if the lymphocytic transfer is duringparturition, since only the young foetus is affected

in this way. Certainly the most likely time fortransfer is when the placenta is separated. It ispossible that accidental and abnormal separationand transfer may occur earlier in pregnancy, andthat this might occasion foetal abnormality, but thisis pure conjecture.Our observations do support the belief that the

volume of maternal blood transferred is small, sinceeven when the mother's blood is heavily infested withmicrofilaria, there are none in the cord blood.

Attention has been directed to the observation thatmore than one morphological entity may proliferatein cultures of activated and segregated lymphocytes.In one case four types were found: (1) the granularpredominant type, 30 ,u in diameter; (2) a muchdarker non-granular cell, 30 ,t in diameter (Fig. 9);(3) a small cell, 6,u in diameter, indistinguishablefrom a small lymphocyte (Fig. 10); (4) myelocytes,segregated along with the lymphocytes. All were inactive division. There is evidence that segregatedlymphocytes may develop along more than onechannel.

It is not known what the end result, in the body,of the activated cell is. On one occasion these cellswere within a macrophage; this may be their normaldestination. In vitro they disintegrate within a fewweeks; but so do most adult mammalian explants.The importance of studies on lymphocytic trans-

formation or activation is great. First, the fact thatone of the commonest mammalian cells was untilrecently believed to be an 'end' cell, with no knownfunction, and is now known to be a resting cell withunlimited potentiality, is a major discovery. Second-ly, it is hoped, with optimism, if up to the presentwithout evidence, that its study in vitro may provehelpful in selecting donors for organ transplantation.Lastly, the basic problem of carcinogenesis is thenature of cell stimulation leading to unnaturalmultiplication, and any information about cellstimuli must take its place in the forefront of cancerresearch.

These discoveries are so recent that it would beuncharitable to suggest that divergence in techniquemakes it impossible to correlate results. If wecompare an analogous biological test, the Rideal-Walker test, for antiseptic activity, we find thata standard broth culture medium, a standardplatinum loop to measure the inoculum, pure phenol,and a stipulated crystallizing point, a standardculture of S.typhi and a rigid technical method areessential in order to reproduce the results. Indeed,any bacteriologist knows that duplicate results areoften unobtainable. Even so, the test only gives therelative value of antiseptics under conditions inwhich, in fact they would never be used.

Until technique is standardized, the practical

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FIG. 9. 'Dark' cells (five-day culture).

application of studies of lymphocytic behaviour willbe impossible. There must be agreement in the firstplace on whether segregated lymphocytes or buffycoats containing all leucocytes are to be used. Iflymphocytes alone are to be used, the method ofsegregation and the permissible numbers of othercells must be defined. There is no such thing as apreparation with lymphocytes alone. The nature ofthe culture medium must be defined; in fact thiscannot at present be precisely achieved, as someanimal serum must be used and no two sera are ofidentical composition. The shape of the containermust be defined as the lymphocytes always settle tothe bottom, where, if the area is sufficiently con-stricted, they cannot survive. The number oflymphocytes per c.mm. and the total volume of cellsuspension must be defined, since these determinethe thickness of the cell carpet. Certain preparationscannot be tested, e.g., those containing microfilaria.The duration of cell culture must be agreed; and ifmixtures of cells from two individuals are studiedthe proportion of each individual's cells must beagreed. During culture, it must be agreed thatcontainers are kept static or rotated; and if kept

FIG. 10. Small lymphocyte in mitosis (five-day culture).

static, whether they are inverted or shaken at anytime.

Finally, and most urgently, there must be agree-ment, if it is at all possible, what a 'transformed' or'activated' cell is. This will take a long time. We mayrecall that no cell is more frequently mentioned inpapers on lymph node pathology than the 'reticulumcell'. Gall (1958), however, believed it to be a mythand illustrates 17 completely different 'reticulumcells' collected from authoritative literature. Wehavetried to define 'activated' cells by all the criteria wewere able to employ. Even then two things are clear:'activation' involves a continuous series from thestandard resting cell to the very large end result;and, secondly, completely different cells appear anddivide by our techniques in small numbers; butpossibly by a varied technique they might pre-dominate. The use of lymphocytic 'transformation'to select organ donors, however desirable and how-ever eventually possible, is not at present a practicalproposition.One suggestion which is purely hypothetical, may

be made. Lymphocytes apparently react only withlymphocytes: in our experiments othercellcultures

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had no effect. If graft reaction and rejection isrelated to lymphocyte activation the process may beinitiated by the lymphocytes in the graft, and not bythe epithelial or glandular cells. It is simple to killlymphocytes without damaging other cells; Colcemid(Ciba), for example, is exceedingly effective. Itwould be of interest to test whether treating graftswith Colcemid to remove all donor lymphocytes hasany effect on 'takes'.

This work was undertaken with the help of a full-timegrant from the British Empire Cancer Campaign forResearch.Thanks are due to Professor M. S. R. Hutt for generous

facilities and helpful advice, and to Professor R. R.Trussell and the staff of the obstetrical department ofMulago Hospital. We are particularly grateful toSister Joyce Hoyes for her forbearance. Dr. Jean T.Holland, M.O. i/c Uganda Blood Transfusion service,and Dr. Elvira C. Arya gave essential advice and help inthe collection of blood samples.

Our greatest debt is to the many blood donors whogenerously and anonymously contributed these samples.

Dr. M. C. Williams of the East African Virus ResearchInstitute and Dr. T. M. Bell of the Imperial CancerResearch Fund gave much help in the collection ofanimal blood specimens.The arduous and responsible task of preparing glass-

ware and apparatus was performed by Mr. HerbertWasswa, S/O Kyeyanga.

REFERENCES

Bain, B., Vas, M. R., and Lowenstein, L. (1964). Blood, 23, 108.Coulson, A. S., and Chalmers, D. G. (1964). Lancet, 1, 468.Desai, R. G., and Creger, W. P. (1963). Blood, 21, 665.Gall, E. A. (1958). Ann. N. Y. Acad. Sci., 73, 120.Gordon, S., and Maclean, L. D. (1965). Nature (Lond.), 208, 795.Kasakura, S., and Lowenstein, L. (1965). Ibid., 208, 794.Pulvertaft, R. J. V. (1964). Lancet, 2, 552.

(1965). J. clin. Path., 18, 261.and Pulvertaft, Isobel (1966). Lancet, 2, 892.

Schrek, R., and Donnelly, W. J. (1961). Blood, 18, 561.

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