rom time to time, I am sent col- edition of Maximov and Bloom’s Textbook

ourcd wall charts that illustrate of Histology, were endowed with haem-
current knowledge about lym- atopoietic, phagocytic and fibrocytic poten-
phocyte subsets, their origin, de- cies. This was the prevailing view at the
velopment and interactions together with time 1 entered the field. The other view was
the battery of lymphokincs that control the that lymphocytes were ‘end’ cells, dying a
cellular changes underlying immune re- few hours after having performed their I.IG~~
sponses. Whether or not tltey rire accurate, known functions. Various graveyards were
they paint a large and complex picture, and suggested, notably the gut and skin.
one that must be difficult for instructors to Thus, at the beginning of 1954, these
explain and for students to grasp. I have an small cells became my obsession, and
additional difficulty. This is to conjure up Florey said if 1 could find out where they
the time, 40 years ago, when virtually the went, we might know what they did. He
only property of lymphocytes about which drew my attention to a technique for col-
there was general agreement was that they were motile. lecting lymph and lymphocytes from the thoracic duct of un-
It was Howard Florey, the Professor of Pathology at Oxford anaesthetized rats, which Bollman ef nl.? had described in 1948. With
University at that time, who suggested that I should work on lym- a little practice, this became a routine procedure. However, in 1950,
phocytes. I had returned to Oxford at the end of 1953 after a year at Mann and Higgins3 found that, although very large numbers of
the Pasteur Institute with littlc idea what to do next. The laboratory lymphocytes could indeed be collected initially from a thoracic duct
in Oxford already had a record of work on lymphocytes that, JC- fistula, continuous drainage for several days resulted in a precipi-
cording to Florey, had blunted the wits of a number of his col- tous fall in the output of cells. By 1957 we had shown that, if all the
leagues and he could see no reason why I should be spared a simi- cells from the fistula were reinfused continuously into the blood,
lar fate. As is well known, Florcy led the team that transformed the output remained constant: the lymphocytes that normally dis-
penicillin from a laboratory curiosity into the most powerful agent appeared from the blood were somehow responsible for maintaining
yet available for treating human infections. Florey drew my atten- the output of cells from the thoracic duct’.
tion to the current state of the lymphocyte field and liked to quote The simplest explanation for this finding was a recirculation of
Arnold Rich: ‘The complctc ignorance of the function of this cell is lymphocytes from blood to lymph. This was not a new idea but one
one of the most humiliating and disgraceful gaps in all medical that had not previously been taken seriously; however, it gained
knowledge”. Rich was talking about ‘small lymphocytes’ and the plausibility from an experiment I still remember with some satis-
‘small round cells’ of the histopathologists. ‘Literally nothing of im- faction. Thoracic duct lymphocytes were labelled irl z,ifrowith in-
portance’, he concluded, ‘is known regarding the potentialities of organic “?I’,thoroughly washed and then reinfused into the blood of
these cells’. This was tltc situation in 1953, the same rat; a substantial wave of cell-associated ‘?I’ appeared in
the lymph of these animals, overlapping the rise and fall of a wave
of increased cell output. Tritiated thymidine ([‘H]TdR) had recently
The recirculation of lymphocytes become available commercially and this enabled us to show that the
Florey suggested that I should work on the problem known at that increased output was no: due to the Troduction of new cells: over
time as ‘the mystery of the disappearing lymphocytes’. The prob- 95% of the lymphocytes in the wave of lymph-borne cells were un-
lem could be stated very simply: sufficient lymphocytes cnier the labelled when tritiated d~ymidine was given along with the cellular
blood each day from the major lymphatic ducts to replace ,-ll those infusion; the remaining 5%, consisting of large lymphocytes, were
in the blood many times over. For example, in the rat, all the lyil, heavily labelled. The larger lymphocytes were new cells and provided
phocptes in the blood are replaced about ten times daily by those an intental control for the effectiveness of the procedure.
entering from the thoracic duct. The fate of these disappearing lym- The paper describing these results was published in 1959 (Ref. 5)
phocytes was accounted for by two rival theories. The first regarded but not before one referee had recommended its rejection on the
lymphocytes as primitive stem cells which, according to the 1949 grounds that all the experiments could be explained by an exchange

of ??I’between the transfused cells and those
that were recovered from the thoracic duct.
The referee was not alone in their criticisms.
At a haematology meeting in the USA In
Hozonrd Florq (1953).
1959 (Ref. 6), there was a discussion about
the uptake and persistence of radioactivity in
small lymphocytes after giving [“H]TdR iiz CZU. The labelling data
indicated a surprisingly long lifespan for blood lymphocytes which,
of course, supported the idea of recirculation -no other rxplanation
could 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 in
lymphoid tissue during the formation of new lymphocytes. For a
short while, this idea of DNA re-utilization was taken sufficiently
seriously to urge caution in accepting the interpretation of my own
experiments in which the cells comprising the \vdve of increased
output from the thoracic duct had failed to label with [7H]TdR. 1 re-
turned from the USA with only a small concession: if recirculation
occurred at all, it only happened on a small scale.
Recirculation still seemed to me to be the simplest explanation of
all our experiments, but it was necessary to: (I) show that it occurred
on a large enough scale to account for the turnover of the blood
lymphocytes; and (2) trace the pathway of individual cells along
their proposed route from blood to lymph. This was accomplished
by labelling the RNA of thoracic duct cells irk &o with a tritiated
precursor and then determining their location by autoradiography
at various times after intravenous infusion. The labelled cells be-
haved as we had predicted. They migrated from the blood into the
cortex of the lymph nodes, passed into the lymph sinuses and then
reappeared in large numbers in the thoracic duct lymph. High con-
centrations of labelled cells were also found in the Peyer’s patches
but none in the thymus and few in nonlymphoid tissues. Large
numbers migrated into the white pulp of the spleen from which
they were known to re-enter the blood directly. The cells that recir-
culated into the lymph were the small lymphocytes; the minority of
larger, dividing lymphocytes in which the DNA could be labelled,
did not reappolr in the lymph but migrated into the lamina propria
of the small intestine, where they had assumed the morphology of
plasma ceils. This last observation, worked on by Julie Knight, was
the basis of subsequent studies on local immunity in the gut.
IMMUNOLOGY TODAY
The autoradiographic study also located
ihe precise route taken by thesmall lympho-
cytes in their journey from the blood illto
the lymph nodes. At short intervals after in-
travenous infusion, labelled cells were seen
to be migrating across the end&helium of
an unusual set of small blood vessels in the
cortex of the lymph nodes and the Peyer’s
patches. These ‘postcapillary venules’ had
the property of mediating a selective migra-
tion of lymphocytes (and, under normal
conditions, no other class of leukocyte) from
the blood. Vincent Marchisi had been work-
ing with Florcy on the migration of cells
from blood vessels during acute inflamma-
tion and had produced a series of e~,z~.tron
micrograph pictures of poly~~iorphonucle~~r
leukocytes passing between the enduthelial
cells of venules in inflamed tissues. Marchisi
did a similar study on serial sections
through normal rat ly.nph nodes and identified lymphocytes at all
stages in their passage across the endothrlium of the venules. This
included an accumulation under the endothelium, a location first
occupied by the labelled cells soon after their intravenous infusion.
Ci com’se, the electron micmgraphs did not reveal the direction of
lymphocyte 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. The
appearance was quite different from that of polymorphouucledr
leukocytes passing between the endothelial cells of venules in in-
tlamed lymph nodes. Such an intracellular mlgr,ltion is certainly
unorthodox and this point has still to be satisfactorily resolved. The
selectivity of postcapillary venules, now known as ‘high endothelial
venules’, presumably means that the normal traffic through them is
mediated by a repertoire of ligands on lymphocytes different from
that on polymorphonuclear leukocytes.
Two papers in 1964 (Refs 7, 8) appear to have convinced mosl
people that small lymphocytes recirculate from blood to lymph by
way of the lymph nodes and that recirculation explained the turn-
over of the blood lymphocytes. The mystery of the disappearing
lymphocytes was solved: they did not disappear at all but, rather,
continually reappeared in the lymph. However, Florey turned out
to be wrong about one aspect-that discovering the fate of the blood
lymphocytes would uncover their function. Unfortunately the dem-
onstration that lymphocytes recirculated from blood to lymph gave
no clues to their function.
immunology
At t]letimetherecirculation story was developing, there was little
in the literature to convince anyone that small lymphocytes had im-
munoIogical functions. In 1959, Burnet wrote that: ‘An obj~cti~c
survey of the facts could well lead to the concIusion that there was
1996
P -11”
IMMUNOLOGY TODAY
no evidence of immunological activity in small lymphocytes’“.
Certainly, by 1939, we knew from the work of McMasteP that
the lymph nodes were the site of antibody formation after regional
antigenic stimulation, and Coons”, using his technique of im-
mmofluorescence, had shown that plasma cells make antibody
in both the primary and the secondary response. However, there
was still no place for small lymphocytes. Coons thought that the
precursor of the plasma cell might be a large undifferentiated cell
he called a primitive reticular cell. The idea that lymph nodes
were the seat of immunological responses was further strengthened
by the fact that the production of antibody, delayed hypersensitivity
and the reactions against both tumour and normal tissue
allografts could be transferred to suitable recipients by means of
lymph node cells from immunized donors. However, even though
small lymphocytes are a major component of lymph nodes, the cell
types responsible for such adoptive immunization had not been
identified.
The view that Burnet expressed in 1959 had to be updated a few
years later, when an immunological role for small lymphocytes was
unequivocally demonstrated in work influenced by a long-standing
friendship with Peter Medawar. Like others, Peter Medawar was
initially rather sceptical about recirculating, long-lived lbmpho-
cytes and, at his instigation, we did an experiment together to see if
a nucleoprotein fraction would substitute for living lymphocytes in
maintaining the output of cells from the thoracic duct. The results
were negative and he later regarded recirculation as an agreeable
idea, likening the blood lymphocytes to a ‘chorus of soldiers in a
provincial production of Faust - they make a brief public appear-
ance and then disappear behind the scenes only to re-enter by the
same route’“.
Medawar introduced me to the main players in the field of trans-
plantation biology and, particularly, to the work of Simonsen, and
of Billingham and Brent, on graft-versus-host (GVH) reactions. It
had a!,ready been shown that these reactions could be induced by
injecta,ng allogeneic lymph node cells or blood leukocytes into neo-
natal recipients, so suspicion was naturally falling on some kind of
lymphocyte as the inducing cell type. In 1958, I discussed with
Medawar the idea of coifirming this suspicion by injecting thoracic
duct cells. However, the demonstration in 196041 by a number of
US that thoracic duct cells could, indeed, induce GVH reactions
i
LL.nducing
left the precise identity of ti-- cell type unknown. Some
thought that large, dividing lymphocytes might be responsible be-
cause donor cell proliferation in the lymphoid tissue of the host was
a feature of the early stages of the disease. We had a simple way of
resolving this problem by testing the ability of small lymphocytes,
purified from the thoracic duct lymph of parental-strain rats, to
cause GVH reactions in adult F, hybrid rats. Such inocula induced
a vicious reaction in the recipients, with progressive wasting and
death after a time that depended on the cell dose. By contrast, inoc-
ula containing mainly large, dividing lymphocytes failed to induce
the disease.
At first, it was not at all clear how the small lymphocytes were
exerting their devastating effect, particuIarIy as one of the current
views, based on infusing [jH]-TdR in zlizn, was that small lympho-
cytes were long-lived ‘end ’ cells. This idea was demolished when
we showed, by injecting radioactively labelled lymphocytes into F,
rats, that during the early stages of the resulting GVH reaction, the
donor small lymphocytes enlarged and began dividing. The donor
origin of these dividing cells was confirmed by means of a chromo-
some marker’~? at last, small lymphocytes did something. Also in
1961, Jacques Miller published his work on the lymphopenia and
immune deficiency of neonatally thymectomized mice; this was the
first of his many contributions t3 knowledge about the function of
the thymus in immunity and about what subsequently became
known as T lymphocytes. A quite independent line of enquiry during
this period also showed that small lymphocytes could be provoked
into 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 the
dividing cells were derived from small lymphocytest7.
Our subsequent experiments on alloreactivity drew heavily on
Medawar’s analysis of the allograft reaction and of itnmunological
tolerance. 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 in
their ability to induce GVH reactions]” and long-standing skin allo-
grafts on tolerant animals were destroyed following an injcsction of
small lymphocytes from normal donors“‘. Bill Ford also contributed
much to the understanding of the alloreactivity of lymphocytes,
and the phenomenon of lymphocyte migration.
It seemed to us unlikely that the immunological activity of small
lymphocytes was restricted to alloreactivity so, concurrently with
the work on GVH reactions, Douglas McGregor had begun studies
on their possible role in the induction of antibody responses.
Support for this possibility came from studies in rats depleted
of recirculating lymphocytes by prolonged drainage of cells from
a thoracic duct fistula. Such animals showed a profound depression
of pritnary antibody responses to tetanus tnxoid and to sheep
erythrocytes, which could bt: restored to normal by injections
of small lymp!locytes from the thoracic duct. Similarly, the primary
response of X-irradiated rats to sheep ervthrocytes could be re-
stored by small lymphocytes from normal, but not from specifically
tolerant, donors. On the other hand, immunized rats that had
been drained of recirculating cells still responded normally to
antigenic challenge: secondary responses, unlike the primary re-
sponses we had studied, did not seem to be sensitive to lymphocyte
depletion. These findings, published in 1963 (Ref. 20), were con-
firmed three years later while working with Jonathan Uhr in New
York, 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 immunized
with 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 cellular
ingredients for mounting a secondary response were present both
in the recirculating pool and in lymphocyte-depleted lymphoid
tissue.
 IMMUNOLOGY TODAY

-r&e bnction of lym generation of antibody diversity, the structure of the surface mol-
The work with Dou$~ McGregor was sufficiently advanced by 1962 ecules of lymphocyte subsets, and the role of these moiecules and
for us to suggest a general immunological function for small lym- of the major histocompatibillty complex in cell cooperation and
phocytesz2. We had interpreted the experiments on GVH reactions as antigen recognition. However, 1 have to admit to one small disap-
showing that small lymphocytes had initiated the immune response pointment: to my knowledge it is still not possible to describe the
following interaction with the foreign histocompatibility antigens of evolution of any immune response in terms of the highly dynamic
the host. We extended this notion to suggest that these small, recircu- structure of lymphoid tissue ill zlizl~and of the migratory pathways
lating cells, not yet in cell division, are the population that endows an of cooperating lymphocytes within it. Maybe my next colol-red wall
animal with its immune respomiveness. Furthermore, following chart will have the answers.
their stimulation with antigen, they would be triggered into division
and differentiation to produce the various effecters of immune re-
sponses, including antibody-forming cells. The weakness of the ar- James Gowans isnf i3 CHI~IIZO~
Ijili, O.+I{, J_IK 0x2 gf+X.

gument was the absence of any firm evidence that small lymphocytes
eferences
would give rise to plasma cells, although we thought this very likely
1 Rich, A.R. (193h) .&I./I, Pnfiwl. 22, 228-254
Such a function at last gave a rflisnli fYPtrefor lymphocyte recircu-
2 Bollman, J.L., Cain, J.C. and Grindley J.H. (lY#) J Ir?h C/N. MP~. 33,
lation: it would allow specifically reactive cells to be selected from the 1349-1352
large recirculating pool into regional lymphoid tissue following lo- 3 Mann. J.D and H&gin%G.M. (1950) Blw0 5, 177-190
calized autigenic stimulation. A selection of this kind was subse 4 Gowans, j.L. (1957) Br. j. E.xp. Pni/r~)l. 38. 67-Z
quently demonstrated ill P~UOfor histocompatibility antigens2’ and 5 Gowans. J.L. (1959) I. Piysiol. IJb, 54-69
for antigens yielding conventional antibody responses2”,25.Thus, the 6 Stohlman, F., ed. (1959) T/w Kiwhrs of Cchh Pdifmfi~w, GITXW and
small Ijrmphocyte was the antigen-sensitive cell that met the require- Stratton
ments of Bumet’s clonal selection theory. 7 Gowans, J.L. and Knight, E.J. (1964) Pmt. X. Soi. Lor~dorzSu 6 159,
257-282
The dispute about the cellular origin of plasma cells still needed
8 Marchesi, V.T. and Gowans, J.L. (1964) Pwr. R. Soi. Lomtw jr,: B 159,
resolving. Our obsession with this problem continued into the pe-
283-290
riod \&en the focus of interest in the field had shifted to studies on
9 Burnet, EM. (1959) in 7%~~Ckwrl S&h~lrr Thy of Aq~md Imrnuuify.
cell colIabora+ 3n, but we were anxious to complete the picture. Our
p. 110, Cambridge University Press
predictions were confirmed by Susan Roser (nee Ellis) and Jonathan 10 McMaster, l?D. and Hudack, 5.5. (1935) 1, Erp Mcci. bl, 783 +O5
Howard in transfer experiments in which donor and recipient cells 11 Leduc, E.H., Coons, A.H. and Connolly, J.M. (1%) J, Evp !&<1. 102,
could be distinguished in suitably chosen irradiated hosts by the 61-72
use of alloantisera: cells’making a primary antibody to sheep eryth- 12 Medarvar, PB. and Medarvar, J.S. (1977) in T/:c L~fbSuwn~, p. 138.
rocytes and a secondary response to tetanus toxoid were, in both Wildwood House

cases, derived from the donors’ small lymphocy&+?“. So these 13 Goxrans, J.L., Gesner, B.M and ILliCrqp, O.D. (1961) in Bi&qi;t~l

versatile cells not only initiated reactions against alloantigens, but Acfri$ I$ flw Lrucqfr~ (Wolstenholme, G.lz.W. and O’Connor, M., ed.,,
pp. 32-10, Churchill
they were also triggered by antigen to generate antibody-producing
14 Guwans, J.L. (1962) Anr~. Nm Ym’kAd. Sci. 99, 432-I-l5
plasma cells.
15 Miller, J.F.A.2 (1961) Lnr~cct ii, 74R719
Of course we never did complete our picture. We could not
16 Nowell, PC. (1960) Cnurer Res. 20, 462-166
explain what we had termed the ‘functional heterogeneity’ that
17 Marshall, W.H. and Robert, K.B. (1963) Q. /. Erp. @sit?!. 48, 146-155
allowed small lymphocytes to engage in all classes of immune 18 McCullagh, f?J, and Gowans, J.L. (1966) in 7%~Lp$roc!/fc ill /I?II~~I~II~~~~~/
response. We made no contribution to the solution of this problem wzd Hncruopoicsb(Yoffey, J.M., ed.), pp. 234-2-11, Edward Arnold
and it is for this reason that I have deliberately avoided any refer- 19 McGregor, D.D. and Go~van~, J.L. (1965) Prq. .411rr;~.1/
Y, l-78
ence to T and B lymphocytes in interpreting our old work. TO my 20 McGregor, D.D. and Gowans, J.L. (19b3) /. E.yp Md 117, 303-320
mind, the first persuasive evidence for the existence of two classes 21 Gowans, J.L.and Uhr, J.W. (1966) I. E-~/J.Md. 121, 1017-1030

of lymphocytes came in 1968 from the work of Mitchell and Miller”. 22 Gowans, J.L., McGregor, D.D.. Cowcn, D.M. and Ford, C.E. (1962)

In 1971, the idea that thymus-derived (T) lymphocytes ‘helped’ the Nnhrw 196, 651-655
23 Ford, W.L. and Atkim, R.C. (19711 ,X~j,lf.
?&w Blof. 23J,liR-1tiO
development of marrow lymphocytes was used by Mitchison to ex-
24 Sprent, J,, hliller, 1.F.A.J’. and Mitchell, G.F. (1971) Cc/l. Iwlfi~~~f. 2.
plain the effect of prior carrier immunization in increasing the pro-
171-181
duction of antihapten antibody following challenge with a hapten-
25 Rowley D.A., Gowans, J.L., Ford, W.L., Atkins, R.C. and Smith. ME.
carrier conjugate3”. Cooperation between B and T lymphocytes had
(1972) /. E-v!‘.II%?/. 136, 499-513
now become the topic of the day. 26 Ellis, ST., Gowans, J.L. and Howard. ].C. (1969) in A~itil;it#w 1.f
Ch~~,rtafl~~~myirl
(Vol. 15) (Sorkin, E., ed.1. pp. 50-35, Karger
27 Howard,J.C.and Gowans,J.L. (1972) Pr~i. R. SOC.
LOII~OII
5~ B 182. lYi-2OY

Concluding remarks 26 Ellis, S.T, and Gowans, J.L. (1973) PIIX. R. S0c. L(IIII/~I!SU. B 183, 123-13’)

Since these early studies, the lymphocyte has risen to become one of 29 Mitchell, G.F. and Miller, J.F.A.P. (3968) J. I?\/‘. Med. 128, H21-s37
30 Mitchjson x.,2. \.,,
11071, F*(r.,,.
L, L. . I 1., l!L27
I .I3&rl!trl!!!l.,
__
the most studied of all mammalian cells. We now know about the