Res. Irnmunol.

0 INSTITLIT PASTEUR~ELSEVIER Paris 1992

Mouse T-lymphocyte

1992, 143, 691-700

activation

by Urtica dioica agglutinin

I. - Delineation of two lymphocyte subsets M.A. Le Moal, J.-H. Colle, A. Galelli and P. Truffa-Bachi

(*I

UnitP d’lmmunophysiologie mokkulaire, Dkpartement d’lmmunologie, Institut Pasteur, 75724 Paris Cedex 15

SUMMARY Urtica dioica agglutinin (UDA) is a mouse T-lymphocyte-specific mitogen endowed with proliferative characteristics different from ConA, the prototypic T-lymphocyte mitogen. In particular, UDA induces 2-3-fold-reduced thymidine incorporation as compared to ConA. In an attempt to define the basis of this reduced proliferation, we analysed whether UDA binds to a unique subset of T lymphocytes or whether it activates only a T-cell subset. Cytofluorimetric analysis showed that this lectin binds uniformly to all T lymphocytes and does not, on this criterion, distinguish a particular T-cell subset. We next analysed whether UDA provokes the activation of all T lymphocytes. This was carried out by measuring the increase in cell size and the induction of the p55 chain of the IL2 receptor. The analysis showed that, throughout the kinetics of cell activation, only one subset of T lymphocytes increased in size and expressed the p55 chain of the IL2 receptor, suggesting that UDA activates only a subpopulation of T cells. This conclusion was strengthened by the analysis of 5-bromo-2-deoxyuridine (BrdU) incorporation into the DNA of UDA-activated cells. Two populations were easily identifiable : a BrdUnegative subset consisting of all the small p55-negative lymphocytes, and a BrdU-labelled subset including all the large p55-positive cells. BrdU was incorporated in both CD4+ and CD8+ cells, indicating that UDA did not distinguish helper from cytotoxic T lym phocytes. In addition to the p55 chain of the IL2R, all cycling cells expressed the Pgp- I activation marker. The T lymphocytes, which bound UDA but did not proliferate, remained fully susceptible to subsequent stimulation by ConA. In conclusion, the capacity to proliferate upon UDA binding differentiates a UDA-sensitive from a UDA-refractory subset among splenic mouse T lymphocytes.

Key-words: T lymphocyte, Urtica dioica, T subsets.

UDA, Agglutinin,

INTRODUCTION

Polyclonal activators such as mitogenic plant lectins have been successfully used in the analy-

Submitted June 15, 1992, acceptedJuly 18, 1992. (*) Corresponding author.

Cytokine; Activation,

Mitogen, Lectin,

sis of lymphocyte activation (for review, see Peacock et al., 1990). Although lectins appear to bind in approximately equal amounts to the membranes of all lymphoid cells, their pattern

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of activity differs quite markedly. In the search for new mitogens which may define new activation patterns or uncovered T-lymphocyte subsets, we have found that a small molecular weight lectin (8.5 kDa) purified from stinging nettle rhizomes (Peumans et al., 1984), Urtica dioica agglutinin (UDA) induces the proliferation of mouse T lymphocytes (Le Moal and Truffa-Bachi, 1988). However, some features distinguished UDA from ConA, the reference mouse T-lymphocyte polyclonal activator : the proliferative response elicited by UDA is 2-3-fold lower than that induced by ConA, and is also characterized by delayed proliferative kinetics (Le Moal and Truffa-Bachi, 1988). T-lymphocyte activation is achieved by binding of various ligands to specific receptors which transmit the activation signal to the cell nucleus. Con4 and PHA exert their activity through binding to distinct surface molecules. ConA interacts with the carbohydrates of the CD3 complex, whereas PHA binds to carbohydrates on the clonotypic Ti receptor (Kanellopoulos et al., 1985). As these molecules are expressed on the membrane of all T lymphocytes, ConA and PHA have the capacity to activate the whole T-cell population. UDA binding to a signaltransmitting molecule exclusively displayed on a T-cell subpopulation may account for its reduced proliferative potential with respect to ConA. To test this hypothesis, we have determined the specificity of UDA binding to T lymphocytes, the proportion of activated and dividing T lymphocytes and the kinetics of lymphocyte activation. The results show that UDA does not distinguish T-cell subsets by its binding capacity. Nevertheless, this lectin defines two subsets of T lymphocytes based on their sensitivity or insensitivity to the UDA-induced mitogenie signals. UDA-sensitive lymphocytes are recruited among both CD4+ and CD8+ lymphocytes.

BrdU ConA FITC MFI

= = = =

S-bromo-2-deoxyuridine. concanavalin A. fluorescein isothiocyanate. mean fluorescence intensity.

ET AL. MATERIALS

AND METHODS

Reagents and mAb UDA, a gift of Dr. Peumans, and ConA (Miles, Kankakee, IL) were used at the optimal final concentrations of 1 and 2 pg/ml, respectively (Le Moal and Truffa-Bachi, 1988). mAb anti-p55 ILZR chain (clone 5A2 obtained from Dr. J. Theze (Moreau et al., 1987)), were fluoresceinated as described by Goding (Goding, 1976) with FITC (Sigma), after ammonium sulphate precipitation of ascites fluids. FITC-UDA was prepared using the same method. PE-labelled anti-CD8 mAb was obtained from Caltag (South San Francisco, CA). PE-anti-CD4 and FITC-anti-BrdU were purchased from Becton Dickinson (Mountain View, CA). FITC-labelled antiPgp-1 (Trowbridge et al., 1982) was a gift of Dr. Ezine, Hopital Necker, Paris. Mice and cell culture Eight to 12-week old BALB/c female mice obtained from the Institut Pasteur breeding facilities were used. Culture medium consisted of “RPM1-1640” medium (Gibco-BRL, Grand Island, NY) supplemented with 2 mM L-glutamine, 50 Kg/ml streptomycin, 50 II-l/ml penicillin and 5 070 decomplemented foetal calf serum, 5 x 10m5 M 2-ME and 1 mM sodium pyruvate. After lysis of the red blood cells using 0.87 % NH&I in 1:25 PBS, spleen cells (2 x lo6 cells/ml with or without mitogen) were seeded in 25-cm* flasks (Corning, Corning, NY), 10 ml/flask, and placed at 37’C in a humidified atmosphere of 5 070CO, in air. BrdU labefling 5-bromo-2-deoxyuridine (0.01 mM final concentration, Amersham, UK) was added at day 1 in ConA-stimulated cultures and at day 2 in the UDAstimulated cultures. The cells were pulsed for 36 h, washed, and recultured for 24 h. They were then collected and labelled with FITC-anti-BrdU mAb according to the method of Dr. P. Carayon (Carayon and Bord, 1992). Briefly, lo6 cells stained with PEanti-CD4 or PE-anti-CD8 mAb as described below were fixed with 1 % paraformaldehyde in PBS containing 0.01 % Tween-20. After 24 h at 4”C, the cells

PE PHA PI UDA

= phycoerythrin. = phytohaemagglutinin. = propidium iodide. = Urlica dioica agglutinin.

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were washed, treated with 50,000 U/ml of DNase-I (Sigma) in 40 mM Tris pH 8 containing 10 mM NaCl and 0.6 mM MgCl, for 30 min at 37”C, washed and incubated at RT for 45 min in 0.5 ml of PBS containing 10 % BSA and 0.5 % Tween-20 in the presence of an optimal concentration of FITC-antiBrdU mAb. FACS analyses were carried out as described below. Fluorescence staining procedure and FACS analysis For membrane antigen labelling, fresh or cultured cells (2 x 10” cells in 200 ~1) were stained with predetermined optimal concentrations of fluorescent reagent in PBS containing 2 % decomplemented bovine serum and 0.02 % sodium azide for 20 min in melting ice. They were washed free of excess reagent by centrifugation over 50 % bovine serum in PBS. Labelled cells were suspended in 1 ml of the medium containing 2 ~1 of a 0.01 pg/ml PI solution (Fluka, Buchs, Switzerland). After labelling with anti-CD4 or anti-CD8 phycoerythrin-labelled mAb, the cells were stained with FITC-UDA at RT and directly analysed. Data acquisition was performed on a FACScan system (Becton Dickinson). Dead cells and debris were gated out using both SSC/FSC (side scatter/forward scatter) and PI/PE (propidium iodide/phycoerythrin) scatterplots. Analyses were performed using the “Consort-30” software (Becton Dickinson) or the Lysys program (Becton Dickinson).

RESULTS

UDA binds to all T lymphocytes

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FITC-UDA. Binding was immediate and no evolution of the percentage of labelled cells or of the mean fluorescence intensity (MFI) was recorded during this interval. The values presented were taken after 2 min of contact. In figure 1 are reported the percentage of UDA-positive CD4 and CD8 cells versus lectin concentration (panel A). From 0.5 yg/ml of FITC-UDA, all CD4 and CD8 cells were labelled. The histograms presented in figure 1 (panel D and E) were obtained with the optimal mitogenic concentration of 1 pg/ml FITC-UDA; they show that the label intensity was uniform (similar profiles were observed with higher doses of lectin). Mean fluorescence intensity (MFI), which measures FITC-UDA density on the cell membrane, is presented in figure 1 (panel B). A plateau was not reached, even with the highest concentration of FITC-UDA used (5 pg/ml), before cell agglutination prevented FACS analysis. As reported with other lectins (Dutton, 1972 ; Janossy and Greaves, 1971; Stobo et al., 1972), for high rnitogen concentrations, there was no direct correlation between the amount of UDA bound and its proliferative capacity. The peak of proliferation was obtained at a concentration of 1 pg/ml of UDA and higher concentrations resulted in inhibition (fig. 1, panel C). Taken together, these data show that the reduced proliferative capacity of UDA with respect to ConA cannot be explained by the selective binding of this lectin to a restricted subset of T lymphocytes.

In view of the reduced proliferation induced by UDA (Le Moal and Truffa-Bachi, 1988), it was of importance to assess an eventual restriction of UDA binding to a subset of T lympho-

UDA-induced proliferation T-lymphocyte subset

cytes. This was carried out by FACS analysis of splenic cells using fluoresceinated UDA (FITCUDA), a molecule that retains all the mitogenic properties of the lectin (results not shown). Pilot experiments have revealed that UDA was bound by all spleen cells ; therefore, in order to assessUDA binding to T lymphocytes, spleen cells were stained with PE-anti-CD4 or PE-antiCD8 mAb. In preliminary experiments, we checked that these antibodies did not interfere with UDA binding. The evolution of the fluorescence associated with CD4+ and CD8 + cells was monitored for 20 min after addition of

Since binding in itself is necessary but not sufficient for activation (for instance, ConA binds to all mouse lymphocytes, but exclusively triggers the T population), we analysed whether UDA activated a smaller number of T lymphocytes than ConA. Cell size increase and the presence of the inducible ~55 chain of the IL2 receptor were used to characterize activated T lymphocytes. Spleen cells were cultured with the optimal mitogenic concentrations of UDA (1 pg/ml) or ConA (2 yg/ml) and collected from day 1 through day 5. The cells were stained with

is restricted to a

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0 FITC-UDA

.l

1

@g/ml)

10

0

.l

1

10

UDA @/ml)

Fluorescence Intensity Fig. 1. UDA binds to all T lymphocytes. Spleen cells were labelled with PE-anti-CD4 or PE-anti-CD8 mAb. Increasing amounts of FITCUDA were added and the cells were analysed by FACS. The percentage of UDA-positive CD4+ and CD8+ T lymphocytes is plotted against lectin concentration (panel A). The mean fluorescence intensity (MFI) of the FITC-UDA positive cells is shown in panel B. The proliferative response versus concentration of UDA is representedin panel C. In panelsD and E are reported the histograms of FITC-UDA binding (1 pg/ml) to CD4 and CD8 cells, respectively.

FITC-anti-p55 chain mAb in conjunction with either PE-labelled anti-CD4 or anti-CD8 mAb. FACS analysis of cell size (forward light scatter) and IL2R p55 density of CD4+ and CD8+ lymphocytes are reported in figure 2. In the UDA-activated group, two distinct populations were detected by day 2. The first consisted of CD4+ or CD8+ blasts expressing a high level of the ~55 chain, and the second of small lymphocytes with basal p55 chain density. In contrast, in the ConA group, all CD4+ and CD8+ lymphocytes were uniformly of large sizeand expressed high levels of the p55 chain by day 1. These data suggested that only a subset of the

CD4+ and of the CD8+ lymphocytes was activated by UDA. Incorporation of BrdU into DNA enables precise evaluation of the cycling cells and of the cells having completed cycling (Dolbeare et al., 1983). In order to directly demonstrate that the UDA-refractory T lymphocytes never entered into cell cycle, we used BrdU labelling. BrdU was added at day 1 to ConA-activated cells and at day 2 to the UDA group; this different timing takes into account the delayed proliferative kinetics induced by UDA. The pulse was performed for 36 h, a time interval that spanned the peak of the response to both lectins; 24 h

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CD4+ T CELLS

Con

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I

A

UDA

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CD8+ T CELLS

Con

A

UDA

d

-

IL-2R

(~5% Density

(log)

Fig. 2. Definition by FACS analysisof a UDA-sensitive and of UDA-refractory T-cell subset.

Fresh and UDA- or ConA-activated cellswerecollectedon the indicateddays and stainedwith PE-labelledanti-CD4 or anti-CD8 in conjunction with FITC-labelled anti-p55 IL2R. The cellswere analysedby flow cytometry. CD4+ and CD8’ cellswere selectedand the FSC wasplotted versus ILZR expression.The valuesinside the contour graphs indicate the percentageof IL-2R+.

after the pulse, cells were collected, stained with FITC-anti-BrdU and PE-anti-CD4 or anti-CD8 mAb and analysed by FACS. CD4+ and CD8+ cells were selectively gated and the proportion of BrdU+ cells among each subpopulation measured. In the UDA group, 60 070of CD4+

and 75 070of the CD8+ lymphocytes incorporated BrdU (fig. 3, right panel). In contrast, in the ConA group, the totality of the T lymphocytes were labelled by BrdU (fig. 3, left panel). These data demonstrate that only a subset of T lymphocytes proliferates in response to UDA.

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UDA

Con A CD4+

CD4+

CD8+

FITC-anti-BrdU

CDS+

(log)

Fig. 3. Cell proliferation analysesusing BrdU incorporation.

The BrdU pulsewasperformed 1 day after initiation of the culture for ConA and after 2 days for UDA. After a 36-hpulse,the cellswerecollectedand labelledwith PE-labelledanti-CD4 or antiCD8 mAb in conjunctionwith FITC-anti-BrdU mAb and analysedby FACS asdescribedin “Materials and Methods”. The valuesinsidethe contour graphsindicate the percentageof BrdU- and BrdU+ amongthe CD4+ and CD8+ subpopulations.

Phenotypic characterization of UDA-activated T lymphocytes The restricted expression of some antigens permits the definition of the T-lymphocyte subsets. Particularly, the differential expression of Pgp-1 has been used to subset virgin and memory or activated T lymphocytes (Budd et al., 1987; Cerottini and MacDonald, 1989). Accordingly, we investigated the expression of Pgp-1 on UDAresponding cells. Spleen cells cultured in the presence of UDA (1 pg/ml) were collected on day 4. Cells were labelled with FITC-anti-Pgp-1 mAb in conjunction with PE-anti-CD4 or PEanti-CD8 mAb. In each CD4+ and CDS+ compartment, large and small cells were separately analysed for Pgp-1 expression (fig. 4, left panel). The histograms presented in figure 4, right

panel, show that all large (solid line) CD4 and CD8 cells expressed a high level of Pgp-1 at their membrane, whereas the majority of the resting cells (dashed line) was Pgp-l-. The UDA-refractory T-lymphocyte remains sensitive to ConA activation

subset

The finding that a T-lymphocyte subset remained refractory to UDA raises the possibility that binding of the lectin had delivered a negative signal to this subpopulation. In order to test this hypothesis, spleen cells pre-cultured with UDA for 2 days were stimulated either with ConA or with UDA for an additional 24 h. The cells were stained with FITC-anti-p% chain mAb in conjunction with either PE-labelled anti-CD4

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CD4 Density (log)

CDS Density (log)

Pgp-1 Density (log)

Fig. 4. Expressionof Pgp-1 on UDA-activated cells. Cells stimulatedwith UDA for 4 days were labelled with either PE-labelledanti-CD4 or antiCD8 mAb in conjunction with FITC-anti-Pgp-1 mAb. CD4+ or CDS+ cellswere selected(right panel)and two windows,one for the smallcellsand the other for the largecellsweredrawn. Pgp-1 expressionin CD4+ or CD8+ cellsis represented.Dotted line = smallcells; solid line= large cells.

or anti-CD8 mAb. CD4+ and CD8 + cells were positively gated and the contour graphs of cell size versus IL2R ~55 chain were recorded. The percentage of small ~55 - and large p55+ cells remained unchanged in the UDA-restimulated group, indicating that the UDA-refractory population did not proliferate after UDA challenge (fig. 5, right panel; CD4+ cells, upper; CD8+ cells lower). In contrast, stimulation by ConA provoked the disappearance of the resting UDArefractory population ; these cells increased in size and expressed the ~55 chain of IL2R in both CD4+ (upper panel left) and CD8+ (lower panel left), showing that the UDA-refractory population can be stimulated by ConA.

DISCUSSION

The studies described in the present paper have specifically addressed the existence of UDA-defined T-lymphocyte subsets. These studies are an extension of our previous report dealing with the characterization of Tlymphocyte activation induced by this novel mitogen (Le Moal and Truffa-Bachi, 1988). The data reported here show that while UDA binds to all T lymphocytes, only a subset of these cells is activated and proliferates in response to this lectin. Mitogens are powerful tools in the investigation and definition of the genetic and biochem-

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ET AL.

RESTIMULATION UDA

-

BY : Con A

IL-2R (~55) Density (log)

Fig. 5. The UDA-refractory subset is sensitive to ConA stimulation. Cells stimulated by UDA for 2 days were restimulated by either UDA or ConA. FACS analysis was carried out 24 h later. CD4+ and CD8+ cells were gated using PE-labelled anti-CD4 or antiCD8 mAb and FSC was plotted versus ILZR expression. Values inside the contour graphs indicate the percentage of IL2R+ and IL2R- cells.

ical events occurring during lymphocyte activation and proliferation (reviewed in Hadden and Coffey, 1990). In addition to the specific transmitting molecules, mitogenic lectins bind to glycosylated residues shared by many surface molecules and, on this criterion, do not distinguish particular T-cell subsets. UDA conforms to this scheme, as FACS analysis showed that UDA was bound by all splenic T lymphocytes. Furthermore, the sharp and homogeneous fluorescence peak observed at all FITC-lectin concentrations excluded preferential binding to a particular subset that could account for the

lower proliferative capacity of this lectin compared to ConA. Higher UDA densities on the T-cell membrane could be reached, increasing the lectin concentration. However, this did not better cell proliferation; on the contrary, at values higher than 1 pg/ml, proliferation decreased. Similar dose-dependent inhibition was also reported for other mitogenic lectins (Dutton, 1972; Janossy and &eaves, 1971; Stobo et al., 1972). UDA-binding experiments did not uncover features which could account for the differences in the proliferative capacity of UDA and ConA.

T-LYMPHOCYTE

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However, the exclusive proliferation of a Tlymphocyte subset could account for this characteristic. The analysis of two tightly correlated parameters specifying the progression of T lymphocytes through the Gl phase, i.e., cell size increase and induction of the ~55 chain of IL2R, revealed that the UDA group is characterized by the persistence of small T lymphocytes. These cells did not express the ~55 chain and were not found in the ConA group. These data suggested that this lymphocyte subset did not respond to UDA. Alternatively, these cells could originate from cells having completed DNA replication. BrdU incorporation into the DNA of cycling cells allows for the distinction between dividing and resting cells. In addition, the detection of BrdU establishes that the lymphocyte has been through a DNA replication cycle (Dolbeare et al., 1983). BrdU was never detected in the small cells and was exclusively found associated with the blast population. This finding rules out the possibility that these small T lymphocytes derived from cells which had previously completed cell cycle. As expected, the lymphocytes in the ConA group were all labelled with BrdU. This set of experiments establishes that UDA activates only a subpopulation of the T lymphocyte population. Among the other markers specifying activated cells, we have analysed the expression of Pgp-1 on UDA-activated and -refractory T lymphocytes. Although Pgp-1, a 95-kDa glycoprotein of unknown function, is found on all T lymphocytes, the level of its expression can vary according to the cell activation state. In particular, T cells upregulate their surface level of Pgp-1 upon antigenic stimulation (Budd et al., 1987; Cerottini and MacDonald, 1989). After UDA stimulation, T lymphocytes can be easily separated into two subsets with respect to Pgp-1 expression. Pgp- 1lo cells include all the ~55 resting T lymphocytes ; the Pgp-1 hi subset corresponds to the ~55 + blastic T lymphocytes. These subsets coincide with the subdivision of cycling and resting cells defined by BrdU incorporation. The mitogenic phytoagglutinins, with the notable exception of jacalin, which activates hu-

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man CD4+ but not CD8+ lymphocytes (Pineau et al., 1990), do not discriminate a particular T-lymphocyte population. In this respect, UDA is an exception, since it displays the remarkable feature of enabling the delineation of two Tlymphocyte subsets across the CD4/CD8 subdivision. The mitogenic activity of plant lectins is related to the presence of “receptors” capable of transmitting an activation signal to the cell nucleus (Kanellopoulos et al., 1985). The prototypic T-lymphocyte mitogens, ConA and PHA, exert their activity through binding to different surface molecules : ConA interacts with the carbohydrate moieties of the CD3 complex, whereas PHA binds to carbohydrates on the clonotypic Ti receptor (Kanellopoulos et al., 1985). These transducing molecules are present on all T cells. The restriction of the UDAinduced proliferation to a discrete T-lymphocyte subset must be associated with the mandatory expression on this subset of a UDA-specific signal-conveying transmembrane molecule absent on the UDA-refractory population. The finding that even though all T lymphocytes bind to UDA, this lectin does not induce an inhibitory signal in the UDA proliferation-refractory subset, shows that UDA binding to activationirrelevant molecules does not alter the cellular potential to engage in division after ConA activation. Finally, UDA induces the proliferation of both CD4+ and CD8+ lymphocytes, and UDA-refractory cells are found in both subpopulations ; this indicates that the expression of the signal-conveying molecule has no relation to the helper/cytotoxic functional cell subdivision. The UDA-driven proliferation of a subset of T cells is related to the specific presence of a signal-conveying molecule ; the search for such a receptor is under way. Its characterization may enable the biochemical and functional identification of UDA-sensitive and UDA-refractory subsets. Acknowledgements This investigation was supported in part by the Institut Pasteur, CNRS (LA 040 359) and Association pour la Recherche sur le Cancer (ARC 6244).

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The authors gratefully acknowledge Dr. W. Peumans for the generous gift of UDA. We thank Dr. P. Carayon for providing the technique of BrdU labelling before publication. We are indebted to Drs. G. Langsley and A. Dautry for helpful discussion, and to Drs. P. Cracker and I. Motta for critically reading the manuscript. We thank Mr. R. Perret for technical help.

Activation I. -

des lymphocytes T par I’agglutinine de la grande ortie Mise en evidence de deux sous-populations

L’agglutinine de la grande ortie (UDA) est un activateur polyclonal des lymphocytes T murins qui se differencie de la concanavaline A (ConA), le mitogene classique de ces cellules, par toute une serie de proprietes. En particulier, la proliferation induite par I’UDA, mesuree par I’incorporation de thymidine tritiee, est de 2-3 fois plus faible que celle obtenue avec la ConA. Cette proliferation reduite n’est pas due a une fixation selective sur certains lymphocytes mais a l’activation d’une sous-population discrete de lymphocytes T. En effet, I’UDA se fixe de maniere identique a tous les lymphocytes. L’etude de l’expression de la chaine ~55 du ricepteur de I’IL2 et l’augmentation de la taille cellulaire nous ont permis de montrer que seule une sous-population de lymphocytes T CD4+ et CD8+ entre dans le cycle cellulaire. Cette observation est corroboree par l’analyse de l’incorporation de bromodtsoxyuridine : une fraction seulement des lymphocytes T CD4+ et CD8+ incorpore ce nucleotide dans son ADN. De plus, l’apparition du marqueur membranaire defini par l’anticorps Pgp-I est strictement correlee avec l’augmentation en taille et la presence de la chaine ~55 du rtcepteur de l’IL2. L’UDA dtfinit clairement deux sous-populations de lymphocyted T, celle qui prolifere apris fixation de I’UDA et celle qui ayant fix6 la lectine n’entre pas dans le cycle cellulaire.

Mofs-cl&s: Lymphocyte T, UDA, Agglutinine, Cytokine; Activation, Mitogene, Lectine, Urtica dioica, Sous-populations T.

References Budd, R.C., Cerottini, J.C., Horvath, C., Bron, C., Pedrazzini, T., Howe, R.C. & MacDonald, H.R. (1987), Distinction of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein con-

ET AL. comitant with antigenic stimulation. J. Immunol., 138, 3120-3129. Carayon, P. & Bord, A. (1992), Identification of DNAreplicating lymphocyte subsets using a new method to label the bromo-deoxyuridine incorporated into the DNA. J. immunol. Methods, 147, 225230. Cerottini, J.C. & MacDonald, H.R. (1989), The cellular basis of T-cell memory. Ann. Rev. Immunol., 7, 77-89. Dolbeare, F., Gratzner, H., Pallavicini, M.G. & Gray, J.W. (1983), Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc. natl. Acad. Sci. (Wash.), 80, 5573-5577. Dutton, R.W. (1972), Inhibitory and stimulatory effects of concanavalin A on the response of mouse spleen cell suspensions to antigen. - I. Characterization of the inhibitory cell activity. J. exp. Med., 136, 14451460. Coding, J.W. (1976), Conjugation of antibodies with fluorochromes : modifications to the standard methods. J. immunol. Methods, 13, 215226. Hadden, J.W. & Coffey, R.G. (1990), Early biochemical events in lymphocyte T activation by mitogens. Zmmunopharmacology Rev., 1, 273-376. Janossy, G. & Greaves, M.F. (1971), Lymphocyte activation. - I. Response of T and B lymphocytes to phytomitogens. Clin. exp. Immunol., 9, 483-498. Kanellopoulos, J.M., De Petris, S., Leca, G. & Crumpton, M.J. (1985), The mitogenic lectin from Phaseolus vulgaris does not recognize the T3 antigen of human T lymphocytes. Eur. J. Immunol., 15, 479-486. Le Meal, M.A. & Truffa-Bachi, P. (1988), Urtica dioica agglutinin, a new mitogen for murine T lymphocytes: unaltered IL-l production but late IL-2 mediated proliferation. Cell. Immunol., 115, 24-35. Moreau, J.L., Nabholz, M., Diamantstein, T., Malek, T., Shevach, E. & Thize, J. (1987), Monoclonal antibodies identify three epitope clusters on the mouse ~55 subunit of the IL-2 receptor: relationship to the IL-2 binding site. Eur. J. Immunol., 17, 929-935. Peacock, J., Colsky, A. & Pinto, V. (1990), Lectins and antibodies as tools for studying cellular interactions. J. Immunol. Meth., 126, 147-157. Peumans, W., De Ley, M. & Broekaert, W. (1984), An unusual lectin from stinging nettle (Urtica dioica) rhizomes. FEBS Letters, 177, 99-103. Pineau, N., Aucouturier, P., Brugier, J.C. & Preud’homme, J.L. (1990), Jacalin: a lectin mitogenit for human CD4 T lymphocytes. Clin. exp. Immunol., 80, 420-425. Stobo, J.D., Rosenthal, A.S. & Paul, W.E. (1972), Functional heterogeneity of murine lymphoid cells. I. Responsiveness to and surface binding of concanavalin A and phytohemagglutinin. J. Immunol., 108, 1-17. Trowbridge, I.S., Lesley, J., Schulte, R., Hyman, R. & Trotter, J. (1982), Biochemical characterization and cellular distribution of a polymorphic, murine cellsurface glycoprotein expressed on lymphoid tissues. Immunogenetics, 15, 299-312.