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Characterization of in vitro differentiated secondary lymphocytes
Experimental Cell Research 100 (1976) 281-290
CHARACTERIZATION
OF IN VITRO
SECONDARY
DIFFERENTIATED
LYMPHOCYTES
Quantitative and Ultrastructural
Study
M. KALINA Department
of Cell Biology and Histology, Tel-Aviv University,
Sackler School Israel
of Medicine,
SUMMARY Blast cells derived from rat lymphocytes by stimulation with concanavalin A (ConA) or pokeweed mitogen (PWM), or by sensitization on xenogeneic fibroblast monolayers, transformed into secondary small lymphocytes following their transfer to syngeneic monolayers devoid of mitogen or sensitizing antigen. This transformation resulted in the disappearance of morphological blast characteristics such as euchromatic nuclei, prominent nucleoli and the aggregation of ribosomes into polysomes. Secondary lymphocytes resembled non-stimulated cells, but differed from them in possessing a slightly larger cytoplasm containing large numbers of lysosomal bodies, interchromatin granules within the nuclei, nucleoli containing homogeneous fine granulo-fibrillar material and a relatively developed Golgi apparatus. Upon re-exposure to the stimulating mitogen or the sensitizing phenotype, the secondary lymphocytes rapidly transformed into blast cells with cytotoxic activity.
There is an increasing amount of evidence linking small to medium-sized lymphoid cells with allograft rejection [l, 21. There are also indications that small lymphocytes are effector cells capable of specific lysis of target cells in vitro [3,4]. Berke & Levey [5] have provided data suggesting that the lymphoid cells responsible for target cells destruction in vitro might also be the effector cells involved in graft rejection in vivo. To analyse the behavior and properties of these small lymphocytes, one needs a pure population of such cells, preferably in an in vitro system. It has been shown by Ginsburg et al. [6] and Hollander & Ginsburg [4] that blast cells obtained by stimulation of rat lymphocytes with phytomitogen, redifferentiate 19-761805
without the mitogen to small-sized cytotoxic lymphocytes, termed secondary lymphocytes. These authors concluded that the secondary lymphocytes obtained by stimulation with pokeweed mitogen (PWM) represented a pure population of memory cells specific to PWM. In similar fashion, the in vitro generation of memory cells reactive to transplantation antigens has been demonstrated by Hollander et al. [4]. Secondary small lymphocytes possessing immunological memory were obtained also by Andersson & Hayry [7] and Macdonald et al. [8], using mixed lymphocyte reaction (MLR) cultures. The present study was undertaken in an attempt to characterize some of the ultrastructural features of rat secondary lymphoE.rp Cell
Res
100 (1976)
282
M. Kalina
cytes, obtained after in vitro stimulation of Growth of blast cells on syngeneic rat lymphocytes with Concanavalin A fibroblast monolayer (ConA), or PWM, or by antigenic sensitiza- The blast cells from both mitogen and antigen stimution with xenogeneic fibroblast monolayers. lated cultures were harvested. washed. resusnended in
MATERIALS
AND METHODS
Animals Lymphocytes were obtained from Lewis strain rats. Embryos for preparation of fibroblast monolavers were obtained either from Lewis strain rats or from C,H/eb mice.
Mitogens A stock solution of PWM (Grand Island Biological Company) containing 1 mg dry weight/ml was kept frozen at -20°C. ConA (Miles-Yeda Ltd., Rehovoth) was kept as stock solution of 1 mg/ml at 4°C.
Fibroblast
monolayer-s
Monolayers from rat and mice embryos were prepared and maintained as previously described [9]. Monolayers used to maintain growth and differentiation of blast cells and secondary lymphocytes were prepared according to Hollander & Ginsburg [3] by plating 5x 10’ tibroblasts in 10 ml of LA medium (5 % lactalbumin hydrolysate in Earle’s solution with 5% calf serum) onto 100 mm Nunc Petri dishes.
Stimulation of rat lymphocytes PWM or ConA
Adherence of lymphocytes monolayers
to target
For assaying adherence the method of Kalina & Hollander [12] was employed. 5X lo6 secondary lymphocytes in 3 ml medium were plated onto the target tibroblast monolayers. When the stimulating agent was a mitogen, it was also added to the medium. The nonadherent lymphocytes were harvested intermittently by pipetting, the monolayer then washed with an additional 3 ml of medium and the washings together with the earlier harvests representing the non-adherent fraction. The remaining monolaver and adhering Ivmuhocytes were overlaid with trypsin solution and incubated for 30 min following which the cell susnension representing the adherent fraction was aspirated. Lymphocytes in both fractions were counted in hemocytometer and mean values were obtained from eight counts of each sample. Adherence and non-adherence were expressed as percent of the total number of lymphocytes.
with
Lymphocyte suspensions were prepared from Lewis rat lvmnh nodes accordine to Ginsburg & Sachs rIOi. The-ceils were centrifuged and resuspended in buibecco’s modified Eagle’s medium containine 15% horse serum (GDBCO)-and 5 pg of PWM or 15 pg of ConA per ml of medium. Lymphocytes 30X lo6 in 4 ml of medium were cultured in 60 mm Nunc plastic Petri dishes and incubated for 4 (PWM) or 3 (ConA) davs at 37°C in a humidified incubator with a flow of 7 %-CO, in air. In some experiments the blast cell populations were purified up to 98% by using bovine serum albumin gradient according to Raidt et al. [ 1I].
Stimulation of rat lymphocytes by xenogeneic (C3H mice) fibroblast monolayers Suspensions of rat lymphocytes were prepared as above-described and 30x lo6 lymphocytes in 4 ml Dulbecco modified Eagle’s medium containing 15% horse serum were plated onto each of the C,H sensitizing tibroblast monolayers. The cultures were then incubated for 4 days at 37°C in a humidified incubator with a flow of 7 % CO, in air. Exp Cell Res 100 (I 976)
fresh medium, and plated onto Lewis rat hbroblast monolayers in 100 mm Petri dishes, 5x 106 cells in 10 ml medium/dish. In order to obtain suspensions of secondary lymphocytes these cultures were then incubated for at least 4 days. In some experiments the secondary lymphocytes were kept in culture up to 21 days, the medium changed every 3 days.
Assays of cell transformation cell lysis
and target
Cell transformation was assessed by measuring the uptake of [3H]TdR as described by Ginsburg et al. [6]. The extent of target cell lysis was assayed by the release of 51Cr. 3x lo6 lymphocytes in 1.5 ml medium were plated on syngeneic or xenogeneic 51Cr-labelled target fibroblasts. The cultures were then incubated at 37°C for 20 h and the amount of radioactivity released into the medium measured according to Berke et al. [9].
Electron microscopy 8 x lo6 lymphocytes were centrifuged and the obtained thin pellet fixed for 2 h in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, post-fixed for 1 h in 1% osmium tetroxide, stained in block with 0.5% uranyl acetate, dehydrated, and finally embedded in Epon 812. In some experiments the monolayer with the adherent lymphocytes was processed in the same way. Ultrathin sections were cut on a Reichert Ultramicrotome, stained with many1 acetate and lead nitrate, and examined with a JEM 100B electron microscope.
Ultrastructure of secondary lymphocytes
Fig. 1. Abscissa: time (days, I, 2, 3, etc.); ordinure: cpmx 103/106cells (10, 20, 30, etc.). Kinetics of 13H]TdR uptake by Lewis rat lymph node lymphocytes stimulated with ConA (O---O), PWM (A---A) or sensitized against C,H fibroblast monolayers (S--m), and restimulation of ConA (O-O), PWM (A-A) or C,H sensitized (m-m) rat secondarv lymphocytes. Conditions for primary stimulation and secondary lymphocytes preparation as well as radioactivity determination is as described in Materials and Methods. Secondary lymphocytes were restimulated 6 days after removal of the stimulating mitogen or sensitizing antigen.
283
by the non-stimulated cells. These kinetics of rat secondary lymphocytes restimulation are in agreement with those obtained by Hollander & Ginsburg [3] and by Hollander et al. [4], who have also shown that ConA, PWM- and antigen-stimulated secondary lymphocytes are capable of massive adherence to fibroblast target cells as well as of cytotoxic activity. Adhesion and cytotoxicity of the secondary lymphocytes in the present study are summarized in table I.
Ultrastructural characterization of ConA-, PWM- and antigen-stimulated secondary lymphocytes The three different populations of rat secondary lymphocytes were observed under the electron microscope and compared to a non-stimulated population of rat lymphocytes and to blast cells stimulated by the two mitogens and by antigens of C,H fibroRESULTS blasts. The secondary lymphocytes obQuantitative characteristics of ConA, obtained from the three types of stimulaPWM- or antigen-stimulated secondary tion were morphologically similar and we lymphocytes shall therefore refer to them simply as secCultures of secondary lymphocytes ob- ondary lymphocytes. tained by the above three procedures were evaluated from the following standpoints: (a) thymidine uptake and blast transformation; (b) adhesion to target fibroblasts; (c) cytotoxicity. Fig. 1 shows the kinetics of thymidine uptake by the three populations of secondary lymphocytes upon re-stimulation with the mitogen or antigen initially used to stimulate these cells. The percentages of blast cells in the various cultures are shown in fig. 2. Noteworthy is the Fig. 2. Abscissa: time (days, I, 2, 3, etc.); ordinate: rapid transformation obtained with sec- % of blast cells (O-100%). of morphological transformation of Lewis ondary lymphocytes as compared with that ratKinetics lymph node lymphocytes stimulated with ConA observed with non-stimulated cells. Thus fO---0): PWM (A---A): or sensitized against C,H fibroblast monolayers (i---m); and restimulation “of the percentage of blast cells in the sec- ConA (0-O): PWM (A-A); or C,H sensitized ondary lymphocyte population reaches (B-m) rat secondary lymphocytes. Percentage of cells was determined by differential counting in maximum 1 day after the onset of restimula- blast hemocytometer. Experimental conditions as described tion as compared with the 34 days required in fig. 1. E.rpCell Res 100(1976)
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M. Kalina
Table 1. Adherence and cytotoxic activity of secondary lymphocytes Class of lymphocyte Secondary-ConA Secondary-ConA Secondary-PWM Secondary-PWM Secondary C3HC Secondary C,H Non-stimulated Non-stimulated Non-stimulated
Mitogen added
Target monolayer
Percentage adherence”
Percentage “‘Cr release*
+ConA -ConA +PWM -PWM
Lewis Lewis Lewis Lewis W BALB/c Lewis Lewis Lewis
65.4 9.2 85.3 5.7 36.2 17.5 N.D. 7.5 2.2
32.0 6.6 57.3 8.2 45.9 16.0 6.0 5.5 1.0
+ConA +PWM Not added
Adherence and 51Crrelease were assayed on the same sample of cultured secondary lymphocytes divided into two portions as described in Materials and Methods. (1 Assayed after 6 h adhesion. * Assayed after 20 h; lysis is expressed as the percent of total radioactivity released into the medium, after correction for spontaneous 51Crrelease in control plates. c Lewis rat lymphocytes sensitized against C,H monolayers. N.D., not determined.
Figs 4-6 are low magnification electron Fig. 3 is a light micrograph of populations of non-stimulated, PWM-stimulated and micrographs of populations of non-stimuPWM-secondary lymphocytes. As can be lated (fig. 4), PWM-stimulated blasts (fig. 5) seen, the diameter of secondary lympho- and of PWM-secondary lymphocytes (fig. cytes is almost the same as that of non- 6). The two most pronounced ultrastrucstimulated cells. In previous calculations tural changes evident after differentiation based on cell volume, typical small lympho- from the blast form to secondary lymphocytes were found to be 6.5 pm, blast cells cyte pertain to the nucleus and to the ar10.5 pm and secondary lymphocytes 7.3 rangement of the ribosomes. The nucleus of the secondary lymphocyte is smaller, pm in average diameter [6].
3. A light micrograph of populations of (a) Lewis rat non-stimulated cells; (b) PWM-stimulated blasts; and (c) PWM-secondary lymphocytes. Note the resemblance in size of cells and nuclei in (a) and (c) in
Fig.
Exp Cell Res I00 (1976)
comparison with (b). A few large cells in (c) have not yet completely differentiated into small secondary lymphocytes. Toluidine blue stained Epon section. x1000.
Ultrastructure
ofsecondary
lymphocytes
285
Figs 4-6. Low magnification electron micrographs of (fig. 4) non-stimulated; (fig. 5) PWM-stimulated (4 days) blast cells; and (fig. 6) PWM-secondary lymphocytes 6 days after removal of the mitogen. Note the prominent nucleoli in the blast cells (NJ~) which disappear in the secondary lymphocytes. The latter cell population appears morphologically homogeneous with small relatively heterochromatic nuclei similar to the non-stimulated lymphocytes (fig. 4). The cytoplasm, however, is more developed in secondary lymphocytes and even at that magnification a large number of vacuoles can be observed (arrows). Fig. 4, x4000; fig. 5, x3400; fig. 6, x3900. Exp Cell Rrs IO0 (1976)
286
M. Kalina
Fig. 7. A part of a &H-sensitized
rat secondary lymphocyte on the sixth day of differentiation from the blast form. Note the nucleolus (NL) containing granular and fibrillar material and areas of condensed chromatin within the nucleus. Interchromatin granules (IG) are evident in the nucleus. Vacuole containine various debris (0) can be seen in the cytoplasm. G: Golgi apparatus. x 14500.
Fig. 8. Parts of two cells from C,H-sensitized blast
cells culture on the 2nd day of differentiation to secondary lymphocytes. Note the typical arrangement of polysomes in the blast cells (I?) in comparison with the discrete ribosomes which are scattered throughout the cytoplasm of secondary lymphocytes (S). x 19000.
more heterochromatic and lacks the large blasts as well as of secondary lymphocytes and complex nucleoli typical for blasts (figs could be observed in the cultures. Sec5, 6, 7, IO). As for the typical arrangement ondary lymphocytes were in many respects of polysomes in blast cells, this gives way morphologically similar to non-stimulated in the secondary lymphocytes to discrete cells, i.e. relatively heterochromatic nuribosomes which are scattered throughout cleus, small nucleolus and discrete free the cytoplasm in similar fashion to the ribo- ribosomes (figs 4, 6, 9, 10). However, they somes in non-stimulated cells (fig. 8). differed from non-stimulated cells in having Observations made at different intervals a relatively large number of interchrofollowing the removal of the stimulating matin, electron dense granules, which are agent (from 4-2 1 days) showed no change in rare in the non-stimulated cells (figs 7, 10, 1l), a large number of large bodies rethe morphology of secondary lymphocytes and populations of these appeared ultra- sembling lysosomes (figs 6, 7, 11) and a structurally homogeneous. At 2-3 days, a seemingly better developed Golgi apparatus mixed population of large and medium (figs 7, 10). The nucleoli of secondary lymExp Cell Rrs 100 (1976)
Ultrastructure
Fig. 9. Electron micrograph of Lewis rat lymphocyte typical of cells in non-stimulated culture. Note the ring-like nucleolus (NL) and the condensed state of most of the chromatin. x 11000. Fig. IO. A &H-sensitized Lewis rat secondary lymphocyte (6th day). Note the small homogeneous nucleolus (NL) and the condensed state of part of the chromatin. Interchromatin granules can be seen in the nucleus (arrow). G, Golgi apparatus. X 11 500.
Fig. II.
of secondary lymphocytes
287
A PWM-secondary lymphocyte (6th day). Note the large vacuoles (0) in the cytoplasm, and the condensed state of the chromatin. x 13 000. Fig. 12. PWM-secondary lymphocytes restimulated in suspension for 12 h with PWM. Some blast features, such as euchromatic nuclei and polysomes, are evident. x3 500.
288
M. Kalina
Fig. 13. A PWM-secondarv lvmphocvte (L) attached to a fibroblast (F) from cell culture which was restimulated with PWM for 2 h on tibroblast monolaver. Note the blast-like euchromatic nucleus and prominent nucleolus evident at that short time of restimulation.
Fig. 14. A higher magnification of the contact region in
fig. 13; the ribosomes in the lymphocytes (L) are already aggregated to form polysomes. x24000.
x6000.
phocytes, although similar in size to that of non-stimulated cells, differ from the latter in some morphological features. They lack the ring-type appearance (fig. 9) and central location typical of non-stimulated cells [ 151; instead, these nucleoli appeared relatively homogeneous in density and contain fine granular and fibrillar material (fig. 10). In most secondary lymphocytes, the cytoplasm appeared to be more developed than in non-stimulated cells and the ratio of cytoplasm to nucleus was slightly higher. Restimulation
of secondary lymphocytes
primary response of the lymph-node cells (fig. 5). Samples of cells taken at 2.5, 5 and 10 h after the onset of stimulation showed an increase in the number of cells with blast characteristics, i.e., displaying cell enlargement, aggregation of ribosomes into polysomes and euchromatic nucleus (fig. 12). When ConA- and PWM-secondary lymphocytes were restimulated on syngeneic tibroblast monolayer, a rapid and massive adherence of the cells onto the monolayer was observed (table 1). In these experiments, most of the adherent cells showed blastlike appearance even 2-3 h after the onset of stimulation (figs 13, 14).
A restimulation of secondary lymphocytes with the appropriate stimulant resulted in a DISCUSSION rapid transformation into blast cells, reaching a peak after 24 h (figs 1, 2). Morpho- The three classes of secondary lymphologically these blast cells were indis- cytes studied by us were obtained from tinguishable from blast cells obtained in the primary rat lymphocytes by exposure of the E.rp Cdl
Rps
100 (IY76)
Ultrastructure
latter to three different modes of activation: (a) A non-specific stimulation of T cells by ConA which polyclonally represented activation of the majority of T lymphocytes [ 131; (b) stimulation with PWM, which represented a specific activation of a small subpopulation (l-3 %) of the cells [3]; in the rat, this subpopulation was found to consist of T cells only [14]; (c) a specific stimulation of a subpopulation of rat lymphocytes reactive against transplantation antigen [4]. The secondary cells obtained by the latter two modes of activation displayed the properties of memory cells. Thus, upon their re-exposure to Iibroblasts together with PWM, or re-exposure tofibroblasts of the sensitizing phenotype, the secondary lymphocytes adhered to the libroblast monolayer and rapidly transformed into blast cells with cytotoxic activity (see table 1, figs 1, 2). Hollander et al. [4] and Hollander [15] using density gradient as well as autoradiography, have shown that secondary lymphocytes, capable of both accelerated blast transformation and targetcell killing, are the progeny of the blast cells obtained by primary sensitization. Some of the experiments presented here were done on blast cells fractionated on a bovine serum albumin density gradient and the secondary lymphocytes reverted from them. This will exclude the possibility that some of the secondary lymphocytes belong to a pool of non-stimulated cells, refractory to the stimulation. The three populations of secondary lymphocytes were morphologically indistinguishable, each comprising a homogeneous population of small lymphocytes. These cells lacked the typical blast characteristics, i.e., polysomes, large euchromatic nuclei and prominent nuclei [16, 171. Morphologically they resembled in many ways the non-stimulated lymphocytes. However
Qfsecondary
lymphocytes
289
a few ultrastructural differences between non-stimulated and secondary lymphocytes were noted, mainly a high number of large lysosomal bodies, interchromatin granules and a relatively developed Golgi apparatus in the latter cells. The nucleolus appeared to be composed of a relatively homogeneous tine fibrillar and granular material, and was often located at the periphery of the nucleus instead of the strict ring type central nucleolus of non-stimulated cells [16]. No characterization of the lysosomal bodies can be given at present since it has been shown that the biogenesis of lysosomes in mitogen-stimulated blast cells is correlated to endocytic activity [18]. However, these bodies containing various cell components, such as remnants of membranes or ribosomes, may represent autophagic vacuoles. Although the possibility that formation of these lysosomes is an in vitro phenomenon cannot be excluded, it seems unlikely since non-stimulated cells cultured under the same conditions did not show this type of bodies. The ratio of cytoplasm to nuclei appeared slightly higher in secondary lymphocytes in comparison to non-stimulated cells. In some ultrastructural respects the secondary lymphocytes resembled the cells described by Matter et al. [ 191 as T,-differentiated lymphocytes, i.e., the immunologically differentiated lymphocytes in the lymph nodes, thymuses and spleens of mice. Secondary lymphocytes, upon re-stimulation, rapidly differentiate into morphologically characteristic blasts (2 h after re-stimulation of PWM secondary cells on syngeneic monolayer). These responsive cells resemble the “graft rejection cells” described by Wiener et al. [l] in areas of graft rejection. Secondary small lymphocytes were also obtained by Andersson & Hayry [7] as well as by Macdonald et al. [8], using the mixed Exp CdRes
100 (1976)
290
M. Kalina
lymphocyte reaction (MLR) technique. These authors found that their cells were capable of rapid secondary transformation specific to the sensitizing antigen, and also displayed high cytotoxic activity and immunological memory. They did not, however, present a morphological description of the cells. The morphology of secondary lymphocytes is still problematic. Moreover, although dissimilar in certain aspects, the non-stimulated and secondary lymphocytes are not easily distinguishable on morphological grounds alone. Cytochemical markers now being investigated by us may perhaps enable a better morphological definition of these cells. Another unresolved problem is the differentiation between killer and non-killer cells. Although our populations of secondary lymphocytes appeared homogeneous, we could not ascertain that all the cells in these populations were killer cells. Therefore the morphological identification of killer cells in a functionally heterogeneous population is a problem that remains to be solved. The technical assistance of Mrs S. Riklis is gratefully acknowledged. The work was supported by a grant from the Office of Chief Scientist Ministry of Health, Israel.
Exp Cell Res 100 (1976)
REFERENCES 1. Wiener, J, Spiro, D & Russell, P S, Am j path0144 (1964) 319. 2. Gowans, J H, McGregor, D D, Cowen, D M & Ford, C D, Nature 196(1962) 651. 3. Hollander, N & Ginsburg, H, J exp med 136(1972) 1344. 4. Hollander, N, Ginsburg, H & Feldman, M, J exp med 140(1974) 1057. 5. Berke, G & Levey, R H, J exp med 134 (1971) 1062. 6. Ginsburg, H, Hollander, N & Feldman, M, J exp med 134(1971) 1062. 7. Andersson, L C & Hayry, P, Stand j immunol 3 (1974) 461. 8. Macdonald, H R, Engers, H D, Cerrotini, J C & Brunner, K T, J exp med 140(1974) 703. 9. Berke, G, Ax, W, Ginsburg, H & Feldman, M, Immunologv 16 (1969) 643. 10. Ginsburg,% & Sachs, L, J cell camp physiol 66 (1965) 199. 11. Raidt, D J, Mishell, R I & Dutton, R W, J exp med 128(1968) 681. 12. Kalina, M & Hollander, N, Immunology 29 (1975) 909. 13. Greaves, M & Janossy, G, Transplant rev 11 (1972) 87. 14. Meuwissen, H J, Van Alten, P A & Good, R A. Transplantation 7 (1969) 1. 15. Hollander, N, Ph.D. Thesis, Weizmann Institute of Science (1975). 16. Biberfeld, P, Acta pathol microbial stand, suppl. 223 (1971) 1. 17. Douglas, S D, Transplant rev 11 (1972) 39. 18. Biberfeld, P, J ultrastruct res 37 (1971) 41. 19. Matter, A, Lisowska-Bernstein, B, Ryser, J E, Lamelin, J P & Vassalli, P, J exp med 136 (1972) 1008. Received February 12, 1976 Accepted February 16, 1976
CHARACTERIZATION
OF IN VITRO
SECONDARY
DIFFERENTIATED
LYMPHOCYTES
Quantitative and Ultrastructural
Study
M. KALINA Department
of Cell Biology and Histology, Tel-Aviv University,
Sackler School Israel
of Medicine,
SUMMARY Blast cells derived from rat lymphocytes by stimulation with concanavalin A (ConA) or pokeweed mitogen (PWM), or by sensitization on xenogeneic fibroblast monolayers, transformed into secondary small lymphocytes following their transfer to syngeneic monolayers devoid of mitogen or sensitizing antigen. This transformation resulted in the disappearance of morphological blast characteristics such as euchromatic nuclei, prominent nucleoli and the aggregation of ribosomes into polysomes. Secondary lymphocytes resembled non-stimulated cells, but differed from them in possessing a slightly larger cytoplasm containing large numbers of lysosomal bodies, interchromatin granules within the nuclei, nucleoli containing homogeneous fine granulo-fibrillar material and a relatively developed Golgi apparatus. Upon re-exposure to the stimulating mitogen or the sensitizing phenotype, the secondary lymphocytes rapidly transformed into blast cells with cytotoxic activity.
There is an increasing amount of evidence linking small to medium-sized lymphoid cells with allograft rejection [l, 21. There are also indications that small lymphocytes are effector cells capable of specific lysis of target cells in vitro [3,4]. Berke & Levey [5] have provided data suggesting that the lymphoid cells responsible for target cells destruction in vitro might also be the effector cells involved in graft rejection in vivo. To analyse the behavior and properties of these small lymphocytes, one needs a pure population of such cells, preferably in an in vitro system. It has been shown by Ginsburg et al. [6] and Hollander & Ginsburg [4] that blast cells obtained by stimulation of rat lymphocytes with phytomitogen, redifferentiate 19-761805
without the mitogen to small-sized cytotoxic lymphocytes, termed secondary lymphocytes. These authors concluded that the secondary lymphocytes obtained by stimulation with pokeweed mitogen (PWM) represented a pure population of memory cells specific to PWM. In similar fashion, the in vitro generation of memory cells reactive to transplantation antigens has been demonstrated by Hollander et al. [4]. Secondary small lymphocytes possessing immunological memory were obtained also by Andersson & Hayry [7] and Macdonald et al. [8], using mixed lymphocyte reaction (MLR) cultures. The present study was undertaken in an attempt to characterize some of the ultrastructural features of rat secondary lymphoE.rp Cell
Res
100 (1976)
282
M. Kalina
cytes, obtained after in vitro stimulation of Growth of blast cells on syngeneic rat lymphocytes with Concanavalin A fibroblast monolayer (ConA), or PWM, or by antigenic sensitiza- The blast cells from both mitogen and antigen stimution with xenogeneic fibroblast monolayers. lated cultures were harvested. washed. resusnended in
MATERIALS
AND METHODS
Animals Lymphocytes were obtained from Lewis strain rats. Embryos for preparation of fibroblast monolavers were obtained either from Lewis strain rats or from C,H/eb mice.
Mitogens A stock solution of PWM (Grand Island Biological Company) containing 1 mg dry weight/ml was kept frozen at -20°C. ConA (Miles-Yeda Ltd., Rehovoth) was kept as stock solution of 1 mg/ml at 4°C.
Fibroblast
monolayer-s
Monolayers from rat and mice embryos were prepared and maintained as previously described [9]. Monolayers used to maintain growth and differentiation of blast cells and secondary lymphocytes were prepared according to Hollander & Ginsburg [3] by plating 5x 10’ tibroblasts in 10 ml of LA medium (5 % lactalbumin hydrolysate in Earle’s solution with 5% calf serum) onto 100 mm Nunc Petri dishes.
Stimulation of rat lymphocytes PWM or ConA
Adherence of lymphocytes monolayers
to target
For assaying adherence the method of Kalina & Hollander [12] was employed. 5X lo6 secondary lymphocytes in 3 ml medium were plated onto the target tibroblast monolayers. When the stimulating agent was a mitogen, it was also added to the medium. The nonadherent lymphocytes were harvested intermittently by pipetting, the monolayer then washed with an additional 3 ml of medium and the washings together with the earlier harvests representing the non-adherent fraction. The remaining monolaver and adhering Ivmuhocytes were overlaid with trypsin solution and incubated for 30 min following which the cell susnension representing the adherent fraction was aspirated. Lymphocytes in both fractions were counted in hemocytometer and mean values were obtained from eight counts of each sample. Adherence and non-adherence were expressed as percent of the total number of lymphocytes.
with
Lymphocyte suspensions were prepared from Lewis rat lvmnh nodes accordine to Ginsburg & Sachs rIOi. The-ceils were centrifuged and resuspended in buibecco’s modified Eagle’s medium containine 15% horse serum (GDBCO)-and 5 pg of PWM or 15 pg of ConA per ml of medium. Lymphocytes 30X lo6 in 4 ml of medium were cultured in 60 mm Nunc plastic Petri dishes and incubated for 4 (PWM) or 3 (ConA) davs at 37°C in a humidified incubator with a flow of 7 %-CO, in air. In some experiments the blast cell populations were purified up to 98% by using bovine serum albumin gradient according to Raidt et al. [ 1I].
Stimulation of rat lymphocytes by xenogeneic (C3H mice) fibroblast monolayers Suspensions of rat lymphocytes were prepared as above-described and 30x lo6 lymphocytes in 4 ml Dulbecco modified Eagle’s medium containing 15% horse serum were plated onto each of the C,H sensitizing tibroblast monolayers. The cultures were then incubated for 4 days at 37°C in a humidified incubator with a flow of 7 % CO, in air. Exp Cell Res 100 (I 976)
fresh medium, and plated onto Lewis rat hbroblast monolayers in 100 mm Petri dishes, 5x 106 cells in 10 ml medium/dish. In order to obtain suspensions of secondary lymphocytes these cultures were then incubated for at least 4 days. In some experiments the secondary lymphocytes were kept in culture up to 21 days, the medium changed every 3 days.
Assays of cell transformation cell lysis
and target
Cell transformation was assessed by measuring the uptake of [3H]TdR as described by Ginsburg et al. [6]. The extent of target cell lysis was assayed by the release of 51Cr. 3x lo6 lymphocytes in 1.5 ml medium were plated on syngeneic or xenogeneic 51Cr-labelled target fibroblasts. The cultures were then incubated at 37°C for 20 h and the amount of radioactivity released into the medium measured according to Berke et al. [9].
Electron microscopy 8 x lo6 lymphocytes were centrifuged and the obtained thin pellet fixed for 2 h in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, post-fixed for 1 h in 1% osmium tetroxide, stained in block with 0.5% uranyl acetate, dehydrated, and finally embedded in Epon 812. In some experiments the monolayer with the adherent lymphocytes was processed in the same way. Ultrathin sections were cut on a Reichert Ultramicrotome, stained with many1 acetate and lead nitrate, and examined with a JEM 100B electron microscope.
Ultrastructure of secondary lymphocytes
Fig. 1. Abscissa: time (days, I, 2, 3, etc.); ordinure: cpmx 103/106cells (10, 20, 30, etc.). Kinetics of 13H]TdR uptake by Lewis rat lymph node lymphocytes stimulated with ConA (O---O), PWM (A---A) or sensitized against C,H fibroblast monolayers (S--m), and restimulation of ConA (O-O), PWM (A-A) or C,H sensitized (m-m) rat secondarv lymphocytes. Conditions for primary stimulation and secondary lymphocytes preparation as well as radioactivity determination is as described in Materials and Methods. Secondary lymphocytes were restimulated 6 days after removal of the stimulating mitogen or sensitizing antigen.
283
by the non-stimulated cells. These kinetics of rat secondary lymphocytes restimulation are in agreement with those obtained by Hollander & Ginsburg [3] and by Hollander et al. [4], who have also shown that ConA, PWM- and antigen-stimulated secondary lymphocytes are capable of massive adherence to fibroblast target cells as well as of cytotoxic activity. Adhesion and cytotoxicity of the secondary lymphocytes in the present study are summarized in table I.
Ultrastructural characterization of ConA-, PWM- and antigen-stimulated secondary lymphocytes The three different populations of rat secondary lymphocytes were observed under the electron microscope and compared to a non-stimulated population of rat lymphocytes and to blast cells stimulated by the two mitogens and by antigens of C,H fibroRESULTS blasts. The secondary lymphocytes obQuantitative characteristics of ConA, obtained from the three types of stimulaPWM- or antigen-stimulated secondary tion were morphologically similar and we lymphocytes shall therefore refer to them simply as secCultures of secondary lymphocytes ob- ondary lymphocytes. tained by the above three procedures were evaluated from the following standpoints: (a) thymidine uptake and blast transformation; (b) adhesion to target fibroblasts; (c) cytotoxicity. Fig. 1 shows the kinetics of thymidine uptake by the three populations of secondary lymphocytes upon re-stimulation with the mitogen or antigen initially used to stimulate these cells. The percentages of blast cells in the various cultures are shown in fig. 2. Noteworthy is the Fig. 2. Abscissa: time (days, I, 2, 3, etc.); ordinate: rapid transformation obtained with sec- % of blast cells (O-100%). of morphological transformation of Lewis ondary lymphocytes as compared with that ratKinetics lymph node lymphocytes stimulated with ConA observed with non-stimulated cells. Thus fO---0): PWM (A---A): or sensitized against C,H fibroblast monolayers (i---m); and restimulation “of the percentage of blast cells in the sec- ConA (0-O): PWM (A-A); or C,H sensitized ondary lymphocyte population reaches (B-m) rat secondary lymphocytes. Percentage of cells was determined by differential counting in maximum 1 day after the onset of restimula- blast hemocytometer. Experimental conditions as described tion as compared with the 34 days required in fig. 1. E.rpCell Res 100(1976)
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Table 1. Adherence and cytotoxic activity of secondary lymphocytes Class of lymphocyte Secondary-ConA Secondary-ConA Secondary-PWM Secondary-PWM Secondary C3HC Secondary C,H Non-stimulated Non-stimulated Non-stimulated
Mitogen added
Target monolayer
Percentage adherence”
Percentage “‘Cr release*
+ConA -ConA +PWM -PWM
Lewis Lewis Lewis Lewis W BALB/c Lewis Lewis Lewis
65.4 9.2 85.3 5.7 36.2 17.5 N.D. 7.5 2.2
32.0 6.6 57.3 8.2 45.9 16.0 6.0 5.5 1.0
+ConA +PWM Not added
Adherence and 51Crrelease were assayed on the same sample of cultured secondary lymphocytes divided into two portions as described in Materials and Methods. (1 Assayed after 6 h adhesion. * Assayed after 20 h; lysis is expressed as the percent of total radioactivity released into the medium, after correction for spontaneous 51Crrelease in control plates. c Lewis rat lymphocytes sensitized against C,H monolayers. N.D., not determined.
Figs 4-6 are low magnification electron Fig. 3 is a light micrograph of populations of non-stimulated, PWM-stimulated and micrographs of populations of non-stimuPWM-secondary lymphocytes. As can be lated (fig. 4), PWM-stimulated blasts (fig. 5) seen, the diameter of secondary lympho- and of PWM-secondary lymphocytes (fig. cytes is almost the same as that of non- 6). The two most pronounced ultrastrucstimulated cells. In previous calculations tural changes evident after differentiation based on cell volume, typical small lympho- from the blast form to secondary lymphocytes were found to be 6.5 pm, blast cells cyte pertain to the nucleus and to the ar10.5 pm and secondary lymphocytes 7.3 rangement of the ribosomes. The nucleus of the secondary lymphocyte is smaller, pm in average diameter [6].
3. A light micrograph of populations of (a) Lewis rat non-stimulated cells; (b) PWM-stimulated blasts; and (c) PWM-secondary lymphocytes. Note the resemblance in size of cells and nuclei in (a) and (c) in
Fig.
Exp Cell Res I00 (1976)
comparison with (b). A few large cells in (c) have not yet completely differentiated into small secondary lymphocytes. Toluidine blue stained Epon section. x1000.
Ultrastructure
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Figs 4-6. Low magnification electron micrographs of (fig. 4) non-stimulated; (fig. 5) PWM-stimulated (4 days) blast cells; and (fig. 6) PWM-secondary lymphocytes 6 days after removal of the mitogen. Note the prominent nucleoli in the blast cells (NJ~) which disappear in the secondary lymphocytes. The latter cell population appears morphologically homogeneous with small relatively heterochromatic nuclei similar to the non-stimulated lymphocytes (fig. 4). The cytoplasm, however, is more developed in secondary lymphocytes and even at that magnification a large number of vacuoles can be observed (arrows). Fig. 4, x4000; fig. 5, x3400; fig. 6, x3900. Exp Cell Rrs IO0 (1976)
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Fig. 7. A part of a &H-sensitized
rat secondary lymphocyte on the sixth day of differentiation from the blast form. Note the nucleolus (NL) containing granular and fibrillar material and areas of condensed chromatin within the nucleus. Interchromatin granules (IG) are evident in the nucleus. Vacuole containine various debris (0) can be seen in the cytoplasm. G: Golgi apparatus. x 14500.
Fig. 8. Parts of two cells from C,H-sensitized blast
cells culture on the 2nd day of differentiation to secondary lymphocytes. Note the typical arrangement of polysomes in the blast cells (I?) in comparison with the discrete ribosomes which are scattered throughout the cytoplasm of secondary lymphocytes (S). x 19000.
more heterochromatic and lacks the large blasts as well as of secondary lymphocytes and complex nucleoli typical for blasts (figs could be observed in the cultures. Sec5, 6, 7, IO). As for the typical arrangement ondary lymphocytes were in many respects of polysomes in blast cells, this gives way morphologically similar to non-stimulated in the secondary lymphocytes to discrete cells, i.e. relatively heterochromatic nuribosomes which are scattered throughout cleus, small nucleolus and discrete free the cytoplasm in similar fashion to the ribo- ribosomes (figs 4, 6, 9, 10). However, they somes in non-stimulated cells (fig. 8). differed from non-stimulated cells in having Observations made at different intervals a relatively large number of interchrofollowing the removal of the stimulating matin, electron dense granules, which are agent (from 4-2 1 days) showed no change in rare in the non-stimulated cells (figs 7, 10, 1l), a large number of large bodies rethe morphology of secondary lymphocytes and populations of these appeared ultra- sembling lysosomes (figs 6, 7, 11) and a structurally homogeneous. At 2-3 days, a seemingly better developed Golgi apparatus mixed population of large and medium (figs 7, 10). The nucleoli of secondary lymExp Cell Rrs 100 (1976)
Ultrastructure
Fig. 9. Electron micrograph of Lewis rat lymphocyte typical of cells in non-stimulated culture. Note the ring-like nucleolus (NL) and the condensed state of most of the chromatin. x 11000. Fig. IO. A &H-sensitized Lewis rat secondary lymphocyte (6th day). Note the small homogeneous nucleolus (NL) and the condensed state of part of the chromatin. Interchromatin granules can be seen in the nucleus (arrow). G, Golgi apparatus. X 11 500.
Fig. II.
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A PWM-secondary lymphocyte (6th day). Note the large vacuoles (0) in the cytoplasm, and the condensed state of the chromatin. x 13 000. Fig. 12. PWM-secondary lymphocytes restimulated in suspension for 12 h with PWM. Some blast features, such as euchromatic nuclei and polysomes, are evident. x3 500.
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Fig. 13. A PWM-secondarv lvmphocvte (L) attached to a fibroblast (F) from cell culture which was restimulated with PWM for 2 h on tibroblast monolaver. Note the blast-like euchromatic nucleus and prominent nucleolus evident at that short time of restimulation.
Fig. 14. A higher magnification of the contact region in
fig. 13; the ribosomes in the lymphocytes (L) are already aggregated to form polysomes. x24000.
x6000.
phocytes, although similar in size to that of non-stimulated cells, differ from the latter in some morphological features. They lack the ring-type appearance (fig. 9) and central location typical of non-stimulated cells [ 151; instead, these nucleoli appeared relatively homogeneous in density and contain fine granular and fibrillar material (fig. 10). In most secondary lymphocytes, the cytoplasm appeared to be more developed than in non-stimulated cells and the ratio of cytoplasm to nucleus was slightly higher. Restimulation
of secondary lymphocytes
primary response of the lymph-node cells (fig. 5). Samples of cells taken at 2.5, 5 and 10 h after the onset of stimulation showed an increase in the number of cells with blast characteristics, i.e., displaying cell enlargement, aggregation of ribosomes into polysomes and euchromatic nucleus (fig. 12). When ConA- and PWM-secondary lymphocytes were restimulated on syngeneic tibroblast monolayer, a rapid and massive adherence of the cells onto the monolayer was observed (table 1). In these experiments, most of the adherent cells showed blastlike appearance even 2-3 h after the onset of stimulation (figs 13, 14).
A restimulation of secondary lymphocytes with the appropriate stimulant resulted in a DISCUSSION rapid transformation into blast cells, reaching a peak after 24 h (figs 1, 2). Morpho- The three classes of secondary lymphologically these blast cells were indis- cytes studied by us were obtained from tinguishable from blast cells obtained in the primary rat lymphocytes by exposure of the E.rp Cdl
Rps
100 (IY76)
Ultrastructure
latter to three different modes of activation: (a) A non-specific stimulation of T cells by ConA which polyclonally represented activation of the majority of T lymphocytes [ 131; (b) stimulation with PWM, which represented a specific activation of a small subpopulation (l-3 %) of the cells [3]; in the rat, this subpopulation was found to consist of T cells only [14]; (c) a specific stimulation of a subpopulation of rat lymphocytes reactive against transplantation antigen [4]. The secondary cells obtained by the latter two modes of activation displayed the properties of memory cells. Thus, upon their re-exposure to Iibroblasts together with PWM, or re-exposure tofibroblasts of the sensitizing phenotype, the secondary lymphocytes adhered to the libroblast monolayer and rapidly transformed into blast cells with cytotoxic activity (see table 1, figs 1, 2). Hollander et al. [4] and Hollander [15] using density gradient as well as autoradiography, have shown that secondary lymphocytes, capable of both accelerated blast transformation and targetcell killing, are the progeny of the blast cells obtained by primary sensitization. Some of the experiments presented here were done on blast cells fractionated on a bovine serum albumin density gradient and the secondary lymphocytes reverted from them. This will exclude the possibility that some of the secondary lymphocytes belong to a pool of non-stimulated cells, refractory to the stimulation. The three populations of secondary lymphocytes were morphologically indistinguishable, each comprising a homogeneous population of small lymphocytes. These cells lacked the typical blast characteristics, i.e., polysomes, large euchromatic nuclei and prominent nuclei [16, 171. Morphologically they resembled in many ways the non-stimulated lymphocytes. However
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a few ultrastructural differences between non-stimulated and secondary lymphocytes were noted, mainly a high number of large lysosomal bodies, interchromatin granules and a relatively developed Golgi apparatus in the latter cells. The nucleolus appeared to be composed of a relatively homogeneous tine fibrillar and granular material, and was often located at the periphery of the nucleus instead of the strict ring type central nucleolus of non-stimulated cells [16]. No characterization of the lysosomal bodies can be given at present since it has been shown that the biogenesis of lysosomes in mitogen-stimulated blast cells is correlated to endocytic activity [18]. However, these bodies containing various cell components, such as remnants of membranes or ribosomes, may represent autophagic vacuoles. Although the possibility that formation of these lysosomes is an in vitro phenomenon cannot be excluded, it seems unlikely since non-stimulated cells cultured under the same conditions did not show this type of bodies. The ratio of cytoplasm to nuclei appeared slightly higher in secondary lymphocytes in comparison to non-stimulated cells. In some ultrastructural respects the secondary lymphocytes resembled the cells described by Matter et al. [ 191 as T,-differentiated lymphocytes, i.e., the immunologically differentiated lymphocytes in the lymph nodes, thymuses and spleens of mice. Secondary lymphocytes, upon re-stimulation, rapidly differentiate into morphologically characteristic blasts (2 h after re-stimulation of PWM secondary cells on syngeneic monolayer). These responsive cells resemble the “graft rejection cells” described by Wiener et al. [l] in areas of graft rejection. Secondary small lymphocytes were also obtained by Andersson & Hayry [7] as well as by Macdonald et al. [8], using the mixed Exp CdRes
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lymphocyte reaction (MLR) technique. These authors found that their cells were capable of rapid secondary transformation specific to the sensitizing antigen, and also displayed high cytotoxic activity and immunological memory. They did not, however, present a morphological description of the cells. The morphology of secondary lymphocytes is still problematic. Moreover, although dissimilar in certain aspects, the non-stimulated and secondary lymphocytes are not easily distinguishable on morphological grounds alone. Cytochemical markers now being investigated by us may perhaps enable a better morphological definition of these cells. Another unresolved problem is the differentiation between killer and non-killer cells. Although our populations of secondary lymphocytes appeared homogeneous, we could not ascertain that all the cells in these populations were killer cells. Therefore the morphological identification of killer cells in a functionally heterogeneous population is a problem that remains to be solved. The technical assistance of Mrs S. Riklis is gratefully acknowledged. The work was supported by a grant from the Office of Chief Scientist Ministry of Health, Israel.
Exp Cell Res 100 (1976)
REFERENCES 1. Wiener, J, Spiro, D & Russell, P S, Am j path0144 (1964) 319. 2. Gowans, J H, McGregor, D D, Cowen, D M & Ford, C D, Nature 196(1962) 651. 3. Hollander, N & Ginsburg, H, J exp med 136(1972) 1344. 4. Hollander, N, Ginsburg, H & Feldman, M, J exp med 140(1974) 1057. 5. Berke, G & Levey, R H, J exp med 134 (1971) 1062. 6. Ginsburg, H, Hollander, N & Feldman, M, J exp med 134(1971) 1062. 7. Andersson, L C & Hayry, P, Stand j immunol 3 (1974) 461. 8. Macdonald, H R, Engers, H D, Cerrotini, J C & Brunner, K T, J exp med 140(1974) 703. 9. Berke, G, Ax, W, Ginsburg, H & Feldman, M, Immunologv 16 (1969) 643. 10. Ginsburg,% & Sachs, L, J cell camp physiol 66 (1965) 199. 11. Raidt, D J, Mishell, R I & Dutton, R W, J exp med 128(1968) 681. 12. Kalina, M & Hollander, N, Immunology 29 (1975) 909. 13. Greaves, M & Janossy, G, Transplant rev 11 (1972) 87. 14. Meuwissen, H J, Van Alten, P A & Good, R A. Transplantation 7 (1969) 1. 15. Hollander, N, Ph.D. Thesis, Weizmann Institute of Science (1975). 16. Biberfeld, P, Acta pathol microbial stand, suppl. 223 (1971) 1. 17. Douglas, S D, Transplant rev 11 (1972) 39. 18. Biberfeld, P, J ultrastruct res 37 (1971) 41. 19. Matter, A, Lisowska-Bernstein, B, Ryser, J E, Lamelin, J P & Vassalli, P, J exp med 136 (1972) 1008. Received February 12, 1976 Accepted February 16, 1976