Scatid. J. Immtttiol. 9, 315-324, 1979
Regulatory Influences on the Response of Rabbit T Cells to Concanavalin A and Phytohaemagglutinin A. R. RIVAS ALCALA, C.T. CHOU, B. CINADER, S. DUBISKI & M. F. FILOSA Institute of Initnunology, University of Toronto. Toronto, Canada
Rivas Alcala. A.R,. Choii, C-T,, Cinader. B,, Duhiski. S. & Fikisa, M,F, Regulatory Influences on the Response of Rabbit T Cells to Concanavalin A and Phytohaemagglutinin, Scuml. J. Immunot. %'i\5-nA. 1979. The proliferative response, induced in rabbit spleen eell,^ by eoncanavalin A (Con A) and phytohaemagglutinin (PHAt. is abolished when T cells are killed with antibody againsi rabbit ihymus lymphocyte aniigen (RTLA) in the presence of complement. The response was examined witb puritied spleen T cells, to whieh various belper cell factions were added: ii could he shown that B cells help ihc responding Tcells, The helper effect in ihe response to PHA is abolished imd ihe response to Con A is reduced hy any manoeuvre which destroys or removes B cells. Help by B cells is given when helper cells have lost prolit'erative capacity as a consequence of mitomycin-C treaiment. Spleen cells adhering to the walls orciiluire luhes help suspended T spleen cells in their response lo Con A, This help could be abolished by complement mediated coll kill with aniihody to rabbit bursal equivalent lymphocyte antigen (RABF'LA), On the other hand, the helper effect in Con A response was increased when T cells were removed. Thus the response of T cells to Con A is regulated by helper B cells and suppressor T cells, Bfmliiiril Cinuilcr. hi.tiinili' of Immunology, Medical .Sih-ncc.i Building. University of Toronto, Toronto. Ontario. Camuki, M5S lAH.
The systematic exploration of the rabbit's immune system has been based on antibodies directed against certain cell tnarkers, particularly against rabbit thymus lymphocyte antigen (RTLA), which characterizes rabbit Tcells [15], and rabbit bursal equivalent lytiiphocyte antigen (RABELA), which characterizes rabbit B cells [7]. Using cotnplement-mcdiated cytotoxic kill, it was shown that the response to such mitogens as eoncanavalin A (Con A) and phytohaemagglutinin (PHA) was being tiiade predominantly by T cells, since a proliferative response to these mitogens depended on the viability of T eells [5. 27, 30], Thus the responses of lymphocytes of the rabbit are sitnilar to those of the mouse [M\. It soon became apparent that the response of T eells to mitogens was modulated by other cell types. When spleen cells were fractionated by various techniques and thymidine uptake was determined in mito-
gen-stimulated fractions, the thymidine uptake yield was always lower than thj c?li yield [30]. It seemed reasonable to conclude that fraciionation, whether based on adherence or cell volume, led to a separation of celis that responded to Con A and other cells which regulated the response. Further evidence for the existence of such regulator cells was obtained as a byproduct of the development of an antiserum specific for the B cell antigen, RABELA: the antiserum had a cytotoxic elTeci on spleen cells, which resulted in a reduced proliferative response after stimulation with Con A. This effect could be reversed by the addition of B eells and it seemed that a regulatory B cell was involved in the response of T cells to Con A [7]. In this paper, purification of spleen T cells (TD cells) will be described. The response of spleen cells to Con A and PHA will be examined as adherent and B cells are removed from
0300-9475/79/0400-0315 $02.00 © 1979 Blackwell Scientific Publications
315
316
A. R. Rivas Akaia et al
spleen cell suspension. Cellular interactions affecting mitogen responsiveness will be investigated and evidence for the existence of helper and suppressor cells will be presented. Ablation and reconstitLttion have been the central technique for the functional definition of lymphoid subpopulations of inbred animals. In outbred animals, this strategy is only admissible if all cells come from the same donor. We have therefore relied on in vitro reconstitution with different fractions of lymphoid cells from one and the same individual rabbit.
MATERIALS AND METHODS Rabbiis. New Zealand white rabbiis (Riemcns Fur Ranches Ltd, St Agathe. Ontario, Canada} of either sex (allotype Ah'jAb') .1-6 months old. were used as donors of cells. Rahhits of allotype Ah''IAh^ were used as donors of sera anil of anlisera and were bred on tlie premises. l^ormal rabbit serum (mature serum, type 2} was obtained from Pel-Freez Biologicals. Inc. (Rogers. Ark., USA). This serum contained predominantly Ab4 allotypic specificity. When normal rabbit serum of Ah'IAh* allotype was needed, il was obtained from rabbiis of this allotype; Atr'jAb^ rabbits were also used as a source of complement. In this case the serum was kept frozen at — 70"C. Coiicaiiavalin A (Lot 100. three times cryslaliized and bophilized) was obtained from Miles-Ycda Lid. Kankakee, 111.. USA. The powder was dissolved in phosphate buffer. pH 7.2 (0.15 M), and aliquots of the solution were kept at - 70 C. Phyiohaemagghiliriin P (Lot 3110-56) was obtained from Difco Laboratories. Detroit, Mich.. USA. The powder was dissolved in phosphate buffer, pH 7.2 (0.15 M) and aliquots of the solution were kept at - 70 'C. Modifu'd McCoy'.\ 5a mciliiim. containing 30",, fetal calf serum (MCM). was purchased from Grand Island Biological Co., Grand Island, N.Y.. USA. Modified CMRL 1066 tissue culture medium [19] was obtained from Ontario Cancer Institute. Toronto, and was thymidine free and supplemented with 0.001",, Lasparagine and with 10 niM Hepes butfer (Grand Island Biological Co.). Minimal essential medium (MEM) (Grand Island Biological Co.) was supplemented with 10"o heat-inactivated fetal calf serum. Isoioiiic imida:ok' buffer (SIM), pH 7.0 [25]. was prepared by dissolving in glass-distilled water. 8.2 g of NaCl. 0.4 g of MgCla 6H2O. 0.373 g of KCI, 8-2 ml of 0.1 N HCI and 0.115 g of imidazole: the volume was adjusted to I litre. Aniibodiex directed against Ab4 allolypic speei/icity. Antisera were raised by isoimmunization of rabbiis of Ab^lAh" allotype [^1. RTI.A oiili.senirn. Heterologous (goat) antiscriim directed against RTLA was prepared and rendered thymus specific by absorption, as previously described [15]. The exclusive specificity of this antiseruni for T
cells has been documented in earlier publications. In addition, we have shown by fluorescent staining that RTLA bearing cells never carry Ig receptors [Szymanska, Dubiski & Cinader. unpublished]. RABELA aiiliseruiii. Heterologous goat antiserum directed against rabbit bursa! equivalent cell antigen was prepared as described by Chou t'l a!. [1\. Briefiy female goats were immunized with appendix cells; sera were heated for 30 min at 56 C and absorbed with rabbit liver cells and erythrocyics, and then with thymocytes and bone marrow cells. The exclusive specificity of this antiserum for B cells has been doeumented in an earlier publication [7]. Cell counts. Cells were counted in a Coulter counter (model F), according to the manufacturer's directions. Rahbii immiinoglobuHns Ug) were prepared from normal sera and from antisera by precipitation with t8"'n sodium sulphate and subsequent chromatography on DEAE-eelluiose [331. The column was equilibrated with 0.0175 M phosphate buffer. pH 7.0. and the nonabsorbed fractions as well as the material eluted with 0.05 M phosphate buffer were collected. The nonabsorbed fraction did not contain contaminant proteins detectable by immunoelectrophoretic analysis; the fraction eluted by 0.05 M phosphate buffer was further purified by ehromatography on Scpliadcx G-200 [I]. The preparations obtained from anti-Ab4 antisera were tested tor antibody activity by gel diffusion. Cyioioxieiiy lesis were based on the methods of Boyse ci al. [6]. Gorer & O'Gorman [17] and Schlesinger [26]. Serial dilutions (0.025 ml) of antisera (equal volume of MCM and antiserum or diluted antiserum) were pipetted into the wells of microtitre plates (Cooke Engineering Co., Alexandria. Va.. USA). Aliquots (0.05 ml) of a cell suspension (10" cells/ml) were added to each well. The mixtures were allowed to stand for 30 min at room temperature before addition of 0.025 ml of complement (rabbil serum diluted with an equal volume of MCM). The plates were kept at 37 C for 60 min in an atmosphere of 5",, CO^ and 95",, air. Thereafter, two-thirds of the supernatant was pipetted off and 0.05 ml of a freshly prepared trypan blue solution was added to each well. Two mixtures, serving as a complement control, contained complement and MCM instead of antiserum. Two mixtures, employed to determine the action of goat scrum, contained a prebleeding which had been absorbed in the same way as the corresponding antiserum. The percentage of stained cells was determined by inspecting 100 200 cells from each well and is recorded as complement mediated cytotoxicity or as cell kill. Tests were disregarded if any control mixture contained more than 20" ^ stained tells. Eniinwralkin of iiuieriiplitigcs. Macrophages were identified by nonspecific esterase staining [32]. Reagenls were obtained from Technieon Instruments Corporation, Tarrytown. N.Y., USA. and some were a gift from this firm. Two different series of mixtures were made in plastie tubes (Falcon Plasties, 2058 tubes. Oxnard. Calif. USA). The first series of mixtures consisted of 6 drops of mono nitrate reagent (TOI-053038). 6 drops of mono dye reagent (TOI-0529-38). 18 drops of nionocyte huder (TOI-0678-38), and 3 drops of mono substrate (TOI-0680-38). The second series of mixtures consisted of 2 drops of a eel! suspension containing 1 > 10" to 2 y 10" cells/ml, 2 drops of heat
Regulation of Response to T-Cett Mitogens maclivatecl fetal calf scrum, and 4 drops of monocyte fix reagenl (TOI-067'^-38). The conlents were mixed and kept at room tcmperattire for 40 s before the first series of mixtures were added; the suspensions were mixed vigorously and were immediately incubated at 37 C for 8 min. The suspension was introduced into an improved Neubauer counting chamber and stained cells were counted under a microscope. DciiTuiimilinn of tbymUline inairporcilian. Detailed procedures have been described elsewhere |27-29]. A suspension of 1 • 10'* nucleated spleen cells was mixed with various doses of Con A. PHA or antiserum in tissue culture tubes (Falcon PlasticsJ. All mitogsns were used in concentrations that resulted in maximum response. The amount of Con A was 300 lig/eulture and that of PHA was 15 iig/culture; ihis optimum was the same for all cell preparations used (not shown). The final volume ofeach culture was 2 ml of CM RL 1066 medium supplemented with heat-inactivated normal rabbit serum. T!ie final serum content was 20" „ for stimulation with Con A and with antibody against allotypic speciticily and 5",, tor stimulation wiih PHA. When cells were cultured wiih antibody against Ab4 allotypic specificity, the allotype of normal serum was Ah-'IAh'': otherwise it was normal rabbit serum !ype 2 (Pel-Free? Biologicals). After 24 h of incubation at 37 C in an atmosphere oC5'!,, CO., and "-IS"„ air. O.I ml of tritiated thymidine(*H-TdR; 2.5 aCi, specific activity 20 Ci/mM, Amersham-Searle Corp.) was added to each culture tube; the incubation was continued for a further 24 h. Thereafter, the harvested cells were washed twice on glass fibre filters (Reeve Angel. N.J.) with two successive cycles each consisting of a wash wiih ice-cold 0.15 M NaCl. 5",, trichloroacetie acid, and absolute methanol. Thymidine incorporation was determined by liquid scintillation counting and expressed as net counts (counts in the presence of mitogen less counts in the absence of the mitogen). Miloinycin-C Ireatmeni of Spleen cells was performed according to Ref, 20. 1 ml of mitomycin-C solution (80 mg/ml; Sigma Chemical Co., St Louis. Mo., USA: Lot 14C-0500) in supplemented MHM was added to I ml of spleen cell suspension (10" eells/ml) in supplemented MFM. Cells were incubated at 37 C for 40 min, washed three times with supplemented MFM. and resuspended in CMRL 1066 for culture. In some instances, cells were killed with antiserum and complement before being washed and treated with mitomycin-C as described above. Mitomycin-C treatment was employed to inhibit proliferative responses of helper cells, i.e. so as to exclude etVects which might be due to a T cell assisted proliferative response of B cells to Con A and PHA. Non-adhercnl cells {NA) were prepared by removal of cells that adhered to or ingested iron core particles, coated with poly L-lysine {Technicon Corp.) as described previously [27]. Rciiiiival iij iinmiinii/rldbiiliii (If;) hcariit); cells hy aniihody coaled hciids. The technique was a modification of that described in Ref. 34. Degalan beads (V26) were obtained from Accurate Chemical and Scientific Corp., Hicksville, N.Y. The beads were washed with giassdistilled water, ihen with sterile phosphate-bufi"ered saline (PBS), pH 7.2, 0.15 M; in each case 10 times the
317
volume of the beads was used for washing. The suspension was then stored in PBS containing 10 ^ M sodium azide. Beads, packed by settling (30 ml), were washed with PBS (100 ml), so as to remove sodium azide, and were introduced into sterile graduated conical bottom tubes (Falcon Plastics, 2070 tubes). Afler the beads had settled at room temperature, the top layer of PBS was removed, and 90 mg of antibody Ig or normal Ig was added in a concentration of 18-24 mg/ml. The beads were gently mixed with the Ig by moving the suspension up and down with a Pasteur pipette. The mixture was next incubated for 40 min in a water bath at 45 | 0.2°C with genile stirring. The preparation was stored at 4'C overnight. On the following day, the beads were washed wiih 200 ml of PBS and transferred to a sterile 25 ml graduated glass pipette (36 ,-.. 0.6 cm), which was closed at the bottom with nylon material and plastic tubing. Thereafter, PBS was replaced by washing with 100 ml of medium CMRL 1066. These operations and the following fractionation were carried out at room tempt ratu re. Non-adherent spleen cells (NA: 2 < 10" to 8 X 10") were suspended in 7.5 ml of medium CMRL 1066 equal to the void volume of the Degalan bead column. The cells were loaded gently on top of the beads, and once they had entered the entire volume of the column, 0.2 ml of medium CMRL 1066 were layered on top of the column and the column was closed for 10 min by clamping the plastic outflow tubing. The cells were then eluted with medium CMRL 1066, and 20 ml were collected. These cells were designated Ty cells and were tested for RABELA- and RTLA-bearing cells (cyiotoxicity assay), for macrophages (non-specific esterase staining), and for mitogen induced thymidine uptake. Fractionation of cell.s hy xeclimenicitiun in ibe earth's gravitational field. Cells were fractionated by velocity sedimentation in a funnel-shaped glass chamber (O. H. Johns, Toronto), 20.7 cm in diameter. The height of the cylindrical portion of the funnel was 13.5 cm, the lotal volume employed was 1860 ml [21, 22]. Nucleated cells (450 • 10*') were loaded as a thin layer on top of a continuous bovine serum albumin (BSA| gradient (0.3 2.0"n). After a sedimentation time of 4 h, cells were collected in thirty fractions of 50 ml each, were counted in a Coulter counter, and were washed in modified CMRL 1066 before being used for functional assays. Tcsl far helper effect wiili nirnebecl lynipltnid cells. Spleen cell suspensions were treated with complement (normal rabbit serum) and either RABLLA or RTLA antiserum, or with a corresponding normal goat serum. The resulting spleen cells were suspended in SIM buffer [25] in a concentration of 2 •' 10'* cells/ml. 2 ml of this suspension were placed in an upright 15 ml culture tube (3033 tissue culture lube. 16 - 125 mm style. Falcon Plastics), and were kept at 37 0.2 C in 5"^ COj and 95",, air for 150 180 min. Thereafter, the non-adherent cells were removed by decantation, followed by washing wiih Dulbecco's [10] buffer at 37 C. Five of the tubes with attached cells were employed to count the number of adherent cells (usually 5 - tO-^); to the remaining tubes 0.4 ml heat inactivated (56 C, 30 min) serum were added: this was followed by Con A and finally either by 1.6 ml of supplemented medium 1066 or by a cell suspension (T^j in ihe same volume.
318
A. R. Riva.s Alcala et al.
O c uQ o CL ^^ W X •o E O a. o
tion net
"(5 o
•
—
5 10 Unfractionated Spleen Cells (net cpm x 10") (b) CO
cn D
—
(D
o
O
E a o
rom net
(J CT
o
1
2 3 NA Cells (net cpm x
4
F I G . I. The etTecis of removal of various cell populations on ihc response of spleen cells to concanavalin A (Con A), (a) Comparison of the response to stimulation with Con A of non adherent spleen cells (NA) and unfractionated spleen cells (S) cells (O) and of highly purified spleen T cells (Tp) and S cells ( • ) . Each point represents the response of an individual animal. The line broken line is the regression line with a slope I; the heavy solid one is the regression line for the plot N A r. S (slope O..'^? -- 0.05) and the broken one the regression line for the plot T D r. S (slope 0.0525 0.0103). (b) Comparison of the responses to stimulation with Con A of NA cells and N A cells passed over Degalan beads coated with normal rabbit immunoglobulin (O) and of NA cells and T,, cells ( • ) . Each point represents the response of an individual animal. The slopes for the respective regression lines are; 0.6 i 0.05 and 0.19 ± 0.04. The broken line is the regression line with the slope I.
RESULTS We have shown previously that thymidine uptake after stimulation with Con A is predominantly due to T cell proliferation [27]. Complement mediated kill of B cells with RABELA antiserum results in reduction of thymidine uptake by 42±23/^. This could be due to loss of helper cells [7]; we therefore proceeded to a vigorous examination of this possi-
bility by purifying T cells and by isolating potential helper cells. It was our first objective to compare the Con A response of unfractionated spleen cells with that of spleen cells from which various subpopulations were removed. The following methods were used to this end: (I) removal of cells that adhered to or ingested iron core particles: the resulting cell preparation was designated NA cells; (2) removal of Ig bearing
Regukilion oj Response lo T-Cell Mitogens
(a)
(b) 40 h
30
X
3
o
20
E
10
319
30
1 I
20
10
FIG. 2. Miiogenic response to Con A; its abrogation by the elimination and reconstitution by the addition of certain cell populations. The target cells were either levied separately or were combined with with the putative adherent helper cells (AH). In each case the number of target cells was 0.5 /^ 10"/ culture. AH cell suspensions were first ineubated with cither antiserum to rabbii biirsal equivalent lymphocyte antigen (RABELA) or with antiserum to rabbit thymus lymphocyte antigen (RTLA) or a corresponding normal preabsorbed goat serum (NGS). These treated cells (4 • 10'') were then allowed to attach to the walls of plastic tubes in which the subsequent culture took place and were, finally, washed free of non-adherent cells. The preparation of attached cells was approximately 10",, of thec^lh; in suspension. Thymidine uptake is shown as vertical bars; vertical lines and dotted areas indicate I standard deviation. Hatching indicates individual cell populations, open bars represent the mixtures of two cell populations. Broken horizontal lines represent arithmetic addilion of component cell populations in a given cell mixture. Olher cell designations: S unfractionated spleen cells; NA non-adherent spleen cells; T,, highly purified spleen T cells.
cells by passing NA cells over aniibody-coaied beads, the resulting ceil preparafion was designated To cells; and (3) removal of Fc bearing cells by passing spleen cells over Ig coated beads. Cell preparations NA and T D were assayed for their content of B and T eells by complement mediated cell kill of RABELA positive B and RTLA positive T eells. In this respect there was no substantial difference between unfractionated spleen cells and NA cells, but To cells contained 85 ± 1 0 % RTLA positive cells and 5 ± 4% RABELA positive cells. The relative B cell responsiveness to T^ cells was also estimated funclionally, in terms of ihymidine incorporation, after stimulation with antibody
against Ab4 and found to be 9 + 8 % of the uptake by unfractionafed stimulated spleen cells. By staining for non-spi^cific esterase, spleen cells, NA cells and T^ cells were found to contain IO±2%, O,7±O,8% and 0.3 + 0,4% macrophiiges. respectively. There was a substantial decrease in thymidine uptake afler Con A stimulation of NA as compared to unfractionated spleen cells. Thus the removal of adherent cells had resulted in little change in T cell content and yet in a reduction in Con A induced thymidine uptake to 1 ±7"/o of that given by the unfractionated spleen cell population, A plot of thymidine incorporation by Con A stimulated unfractionated spleen cells against stimulated NA cells
320
A. R. Rivas Alcala er al.
gives a regression line with a slope that is significantly dilTerent from 1 (Pible that cooperative interaction with adherent B cells was involved, as we have found in the case of rabbit lymphocytes. It would appear to us that apparent species differences may not be attributable to divergences in the helper mechanism of different species, but rather to the test system and to the method of cell identification used by different investigators. In the mouse system, la-positive cells were found to give help in the response to Con A [4, 16, 18]. Tbis help could also be given by soluble la molecules which can be supplied in normal serum [24]. Whether help in the rabbit system is also ladependent remains to be seen. The response of T cells to Con A involves not only the two already mentioned, but also a suppressor T cell. Evidence for an adherent T suppressor cell operating in the response to C on A was obtained from the increased helper elfect of adherent cells tbat were freed of T cells with complement and RTLA aniiserum (Fig. 2). The population containing helper cells also contains T cells that can respond to Con A (AHNGS in Fig. 2); this activity is lost after exposure to RTLA (AHRILA in Fig. 2). However, the helper effect is increased after elimination of
323
viable T cells, as judged by comparing the sum of the individual responses of the components of the mixture with the directly measured response of the mixture. The dilTerence can be judged from the relative distance between the broken line and tbe top of the bar in AHNGS + T|) and AHRTLA + TD of Fig. 2. The broken line
represents in each case the arithmetic addition of the individual responses of the components of the mixture. Suppressor cells of this general type bave been reported in the rat lymphoid system, where a ihymus dependent ceil suppresses proliferative responses of non-adherent spken cells to antigen and to T cell mitogens, paiticularly PHA [3, 11-14]. These cells are not found in purified spleen or peritoneal maerophdges, nor in rats deprived of thymus cells in vivo. It was therefore concluded that suppression was due to T cell rather than B eel! [13]. Subsequent studies led to the view that macrophages were involved in the suppressive effect, since cytotoxic agents, such as carrageenan and cyclophosphamide. reduced the suppressive synergy between tbe rats" suppressor T cell and maerophages [2]. Whether the suppressor cell described in the rai lynipboid response belongs to a cell lineage similar to that involved in our system of the rabbit lymphoid response to Con A remains to be seen.
ACKNOWLEDGMENTS This work was supported by grants from the Medical Research Council of Canada and by the National Cancer Institute of Canada. We are grateful to Mr Ladislav Horvatb for effective technical help and lo Technicon Instruments Corp., Tarrytown, N.Y., for the gift of reagents.
REFERENCES I Aiidrcv^s. P. The gel tillralion behaviour of proteins relaied to their jiiolcculur woighl over a wide range. Bi.H/win. J. 96, 595. 1465, : Bash. J.A.. Singer. A.M. & Waksman, B.H. The suppressive efftci of immunizaiLun on ihe proliferalive responses of rat T cells in vHru. II. Abrogation oT aniigen-induced suppression by selective cyloloxie agents. J. ImniiinoL 116, 1.150. 1976, 3 Bash, J.A, & Waksman. B.H, The suppressive efTecl orimmuni7atioii on the proliferative responses of rat T ceils in vitro. J. liiiiminul. 114, 78 1. 1975.
324
A. R. Rivas Alcala ct al
4 Bick. P.H,. Persson. U,. Smilh. E,. Moller, E. & Hamarstrom, L. Genetic control of lymphocyte activation: lack of response to low doses of concanavalin A in lipopolysaccharide-nonrespondcr mice. J. e.xp. Med. 146, 1146. 1977, 5 Bona, C . ChediJ. L., Damais. C . Ciorharu, R,, Shek, P.N,. Duhiski, S, & Citiader. B, Blast transformalion of rahhil B-dcrived lymphocytes hy a mitogen extracted from Nocardia, J. Immunol. 114, 348, 1975. 6 Boyse. E.A.. Old., L.J. & Chouroulinkov, I, Cylotoxic lesl for demonstration of mouse antibody. Methods med. Res. 10. 39. 1964. 7 Chou, C,-T.. Cinader. B. & Dubiski, S. A membrane antigen of rahhit btirsal eqtiivalent cells. Cell, tmmunol. 28, :i34. 1977. 8 Delespesse, G,, Duchateaii, J., Gausset, Ph. & Govaerts, A. In vitro response of subpopiilalions of human lonsil lymphocytes. I, Cellular collaboration in the proliferative response to PHA and Con A, J. Immnnol. 116, 437. 1976, 9 Dubiski, S, Immunochemistry and genetics of a "new" allolypic specificity A e " of yG immunoglobulins: recombination in somatic cells, / . Immunol. 103, 120. 1969. 10 Dulbecco, R, & Vogt, M, Plaque formation anii isolation of pure lines wiih poliomyelitis viruses, J. exp. Med. 99, 167, 1954, 11 Folch, H. & Waksman. B,H. Regulation of lymphocyte responses in viiro. V. Suppressor acliviiy of adherent and nonadherent rat lymphoid cetls. Cell. Immunol. 9, 12. 1973, 12 Fokh. H, & Waksman, B,H, //( \itro responses of rat lymphocytes following adull thymectomy. II. Increased inhibition by splenic adherent cells of responses lo phytohemagglutinin. Cell. Immunol. 9, 25, 197.1. 13 Folch, H, &. Waksman. B,H. The splenic suppressor cell. I. Activity oTthymus-dependent adherent cells; Changes with age and stress, / . lmtnuni>l. 1(3, 127, 1974. 14 Folch, H., Yoshinaga. M. & Waksman. B,H- Regulation of lymphocyte responses in vitro. III. Inhibition by adherent ceils of the T-lymphocyte response to phytohemagglutinin, J. Immunol. 110, 835. 197.1, 15 Fradelizi. D,P,. Chon, C,-T.. Cinader. B, & Dubiski. S, RTLA. a membrane antigen of rabbit thymus cells. Cell. Immunol. 7, 484. 1973, 16 Frelinger. J,A. la-bearing cells promote the concanavalin A mitogenic response of lu-ncgative T cells. Eur. J. Imnumol. 7, 447. 1977, 17 Gorer, P,A, & O'Gorman. P. Cylotoxic activity of isoaniibodies in mice, Transplont. Bull. 3. 142, 1956. 18 Habu, S, & Raff, M-C, Accessory cell dependence of lectin-induced proliferation of mouse T lymphocytes. Eur.J. Immunol. 7,451. 1977, 19 Healy. G,M,, Fisher. D,C, & Parker, R,C, Nutrition of animal cells in tissue culture, X. Synthetic medium N o . 8 5 8 , Proc.
Soc. e.xp. Biol. Med.
8 9 , 7 1 , 1955,
2U Hodcd, R.J, & Terry. W.D. Comparison of irradiated and milomycin-treated mouse spleen cells as stimulating cells in mixed lymphocyte cultures and in vitro sensilization. J. Immunol. 113, 39. 1974. 21 Miller. R,G, p, 87 in Pain, R,H, & Smilh, B,J, (ed,) A'cir Tcclinic/Ufs in Biophysics ond Cell Biology. John Wiley & Sons, London, 1973, 22 Miller, R,G. & Phillips, R.A. Separation of cells by velocity sedimentation, J. celt. Physiol. 73, 191. 1969. 23 Mills. G,, Moniicone. V. & Paetkau. V, The role ol" macrophages in thymocyle milogenesis. J. Immunol. 117, 1325. 1976, 24 Persson, U,, Hammarstrom. E., Moller, E., Moller. G. & Smilh. C.I.E, The role of adherent cells in B and T lymphocyte activation, Immunol. Rev. 40, 78. 1978, 25 Rabinovitch, M. & deStefano, M,J, Macrophages spreading in viiro. I. Inducers of spreading. Exp. Cell fic.s, 77, 323, 1973. 26 Schlesinger, M. Immune lysis of thymus and spleen cells of embryonic and neonatal mice, J. Immunol. 94,358, 1965, 27 Shek, P,N,. Chou. C.-T.. Dubiski. S. & Cinader. B. Mitogen stimulation of rabbit spleen cells before and after complement mediated cell kill with an aniiserum directed against the thymus antigen RTLA. Int. Arch. Allergy, 46, 753. 1974, 28 Shek, P,N.. Chou, C,-T,, Dubiski. S. & Cinader, B, Volume, adherence properties, membrane antigens and mitogen responsiveness of rabbit lymphoid cell subpopulations, Immunol. Comntiin. 4^S]. 1975. 29 Shek, P.N,, Chou, C-T., Dubiski, S, & Cinader, B, A suppressor cell which interferes with anti-allolype stimulated DNA synthesis of rabbit B cells. Cell. Imnumol. 18, 331, 1975. 30 Shek, P.N., Chou, C,-T,. Dubiski, S, & Cinader, B. Rabbil lymphoid cells. I, T-eell miiogens, cell volume and adherence properties as probes for cellular heterogeneity. Immunology. 30, 549, 1976. 31 Stobo. J,D, PhytohcniLigglulinin and concanavalin A: probes for murine " T " cell activaiion and differentiation. TrcinspL Rev. II, 60. 1972, 32 Tucker. S,B,. Pierre. R,V, & Jordon. R.E, Rapid identification of monocytes in a mixed mononuclear cell preparaiion, Immunol. Mi-lh. 14, 267. 1977, 33 Weir, D,M, (eii,) pp, 6.5 and 7.10 in Hondbouk of Experimenlal Immunology, 2nd edn, Blackwell Seienlific Publications, Oxford, 1973. 34 Wigzell, H,. Suiidquist. K.G. & Yoshida. T.O. Separation of cells according to surface antigens by the use of antibody-coaled columns. Fractionation of cells carrying immunoglobulins and blood grotip antigen. .Scond. J. Immunol. 1, 75, 1972,
Received 9 August 1978 Received in revised form 10 November 1978
Regulatory Influences on the Response of Rabbit T Cells to Concanavalin A and Phytohaemagglutinin A. R. RIVAS ALCALA, C.T. CHOU, B. CINADER, S. DUBISKI & M. F. FILOSA Institute of Initnunology, University of Toronto. Toronto, Canada
Rivas Alcala. A.R,. Choii, C-T,, Cinader. B,, Duhiski. S. & Fikisa, M,F, Regulatory Influences on the Response of Rabbit T Cells to Concanavalin A and Phytohaemagglutinin, Scuml. J. Immunot. %'i\5-nA. 1979. The proliferative response, induced in rabbit spleen eell,^ by eoncanavalin A (Con A) and phytohaemagglutinin (PHAt. is abolished when T cells are killed with antibody againsi rabbit ihymus lymphocyte aniigen (RTLA) in the presence of complement. The response was examined witb puritied spleen T cells, to whieh various belper cell factions were added: ii could he shown that B cells help ihc responding Tcells, The helper effect in ihe response to PHA is abolished imd ihe response to Con A is reduced hy any manoeuvre which destroys or removes B cells. Help by B cells is given when helper cells have lost prolit'erative capacity as a consequence of mitomycin-C treaiment. Spleen cells adhering to the walls orciiluire luhes help suspended T spleen cells in their response lo Con A, This help could be abolished by complement mediated coll kill with aniihody to rabbit bursal equivalent lymphocyte antigen (RABF'LA), On the other hand, the helper effect in Con A response was increased when T cells were removed. Thus the response of T cells to Con A is regulated by helper B cells and suppressor T cells, Bfmliiiril Cinuilcr. hi.tiinili' of Immunology, Medical .Sih-ncc.i Building. University of Toronto, Toronto. Ontario. Camuki, M5S lAH.
The systematic exploration of the rabbit's immune system has been based on antibodies directed against certain cell tnarkers, particularly against rabbit thymus lymphocyte antigen (RTLA), which characterizes rabbit Tcells [15], and rabbit bursal equivalent lytiiphocyte antigen (RABELA), which characterizes rabbit B cells [7]. Using cotnplement-mcdiated cytotoxic kill, it was shown that the response to such mitogens as eoncanavalin A (Con A) and phytohaemagglutinin (PHA) was being tiiade predominantly by T cells, since a proliferative response to these mitogens depended on the viability of T eells [5. 27, 30], Thus the responses of lymphocytes of the rabbit are sitnilar to those of the mouse [M\. It soon became apparent that the response of T eells to mitogens was modulated by other cell types. When spleen cells were fractionated by various techniques and thymidine uptake was determined in mito-
gen-stimulated fractions, the thymidine uptake yield was always lower than thj c?li yield [30]. It seemed reasonable to conclude that fraciionation, whether based on adherence or cell volume, led to a separation of celis that responded to Con A and other cells which regulated the response. Further evidence for the existence of such regulator cells was obtained as a byproduct of the development of an antiserum specific for the B cell antigen, RABELA: the antiserum had a cytotoxic elTeci on spleen cells, which resulted in a reduced proliferative response after stimulation with Con A. This effect could be reversed by the addition of B eells and it seemed that a regulatory B cell was involved in the response of T cells to Con A [7]. In this paper, purification of spleen T cells (TD cells) will be described. The response of spleen cells to Con A and PHA will be examined as adherent and B cells are removed from
0300-9475/79/0400-0315 $02.00 © 1979 Blackwell Scientific Publications
315
316
A. R. Rivas Akaia et al
spleen cell suspension. Cellular interactions affecting mitogen responsiveness will be investigated and evidence for the existence of helper and suppressor cells will be presented. Ablation and reconstitLttion have been the central technique for the functional definition of lymphoid subpopulations of inbred animals. In outbred animals, this strategy is only admissible if all cells come from the same donor. We have therefore relied on in vitro reconstitution with different fractions of lymphoid cells from one and the same individual rabbit.
MATERIALS AND METHODS Rabbiis. New Zealand white rabbiis (Riemcns Fur Ranches Ltd, St Agathe. Ontario, Canada} of either sex (allotype Ah'jAb') .1-6 months old. were used as donors of cells. Rahhits of allotype Ah''IAh^ were used as donors of sera anil of anlisera and were bred on tlie premises. l^ormal rabbit serum (mature serum, type 2} was obtained from Pel-Freez Biologicals. Inc. (Rogers. Ark., USA). This serum contained predominantly Ab4 allotypic specificity. When normal rabbit serum of Ah'IAh* allotype was needed, il was obtained from rabbiis of this allotype; Atr'jAb^ rabbits were also used as a source of complement. In this case the serum was kept frozen at — 70"C. Coiicaiiavalin A (Lot 100. three times cryslaliized and bophilized) was obtained from Miles-Ycda Lid. Kankakee, 111.. USA. The powder was dissolved in phosphate buffer. pH 7.2 (0.15 M), and aliquots of the solution were kept at - 70 C. Phyiohaemagghiliriin P (Lot 3110-56) was obtained from Difco Laboratories. Detroit, Mich.. USA. The powder was dissolved in phosphate buffer, pH 7.2 (0.15 M) and aliquots of the solution were kept at - 70 'C. Modifu'd McCoy'.\ 5a mciliiim. containing 30",, fetal calf serum (MCM). was purchased from Grand Island Biological Co., Grand Island, N.Y.. USA. Modified CMRL 1066 tissue culture medium [19] was obtained from Ontario Cancer Institute. Toronto, and was thymidine free and supplemented with 0.001",, Lasparagine and with 10 niM Hepes butfer (Grand Island Biological Co.). Minimal essential medium (MEM) (Grand Island Biological Co.) was supplemented with 10"o heat-inactivated fetal calf serum. Isoioiiic imida:ok' buffer (SIM), pH 7.0 [25]. was prepared by dissolving in glass-distilled water. 8.2 g of NaCl. 0.4 g of MgCla 6H2O. 0.373 g of KCI, 8-2 ml of 0.1 N HCI and 0.115 g of imidazole: the volume was adjusted to I litre. Aniibodiex directed against Ab4 allolypic speei/icity. Antisera were raised by isoimmunization of rabbiis of Ab^lAh" allotype [^1. RTI.A oiili.senirn. Heterologous (goat) antiscriim directed against RTLA was prepared and rendered thymus specific by absorption, as previously described [15]. The exclusive specificity of this antiseruni for T
cells has been documented in earlier publications. In addition, we have shown by fluorescent staining that RTLA bearing cells never carry Ig receptors [Szymanska, Dubiski & Cinader. unpublished]. RABELA aiiliseruiii. Heterologous goat antiserum directed against rabbit bursa! equivalent cell antigen was prepared as described by Chou t'l a!. [1\. Briefiy female goats were immunized with appendix cells; sera were heated for 30 min at 56 C and absorbed with rabbit liver cells and erythrocyics, and then with thymocytes and bone marrow cells. The exclusive specificity of this antiserum for B cells has been doeumented in an earlier publication [7]. Cell counts. Cells were counted in a Coulter counter (model F), according to the manufacturer's directions. Rahbii immiinoglobuHns Ug) were prepared from normal sera and from antisera by precipitation with t8"'n sodium sulphate and subsequent chromatography on DEAE-eelluiose [331. The column was equilibrated with 0.0175 M phosphate buffer. pH 7.0. and the nonabsorbed fractions as well as the material eluted with 0.05 M phosphate buffer were collected. The nonabsorbed fraction did not contain contaminant proteins detectable by immunoelectrophoretic analysis; the fraction eluted by 0.05 M phosphate buffer was further purified by ehromatography on Scpliadcx G-200 [I]. The preparations obtained from anti-Ab4 antisera were tested tor antibody activity by gel diffusion. Cyioioxieiiy lesis were based on the methods of Boyse ci al. [6]. Gorer & O'Gorman [17] and Schlesinger [26]. Serial dilutions (0.025 ml) of antisera (equal volume of MCM and antiserum or diluted antiserum) were pipetted into the wells of microtitre plates (Cooke Engineering Co., Alexandria. Va.. USA). Aliquots (0.05 ml) of a cell suspension (10" cells/ml) were added to each well. The mixtures were allowed to stand for 30 min at room temperature before addition of 0.025 ml of complement (rabbil serum diluted with an equal volume of MCM). The plates were kept at 37 C for 60 min in an atmosphere of 5",, CO^ and 95",, air. Thereafter, two-thirds of the supernatant was pipetted off and 0.05 ml of a freshly prepared trypan blue solution was added to each well. Two mixtures, serving as a complement control, contained complement and MCM instead of antiserum. Two mixtures, employed to determine the action of goat scrum, contained a prebleeding which had been absorbed in the same way as the corresponding antiserum. The percentage of stained cells was determined by inspecting 100 200 cells from each well and is recorded as complement mediated cytotoxicity or as cell kill. Tests were disregarded if any control mixture contained more than 20" ^ stained tells. Eniinwralkin of iiuieriiplitigcs. Macrophages were identified by nonspecific esterase staining [32]. Reagenls were obtained from Technieon Instruments Corporation, Tarrytown. N.Y., USA. and some were a gift from this firm. Two different series of mixtures were made in plastie tubes (Falcon Plasties, 2058 tubes. Oxnard. Calif. USA). The first series of mixtures consisted of 6 drops of mono nitrate reagent (TOI-053038). 6 drops of mono dye reagent (TOI-0529-38). 18 drops of nionocyte huder (TOI-0678-38), and 3 drops of mono substrate (TOI-0680-38). The second series of mixtures consisted of 2 drops of a eel! suspension containing 1 > 10" to 2 y 10" cells/ml, 2 drops of heat
Regulation of Response to T-Cett Mitogens maclivatecl fetal calf scrum, and 4 drops of monocyte fix reagenl (TOI-067'^-38). The conlents were mixed and kept at room tcmperattire for 40 s before the first series of mixtures were added; the suspensions were mixed vigorously and were immediately incubated at 37 C for 8 min. The suspension was introduced into an improved Neubauer counting chamber and stained cells were counted under a microscope. DciiTuiimilinn of tbymUline inairporcilian. Detailed procedures have been described elsewhere |27-29]. A suspension of 1 • 10'* nucleated spleen cells was mixed with various doses of Con A. PHA or antiserum in tissue culture tubes (Falcon PlasticsJ. All mitogsns were used in concentrations that resulted in maximum response. The amount of Con A was 300 lig/eulture and that of PHA was 15 iig/culture; ihis optimum was the same for all cell preparations used (not shown). The final volume ofeach culture was 2 ml of CM RL 1066 medium supplemented with heat-inactivated normal rabbit serum. T!ie final serum content was 20" „ for stimulation with Con A and with antibody against allotypic speciticily and 5",, tor stimulation wiih PHA. When cells were cultured wiih antibody against Ab4 allotypic specificity, the allotype of normal serum was Ah-'IAh'': otherwise it was normal rabbit serum !ype 2 (Pel-Free? Biologicals). After 24 h of incubation at 37 C in an atmosphere oC5'!,, CO., and "-IS"„ air. O.I ml of tritiated thymidine(*H-TdR; 2.5 aCi, specific activity 20 Ci/mM, Amersham-Searle Corp.) was added to each culture tube; the incubation was continued for a further 24 h. Thereafter, the harvested cells were washed twice on glass fibre filters (Reeve Angel. N.J.) with two successive cycles each consisting of a wash wiih ice-cold 0.15 M NaCl. 5",, trichloroacetie acid, and absolute methanol. Thymidine incorporation was determined by liquid scintillation counting and expressed as net counts (counts in the presence of mitogen less counts in the absence of the mitogen). Miloinycin-C Ireatmeni of Spleen cells was performed according to Ref, 20. 1 ml of mitomycin-C solution (80 mg/ml; Sigma Chemical Co., St Louis. Mo., USA: Lot 14C-0500) in supplemented MHM was added to I ml of spleen cell suspension (10" eells/ml) in supplemented MFM. Cells were incubated at 37 C for 40 min, washed three times with supplemented MFM. and resuspended in CMRL 1066 for culture. In some instances, cells were killed with antiserum and complement before being washed and treated with mitomycin-C as described above. Mitomycin-C treatment was employed to inhibit proliferative responses of helper cells, i.e. so as to exclude etVects which might be due to a T cell assisted proliferative response of B cells to Con A and PHA. Non-adhercnl cells {NA) were prepared by removal of cells that adhered to or ingested iron core particles, coated with poly L-lysine {Technicon Corp.) as described previously [27]. Rciiiiival iij iinmiinii/rldbiiliii (If;) hcariit); cells hy aniihody coaled hciids. The technique was a modification of that described in Ref. 34. Degalan beads (V26) were obtained from Accurate Chemical and Scientific Corp., Hicksville, N.Y. The beads were washed with giassdistilled water, ihen with sterile phosphate-bufi"ered saline (PBS), pH 7.2, 0.15 M; in each case 10 times the
317
volume of the beads was used for washing. The suspension was then stored in PBS containing 10 ^ M sodium azide. Beads, packed by settling (30 ml), were washed with PBS (100 ml), so as to remove sodium azide, and were introduced into sterile graduated conical bottom tubes (Falcon Plastics, 2070 tubes). Afler the beads had settled at room temperature, the top layer of PBS was removed, and 90 mg of antibody Ig or normal Ig was added in a concentration of 18-24 mg/ml. The beads were gently mixed with the Ig by moving the suspension up and down with a Pasteur pipette. The mixture was next incubated for 40 min in a water bath at 45 | 0.2°C with genile stirring. The preparation was stored at 4'C overnight. On the following day, the beads were washed wiih 200 ml of PBS and transferred to a sterile 25 ml graduated glass pipette (36 ,-.. 0.6 cm), which was closed at the bottom with nylon material and plastic tubing. Thereafter, PBS was replaced by washing with 100 ml of medium CMRL 1066. These operations and the following fractionation were carried out at room tempt ratu re. Non-adherent spleen cells (NA: 2 < 10" to 8 X 10") were suspended in 7.5 ml of medium CMRL 1066 equal to the void volume of the Degalan bead column. The cells were loaded gently on top of the beads, and once they had entered the entire volume of the column, 0.2 ml of medium CMRL 1066 were layered on top of the column and the column was closed for 10 min by clamping the plastic outflow tubing. The cells were then eluted with medium CMRL 1066, and 20 ml were collected. These cells were designated Ty cells and were tested for RABELA- and RTLA-bearing cells (cyiotoxicity assay), for macrophages (non-specific esterase staining), and for mitogen induced thymidine uptake. Fractionation of cell.s hy xeclimenicitiun in ibe earth's gravitational field. Cells were fractionated by velocity sedimentation in a funnel-shaped glass chamber (O. H. Johns, Toronto), 20.7 cm in diameter. The height of the cylindrical portion of the funnel was 13.5 cm, the lotal volume employed was 1860 ml [21, 22]. Nucleated cells (450 • 10*') were loaded as a thin layer on top of a continuous bovine serum albumin (BSA| gradient (0.3 2.0"n). After a sedimentation time of 4 h, cells were collected in thirty fractions of 50 ml each, were counted in a Coulter counter, and were washed in modified CMRL 1066 before being used for functional assays. Tcsl far helper effect wiili nirnebecl lynipltnid cells. Spleen cell suspensions were treated with complement (normal rabbit serum) and either RABLLA or RTLA antiserum, or with a corresponding normal goat serum. The resulting spleen cells were suspended in SIM buffer [25] in a concentration of 2 •' 10'* cells/ml. 2 ml of this suspension were placed in an upright 15 ml culture tube (3033 tissue culture lube. 16 - 125 mm style. Falcon Plastics), and were kept at 37 0.2 C in 5"^ COj and 95",, air for 150 180 min. Thereafter, the non-adherent cells were removed by decantation, followed by washing wiih Dulbecco's [10] buffer at 37 C. Five of the tubes with attached cells were employed to count the number of adherent cells (usually 5 - tO-^); to the remaining tubes 0.4 ml heat inactivated (56 C, 30 min) serum were added: this was followed by Con A and finally either by 1.6 ml of supplemented medium 1066 or by a cell suspension (T^j in ihe same volume.
318
A. R. Riva.s Alcala et al.
O c uQ o CL ^^ W X •o E O a. o
tion net
"(5 o
•
—
5 10 Unfractionated Spleen Cells (net cpm x 10") (b) CO
cn D
—
(D
o
O
E a o
rom net
(J CT
o
1
2 3 NA Cells (net cpm x
4
F I G . I. The etTecis of removal of various cell populations on ihc response of spleen cells to concanavalin A (Con A), (a) Comparison of the response to stimulation with Con A of non adherent spleen cells (NA) and unfractionated spleen cells (S) cells (O) and of highly purified spleen T cells (Tp) and S cells ( • ) . Each point represents the response of an individual animal. The line broken line is the regression line with a slope I; the heavy solid one is the regression line for the plot N A r. S (slope O..'^? -- 0.05) and the broken one the regression line for the plot T D r. S (slope 0.0525 0.0103). (b) Comparison of the responses to stimulation with Con A of NA cells and N A cells passed over Degalan beads coated with normal rabbit immunoglobulin (O) and of NA cells and T,, cells ( • ) . Each point represents the response of an individual animal. The slopes for the respective regression lines are; 0.6 i 0.05 and 0.19 ± 0.04. The broken line is the regression line with the slope I.
RESULTS We have shown previously that thymidine uptake after stimulation with Con A is predominantly due to T cell proliferation [27]. Complement mediated kill of B cells with RABELA antiserum results in reduction of thymidine uptake by 42±23/^. This could be due to loss of helper cells [7]; we therefore proceeded to a vigorous examination of this possi-
bility by purifying T cells and by isolating potential helper cells. It was our first objective to compare the Con A response of unfractionated spleen cells with that of spleen cells from which various subpopulations were removed. The following methods were used to this end: (I) removal of cells that adhered to or ingested iron core particles: the resulting cell preparation was designated NA cells; (2) removal of Ig bearing
Regukilion oj Response lo T-Cell Mitogens
(a)
(b) 40 h
30
X
3
o
20
E
10
319
30
1 I
20
10
FIG. 2. Miiogenic response to Con A; its abrogation by the elimination and reconstitution by the addition of certain cell populations. The target cells were either levied separately or were combined with with the putative adherent helper cells (AH). In each case the number of target cells was 0.5 /^ 10"/ culture. AH cell suspensions were first ineubated with cither antiserum to rabbii biirsal equivalent lymphocyte antigen (RABELA) or with antiserum to rabbit thymus lymphocyte antigen (RTLA) or a corresponding normal preabsorbed goat serum (NGS). These treated cells (4 • 10'') were then allowed to attach to the walls of plastic tubes in which the subsequent culture took place and were, finally, washed free of non-adherent cells. The preparation of attached cells was approximately 10",, of thec^lh; in suspension. Thymidine uptake is shown as vertical bars; vertical lines and dotted areas indicate I standard deviation. Hatching indicates individual cell populations, open bars represent the mixtures of two cell populations. Broken horizontal lines represent arithmetic addilion of component cell populations in a given cell mixture. Olher cell designations: S unfractionated spleen cells; NA non-adherent spleen cells; T,, highly purified spleen T cells.
cells by passing NA cells over aniibody-coaied beads, the resulting ceil preparafion was designated To cells; and (3) removal of Fc bearing cells by passing spleen cells over Ig coated beads. Cell preparations NA and T D were assayed for their content of B and T eells by complement mediated cell kill of RABELA positive B and RTLA positive T eells. In this respect there was no substantial difference between unfractionated spleen cells and NA cells, but To cells contained 85 ± 1 0 % RTLA positive cells and 5 ± 4% RABELA positive cells. The relative B cell responsiveness to T^ cells was also estimated funclionally, in terms of ihymidine incorporation, after stimulation with antibody
against Ab4 and found to be 9 + 8 % of the uptake by unfractionafed stimulated spleen cells. By staining for non-spi^cific esterase, spleen cells, NA cells and T^ cells were found to contain IO±2%, O,7±O,8% and 0.3 + 0,4% macrophiiges. respectively. There was a substantial decrease in thymidine uptake afler Con A stimulation of NA as compared to unfractionated spleen cells. Thus the removal of adherent cells had resulted in little change in T cell content and yet in a reduction in Con A induced thymidine uptake to 1 ±7"/o of that given by the unfractionated spleen cell population, A plot of thymidine incorporation by Con A stimulated unfractionated spleen cells against stimulated NA cells
320
A. R. Rivas Alcala er al.
gives a regression line with a slope that is significantly dilTerent from 1 (Pible that cooperative interaction with adherent B cells was involved, as we have found in the case of rabbit lymphocytes. It would appear to us that apparent species differences may not be attributable to divergences in the helper mechanism of different species, but rather to the test system and to the method of cell identification used by different investigators. In the mouse system, la-positive cells were found to give help in the response to Con A [4, 16, 18]. Tbis help could also be given by soluble la molecules which can be supplied in normal serum [24]. Whether help in the rabbit system is also ladependent remains to be seen. The response of T cells to Con A involves not only the two already mentioned, but also a suppressor T cell. Evidence for an adherent T suppressor cell operating in the response to C on A was obtained from the increased helper elfect of adherent cells tbat were freed of T cells with complement and RTLA aniiserum (Fig. 2). The population containing helper cells also contains T cells that can respond to Con A (AHNGS in Fig. 2); this activity is lost after exposure to RTLA (AHRILA in Fig. 2). However, the helper effect is increased after elimination of
323
viable T cells, as judged by comparing the sum of the individual responses of the components of the mixture with the directly measured response of the mixture. The dilTerence can be judged from the relative distance between the broken line and tbe top of the bar in AHNGS + T|) and AHRTLA + TD of Fig. 2. The broken line
represents in each case the arithmetic addition of the individual responses of the components of the mixture. Suppressor cells of this general type bave been reported in the rat lymphoid system, where a ihymus dependent ceil suppresses proliferative responses of non-adherent spken cells to antigen and to T cell mitogens, paiticularly PHA [3, 11-14]. These cells are not found in purified spleen or peritoneal maerophdges, nor in rats deprived of thymus cells in vivo. It was therefore concluded that suppression was due to T cell rather than B eel! [13]. Subsequent studies led to the view that macrophages were involved in the suppressive effect, since cytotoxic agents, such as carrageenan and cyclophosphamide. reduced the suppressive synergy between tbe rats" suppressor T cell and maerophages [2]. Whether the suppressor cell described in the rai lynipboid response belongs to a cell lineage similar to that involved in our system of the rabbit lymphoid response to Con A remains to be seen.
ACKNOWLEDGMENTS This work was supported by grants from the Medical Research Council of Canada and by the National Cancer Institute of Canada. We are grateful to Mr Ladislav Horvatb for effective technical help and lo Technicon Instruments Corp., Tarrytown, N.Y., for the gift of reagents.
REFERENCES I Aiidrcv^s. P. The gel tillralion behaviour of proteins relaied to their jiiolcculur woighl over a wide range. Bi.H/win. J. 96, 595. 1465, : Bash. J.A.. Singer. A.M. & Waksman, B.H. The suppressive efftci of immunizaiLun on ihe proliferalive responses of rat T cells in vHru. II. Abrogation oT aniigen-induced suppression by selective cyloloxie agents. J. ImniiinoL 116, 1.150. 1976, 3 Bash, J.A, & Waksman. B.H, The suppressive efTecl orimmuni7atioii on the proliferative responses of rat T ceils in vitro. J. liiiiminul. 114, 78 1. 1975.
324
A. R. Rivas Alcala ct al
4 Bick. P.H,. Persson. U,. Smilh. E,. Moller, E. & Hamarstrom, L. Genetic control of lymphocyte activation: lack of response to low doses of concanavalin A in lipopolysaccharide-nonrespondcr mice. J. e.xp. Med. 146, 1146. 1977, 5 Bona, C . ChediJ. L., Damais. C . Ciorharu, R,, Shek, P.N,. Duhiski, S, & Citiader. B, Blast transformalion of rahhil B-dcrived lymphocytes hy a mitogen extracted from Nocardia, J. Immunol. 114, 348, 1975. 6 Boyse. E.A.. Old., L.J. & Chouroulinkov, I, Cylotoxic lesl for demonstration of mouse antibody. Methods med. Res. 10. 39. 1964. 7 Chou, C,-T.. Cinader. B. & Dubiski, S. A membrane antigen of rahhit btirsal eqtiivalent cells. Cell, tmmunol. 28, :i34. 1977. 8 Delespesse, G,, Duchateaii, J., Gausset, Ph. & Govaerts, A. In vitro response of subpopiilalions of human lonsil lymphocytes. I, Cellular collaboration in the proliferative response to PHA and Con A, J. Immnnol. 116, 437. 1976, 9 Dubiski, S, Immunochemistry and genetics of a "new" allolypic specificity A e " of yG immunoglobulins: recombination in somatic cells, / . Immunol. 103, 120. 1969. 10 Dulbecco, R, & Vogt, M, Plaque formation anii isolation of pure lines wiih poliomyelitis viruses, J. exp. Med. 99, 167, 1954, 11 Folch, H. & Waksman. B,H. Regulation of lymphocyte responses in viiro. V. Suppressor acliviiy of adherent and nonadherent rat lymphoid cetls. Cell. Immunol. 9, 12. 1973, 12 Fokh. H, & Waksman, B,H, //( \itro responses of rat lymphocytes following adull thymectomy. II. Increased inhibition by splenic adherent cells of responses lo phytohemagglutinin. Cell. Immunol. 9, 25, 197.1. 13 Folch, H, &. Waksman. B,H. The splenic suppressor cell. I. Activity oTthymus-dependent adherent cells; Changes with age and stress, / . lmtnuni>l. 1(3, 127, 1974. 14 Folch, H., Yoshinaga. M. & Waksman. B,H- Regulation of lymphocyte responses in vitro. III. Inhibition by adherent ceils of the T-lymphocyte response to phytohemagglutinin, J. Immunol. 110, 835. 197.1, 15 Fradelizi. D,P,. Chon, C,-T.. Cinader. B, & Dubiski. S, RTLA. a membrane antigen of rabbit thymus cells. Cell. Immunol. 7, 484. 1973, 16 Frelinger. J,A. la-bearing cells promote the concanavalin A mitogenic response of lu-ncgative T cells. Eur. J. Imnumol. 7, 447. 1977, 17 Gorer, P,A, & O'Gorman. P. Cylotoxic activity of isoaniibodies in mice, Transplont. Bull. 3. 142, 1956. 18 Habu, S, & Raff, M-C, Accessory cell dependence of lectin-induced proliferation of mouse T lymphocytes. Eur.J. Immunol. 7,451. 1977, 19 Healy. G,M,, Fisher. D,C, & Parker, R,C, Nutrition of animal cells in tissue culture, X. Synthetic medium N o . 8 5 8 , Proc.
Soc. e.xp. Biol. Med.
8 9 , 7 1 , 1955,
2U Hodcd, R.J, & Terry. W.D. Comparison of irradiated and milomycin-treated mouse spleen cells as stimulating cells in mixed lymphocyte cultures and in vitro sensilization. J. Immunol. 113, 39. 1974. 21 Miller. R,G, p, 87 in Pain, R,H, & Smilh, B,J, (ed,) A'cir Tcclinic/Ufs in Biophysics ond Cell Biology. John Wiley & Sons, London, 1973, 22 Miller, R,G. & Phillips, R.A. Separation of cells by velocity sedimentation, J. celt. Physiol. 73, 191. 1969. 23 Mills. G,, Moniicone. V. & Paetkau. V, The role ol" macrophages in thymocyle milogenesis. J. Immunol. 117, 1325. 1976, 24 Persson, U,, Hammarstrom. E., Moller, E., Moller. G. & Smilh. C.I.E, The role of adherent cells in B and T lymphocyte activation, Immunol. Rev. 40, 78. 1978, 25 Rabinovitch, M. & deStefano, M,J, Macrophages spreading in viiro. I. Inducers of spreading. Exp. Cell fic.s, 77, 323, 1973. 26 Schlesinger, M. Immune lysis of thymus and spleen cells of embryonic and neonatal mice, J. Immunol. 94,358, 1965, 27 Shek, P,N,. Chou. C.-T.. Dubiski. S. & Cinader. B. Mitogen stimulation of rabbit spleen cells before and after complement mediated cell kill with an aniiserum directed against the thymus antigen RTLA. Int. Arch. Allergy, 46, 753. 1974, 28 Shek, P,N.. Chou, C,-T,, Dubiski. S. & Cinader, B, Volume, adherence properties, membrane antigens and mitogen responsiveness of rabbit lymphoid cell subpopulations, Immunol. Comntiin. 4^S]. 1975. 29 Shek, P.N,, Chou, C-T., Dubiski, S, & Cinader, B, A suppressor cell which interferes with anti-allolype stimulated DNA synthesis of rabbit B cells. Cell. Imnumol. 18, 331, 1975. 30 Shek, P.N., Chou, C,-T,. Dubiski, S, & Cinader, B. Rabbil lymphoid cells. I, T-eell miiogens, cell volume and adherence properties as probes for cellular heterogeneity. Immunology. 30, 549, 1976. 31 Stobo. J,D, PhytohcniLigglulinin and concanavalin A: probes for murine " T " cell activaiion and differentiation. TrcinspL Rev. II, 60. 1972, 32 Tucker. S,B,. Pierre. R,V, & Jordon. R.E, Rapid identification of monocytes in a mixed mononuclear cell preparaiion, Immunol. Mi-lh. 14, 267. 1977, 33 Weir, D,M, (eii,) pp, 6.5 and 7.10 in Hondbouk of Experimenlal Immunology, 2nd edn, Blackwell Seienlific Publications, Oxford, 1973. 34 Wigzell, H,. Suiidquist. K.G. & Yoshida. T.O. Separation of cells according to surface antigens by the use of antibody-coaled columns. Fractionation of cells carrying immunoglobulins and blood grotip antigen. .Scond. J. Immunol. 1, 75, 1972,
Received 9 August 1978 Received in revised form 10 November 1978
