Immunology 1977 32 359
Human mixed lymphocyte culture using separated lymphocyte populations
M. R. POTTER & M. MOORE Immunology Division, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester
Received 5 July 1976; acceptedfor publication
Summary. The ability of human blood lymphocyte populations enriched with T or B cells to act as responder and stimulator populations in the oneway mixed lymphocyte reaction (MLR) was investigated. T- and B-cell-enriched populations were obtained by separation of rosette-forming and non rosette-forming cells and T-cell-enriched populations were also obtained by nylon-fibre column filtration. Using cells prepared by rosette sedimentation, control unseparated and T-cell-enriched populations responded well when stimulated by mitomycin C-treated unseparated cells from a second individual; and stimulation by T- and B-enriched populations generally produced some response, although the magnitude was variable. B-cell-enriched populations gave virtually no response regardless of the composition of the stimulating populations. Nylon-column-enriched T-cell populations responded to stimulation by control unseparated cells but not to T cells purified by the same procedure. T-cell enriched populations prepared by the two methods thus had different activities in the MLR despite containing similar numbers of T cells suggesting that other factors, such as the presence of small numbers of accessory cells, are important in determining the magnitude of the MLR.
5
August 1976
INTRODUCTION Blast transformation in mixed lymphocyte culture (MLC) as first described by Bain, Vas & Lowenstein (1964) is an in vitro expression of an immune reaction against genetically determined incompatibilities. Used in this context lymphocyte suspensions from human peripheral blood or the lymphoid organs of laboratory animals commonly comprise an heterogeneous mixture of lymphocytes and a variable number of non-lymphoid cells. The recognition of two major lymphocyte populations and cells which may be identified and separated on the basis of distinctive cell-surface markers, has prompted several investigators to study their respective roles in MLC. While these studies have unequivocally established that the responding cells belong to the thymus-dependent series (Chess, MacDermott & Schlossman 1974, Von Boehmer, 1974, Takiguchi, Adler & Smith, 1971, Plate & McKenzie, 1973, Lohrmann & Whang-Peny, 1974; Dean, Silva, McCoy, Leonard, Cannon & Herberman, 1974) the nature of the stimulator cells has not been clearly defined. We have previously reported that although certain in vitro properties in a heterogeneous mixture of cell types are frequently attributable to specific cell populations (e.g. phytomitogen responsiveness of T lymphocytes) it need not necessarily follow that the behaviour of such populations will be quantitatively similar in isolation (Potter & Moore, 1975).
Correspondence: Dr M. Moore, Immunology Division, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX.
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In order to determine the conditions under which optimal stimulation and responsiveness may be achieved in the MLC we have investigated both the stimulatory and responding capacity of human peripheral blood lymphocyte populations enriched with T or B cells by different procedures and in addition the stimulating capacity of a tissue culture cell line expressing B-cell markers (Bri 8), of human derivation.
MATERIALS AND METHODS Blood lymphocytes The initial lymphocyte-rich cell populations used in all experiments were prepared from peripheral blood by centrifugation on Ficoll-Triosil as reported previously (Potter & Moore 1975). Rosette sedimentation and estimation of T and B lymphocytes T- and B-cell enriched preparations were obtained by formation of rosettes with sheep erythrocytes and separation of rosette forming and non-rosette forming cells on Ficoll-Triosil. Using this method it was possible to obtain populations containing 80-95 per cent T or B lymphocytes, as determined by enumeration of the rosette-forming cells and surface immunoglobulin bearing cells in the respective populations (Potter & Moore 1975). By these procedures blood lymphocytes from two individuals were separated into T- and B-cellenriched populations and a control unseparated population. The three populations obtained from one individual were treated with mitomycin C (25 pg/ml for 30 min at 370) and used to stimulate the three populations obtained from a second individual in one-way mixed lymphocyte cultures.
Nylon fibre column separation T-cell enriched lymphocyte populations (85-95 per cent T cells) were also obtained by incubating lymphocytes previously separated on Ficoll-Triosil, on columns packed with nylon fibre and eluting the non-adherent cells as previously described (Potter & Moore 1975). These populations and controls, which had not been so depleted of adherent cells were also used as responding and stimulating cells in one-way MLC.
Depletion ofplastic adherent cells In some experiments Ficoll-Triosil separated blood
lymphocytes were depleted of plastic adherent cells by incubation in tissue culture flasks for 1 h at 370, and the ability of these cells to respond and stimulate in the MLC was also examined. This procedure did not significantly alter the proportion of T and B lymphocytes in the population.
Mixed lymphocyte culture MLC reactions were performed in Falcon microplates using HEPES-buffered Minimum Essential Medium supplemented with 10 per cent human AB serum. The lymphocytes were used at a concentration 2 x 105 responding cells per well together with 4 x 105 stimulating cells in a total volume of 0-2 ml, which in preliminary experiments were found to be the optimum conditions for this system. Control wells containing responding and stimulating cells only were always included and all reactions were performed in triplicate. The plates were incubated for 5 days at 370 in a humid chamber and 1 pCi of [3H]thymidine (Amersham, specific activity 2-5 CfmMol) was added to each well for the final 6 h of incubation. At the end of the culture period the cells were collected on fibre discs using a SAM 2 multiple culture harvester. The discs were dried and transferred to vials containing scintillation fluid (toluene containing PPO and POPOP) for counting. The results were expressed as mean values of triplicate cultures in c.p.m.
Differential cell counts Differential cell counts were performed on lymphocyte preparations using stained cytocentrifuge preparations. Lymphocyte preparations obtained by Ficoll-Triosil centrifugation contained on average 85 per cent lymphocytes; this value was increased to 90 per cent by removal of plastic adherent cells and 95 per cent by nylon-column filtration. RESULTS Rosette sedimentation Blood lymphocyte preparations were separated into a T-cell-enriched rosette-forming population, a Bcell-enriched non-rosette forming population and a control unseparated population. The three populations obtained from one individual were treated with mitomycin C and used to stimulate the three populations obtained from a second individual in a unidirectional MLC.
361
Human mixed lymphocyte culture 6 - (a)
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Figure 1. MLR between human blood lymphocyte populations separated by rosette sedimentation. Control unseparated, rosette-forming and non-rosette-forming populations from one individual were used as responder populations and the same three populations from a second individual were used as stimulator populations, following treatment with mitomycin C. Cultures were incubated for 5 days and [3H]thymidine incorporation was measured during the final 6 h of culture. Each value represents the mean of triplicate wells in four separate experiments (a, b, c and d). The stimulator populations used were as follows: control unseparated, open columns; rosette forming, hatched columns; non-rosette forming, stippled columns; responder cells only, solid columns.
In three out of four experiments (Fig. la-c) a fairly consistent pattern of response was obtained, but in the fourth experiment there was some variation (Fig. Id). In experiments a, b and c control unseparated cells gave the highest responses when stimulated by mitomycin C-treated control cells, stimulation by the T-cell-enriched populations produced a lower but still substantial reponse while stimulation by the B-cell-enriched populations produced poor stimulation. When the T-cell-enriched populations were used as the responding cells the reactions produced were somewhat higher than those produced in control unseparated cells, but again stimulation by mitomycin C-treated control cells produced the highest responses. The T-cell enriched populations produced a lower level of stimulation and the B-cell enriched populations evoked, at least in two (b and c) of the three tests a feeble reaction. When B-cell-enriched populations were used as the responding cells the reactions were quite different,
0
Control None Control None Non-adherent Non-adherent Responder cells Figure 2. MLR between human blood lymphocyte populations enriched by nylon column filtration (a and b) or by the removal of plastic adherent cells (c and d). Control unseparated and enriched populations from one individual were stimulated by mitomycin C-treated control and enriched populations from a second individual. Cultures were incubated for 5 days and [3H]thymidine incorporation was measured during the final 6 h of culture. Each value represents the mean of triplicate wells in individual experiments. The stimulator populations used were as follows: control unseparated, open columns; depleted (a and b nylon column, c and d plastic adherence), hatched columns; responder cells only, stippled columns.
little or no stimulation was obtained with any of the three stimulating populations. In the fourth experiment (Fig. id) the highest responses were again obtained with the T-cell-enriched responding population; control cells gave a lower response and the B-cell-enriched population a very low response. However the levels of stimulation produced by the different stimulating populations showed one major difference viz, the B-cell-enriched population produced high levels of stimulation while the control and T-cell-enriched populations produced lower but still substantial stimulation. In the case of blood lymphocytes separated by rosette sedimentation therefore the highest responses were consistently observed in the T-cell-enriched populations, unseparated control cells gave a lower response while B-cell-enriched populations were virtually unresponsive. With one exception the most efficient stimulating cells were the control unseparated very
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population. By contrast, the stimulation evoked by T-cell-enriched populations was invariably below the level produced by control cells.
(b) 4' n
-=
Nylon-column filtration Blood lymphocyte preparations were separated into A / a T-cell enriched nylon column filtered population and a control unfiltered population. Nylon-column enriched and control cells from one individual were p treated with mitomycin C and used to stimulate the Ui two populations obtained from a second individual in a one-way MLC as previously described. The T-cell-enriched populations obtained by passage through nylon fibre columns gave slightly lower responses than the control populations, but high levels of stimulation of both responding populations were only obtained with mitomycin C-treated -xoz __-x -x control cells. By contrast mitomycin C-treated x x -~~~~~~ T-cell-enriched populations produced poor stimulaI I IJ I 102 WE I tion of control populations and no significant stimu105 2xlO 5x104 0 x04 105 2X105 lation of T-cell-enriched populations (Fig. 2a, b). Stimulating cells (Bri 8) per well Mixed lymphocyte cultures in which both the Figure 3. Stimulation of separated blood lymphocyte responding and stimulating populations were nylon populations by mitomycin C-treated Bri 8 cells. 2 x lO blood column filtered therefore showed virtually no lymphocytes separated by rosette sedimentation (a and b) or response. by nylon column filtration (c and d) were stimulated by 5 x 104, l0s or 2 x lO Bri 8 cells. Cultures were incubated for .
EX in '1--, O
Removal of plastic-adherent cells The effect of depleting adherent cells from FicollTriosil lymphocyte preparations under conditions where the proportion of T and B cells remained unchanged was also investigated. Blood lymphocyte preparations from two individuals were depleted of plastic adherent cells. Control and depleted populations from one individual were treated with mitomycin C and used to stimulate control and depleted populations from a second individual. Both the control and depleted populations responded when stimulated by mitomycin C-treated control cells (Fig. 2c, d) but when the depleted population was used as the stimulator the control population gave a lower response and the depleted population virtually no response at all. Blood lymphocyte response to Bri 8 Mixed lymphocyte cultures were also performed using blood lymphocyte populations separated by rosette sedimentation and nylon-column filtration as the responding population and mitomycin C-
x
x
o
5 days and [3H]thymidine incorporation was measured during the final 6 h of culture. Each value represents the mean of triplicate wells in individual experiments. The following responder populations were used: control rosetted but unseparated (0); rosette forming (A); non-rosette forming (E); control untreated (-); nylon column (A). The background incorporation by Bri 8 cells cultured alone is also given ( x).
treated cells of a human lymphoid cell line (Bri 8) as the stimulating population. In these experiments the T-cell enriched populations obtained by both methods produced responses that were similar to the control unseparated populations (Fig. 3). In the case of T-cell-enriched populations prepared by rosette sedimentation the response produced was equal to or greater than that of control cells and in the case of nylon-column-filtered cells the response was equal to or slightly less than that of control cells. The B-cellenriched populations gave very little or no response to the cell line. The reactions produced by control and T-cell-enriched populations stimulated by Bri 8 were generally quantitatively greater than those produced in response to allogeneic peripheral blood lymphocytes.
Human mixed lymphocyte culture DISCUSSION The failure of B-cell-enriched populations to respond to stimulation by unseparated allogeneic lymphocytes was in general as consistent a feature of this study as the reproducible response of T-cell-enriched populations. These results are therefore in agreement with a number of other studies, using human and animal lymphocytes, indicating that the principal responding cell in the MLC is a thymus dependent lymphocyte (Chess, MacDermott & Schlossman, 1974; Von Boehmer, 1974; Takiguchi et al., 1971; Plate & McKenzie 1973; Lohrmann & Whang-Peny 1974; Dean et al., 1974). The lack of reactivity of separated B-lymphocyte populations does not rule out the possibility that B cells may contribute to the response produced by a population containing T and B cells, for instance, via stimulation by T-cell factors. The behaviour of the various preparations in MLC however revealed other differences which would appear to have implications not only for the nature of the stimulatory, but also for the responding populations of lymphocytes. Thus the responding capacity of T-cell-enriched populations separated by rosette sedimentation was significantly greater than that for T cells prepared by nylon-wool filtration, even though the absolute number of T cells present in each preparation was comparable. Furthermore, stimulatory capacity was not simply related to the proportion of T and B cells in these populations. In the case of cells separated by rosette sedimentation, mitomycin C-treated control cells most frequently induced highest stimulation but neither T- nor B-cellenriched populations were without effect, although the latter were the more variable in this respect. By contrast, nylon fibre column purified T cells evoked a poor response in control populations and none in T-cell-enriched populations. Thus, T-cell-enriched populations prepared by the two methods possessed different stimulating and responding properties, despite the close similarity of T- and B-cell content. This was most evident when both the responding and stimulating populations were T cell enriched; MLCs containing two rosette-cell-enriched populations resulted in substantial blastogenesis while virtually no interaction occurred when both stimulator and responder cells were enriched by nylon column filtration. If extrinsic damage to cells as an artefact of the separation procedures is discounted, these differences may be attributable to variation in the K
363
number of residual non-lymphoid cells present in the populations. In that event, the number involved must be very small since rosette-purified T cells contained on average 6 per cent non-lymphoid cells (monocytes and polymorphs) and nylon-column purified T cells only 4 per cent non-lymphoid cells. Control populations that stimulated well contained on average 15 per cent non-lymphoid cells. This interpretation was confirmed by data obtained from MLCs between lymphocyte populations depleted of plastic adherent cells. These were invariably poor stimulators, a fact again indicative of an important accessory role for non-lymphoid cells, since removal of plastic adherent cells did not significantly alter the proportion of T and B lymphocytes in the population. However the experimental design precluded any definitive statement as to which population-stimulator or responder-was primarily influenced by the accessory cells. On the basis of these observations we conclude that both T and B lymphocytes may stimulate in the human MLC but that the magnitude of the response depends not only on the number of T and B cells present but also on the presence of adherent cells. Small numbers of non-lymphoid cells may be envisaged to influence the MLC via a number of possible mechanisms. They may directly stimulate the responding population since it has been shown that monocytes and granulocytes can stimulate allogeneic lymphocytes in some systems (Rode & Gordon, 1974; Sharma, Terasaki & Mickey, 1975). Alternatively the presence of non-lymphoid cells may be necessary to permit stimulation by the lymphoid population either by a co-operative mechanism or by supporting the survival of lymphocytes in culture. In the case of B-cell-enriched populations depletion of a non-lymphoid population is unlikely to be a limiting factor since these populations contained slightly more non-lymphoid cells than the control
populations. Other investigators have reported conflicting results concerning the nature of the stimulating population in the MLC. Using mouse lymphocytes separated by electrophoresis it has been demonstrated that both T- and B-cell enriched populations produce stimulation (Von Boehmer, 1974) and thoracic duct lymphocytes (TDL) from athymic nude mice and heterozygous littermates have comparable stimulatory activity (Cheers & Sprent, 1973). Others have prepared T- and B-cell-enriched human lymphocyte populations by rosette sediment-
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ation (Lohrmann, Novikovs & Graw, 1974) and mouse lymphocytes by the action of cytotoxic antisera and complement (Plate & McKenzie, 1973) and in these experiments B cells provided a better stimulus than T cells. However, in experiments using human lymphocytes separated on immunoabsorbent columns it has been reported that T cells produced greater stimulation than B cells (Chess et al., 1974). Moreover, neonatal thymectomy of mice has been shown to reduce the capacity of spleen cells to respond and stimulate in the MLR (Howe, Manziello & Goldstein, 1972). This wide variation is probably due to some extent to the different separation procedures and lymphocyte preparations used, but again suggests that other factors, in addition to the T-B cell composition, are important in determining the magnitude of the MLR. It is known that small numbers of monocytes or macrophages can increase the response of human and rat lymphocytes in the MLR (Rode & Gordon, 1974; Keller, 1975) and that the addition of adherent cells can greatly increase the reaction between allogeneic T-cell-enriched populations (Sondel, Chess & Schlossman, 1975). However it is apparent that relatively small variations in the number of adherent cells in the different preparations may result in quite different reactions; too few or too many adherent cells may depress the MLR in a similar manner to that of PHA responsiveness (Keller, 1975; Folch, Yoshinaga & Waksman, 1973). The experiments performed using mitomycin Ctreated cells of a human lymphoid cell line to stimulate separated blood lymphocyte populations demonstrated that these cells, which carry B-cell surface markers, produce strong stimulation of T-cell-enriched and control unseparated blood lymphocytes. It is possible that the strong stimulating capacity of the B-cell line compared with B-cellenriched blood lymphocyte populations may be associated with changes that have occurred in culture, for example, the transformation to a rapidly dividing cell or the expression of viral antigens. Other workers have reported strong stimulation of blood lymphocytes by lymphoid cell lines (Hardy, Ling & Knight 1969; Han, 1972) and it has been suggested that this is a property of cell lines carrying B-cell surface markers but not cell lines carrying T-cell surface markers (Han & Minowada 1973, Royston, Graze & Pitts, 1974; Pauly, Minowada, Han & Moore, 1975). Blood lymphocytes from patients with chronic lymphocytic leukaemia (CLL),
which in most cases contain a high proportion of cells with B-lymphocyte surface markers, have also been shown to stimulate in the MLR (Ruhl, Vogl, Bochert, Schmidt, Moelle & Schaoua, 1975; Kasakura, 1975; Smith, Browne & Slungaard, 1973). However, there are also reports of a lymphoid cell line (Callewaert, Kaplan, Peterson & Lightbody, 1975) and CLL cells (Insel, Melewicz, La Via & Balch, 1975) with T-cell surface markers producing stimulation in the MLR, so that there seems to be no absolute correlation between the presence of B-cell surface markers and the stimulating capacity of these cells. ACKNOWLEDGMENTS This work was supported by grants from the Medical Research Council and the Cancer Research Campaign. We are indebted to Miss Wendy White for skilled technical assistance. REFEREN CES BAIN B., VAS M.R. & LOWENSTEIN L. (1964) The development of a large immature mononuclear cells in mixed leukocyte cultures. Blood, 23, 108. VON BOEHMER H. (1974) Separation of T and B lymphocytes and their role in the mixed lymphocyte reaction. J. Imnunol, 112, 70. CALLEWAERT D.M., KAPLAN J., PETERSON W.D. & LIGHTBODY J.J. (1975) Stimulation in the mixed leukocyte culture and generation of effector cells in cell mediated lympholysis by a human T lymphoblast cell line. Cell. Immunol. 19, 276. CHEERS C. & SPRENT J. (1973) B lymphocytes as stimulators of a mixed lymphocyte reaction. Transplantation, 15, 336. CHESS L., MACDERMOTT R.P. & SCHLOSSMAN S.F. (1974) Immunologic functions of isolated human lymphocyte subpopulations. II. Antigen triggering of T and B cells in vitro. J. Immunol. 113, 1122. DEAN J.H., SILVA J.S., McCoy J.L., LEONARD C.M., CANNON G.B. & HERBERMAN R.B. (1975) Functional activities of rosette separated human peripheral blood leukocytes. J. Immunol. 115, 1449. FOLCH H., YOSHINAGA M. & WAKSMAN B.H. (1973) Regulation of lymphocyte responses in vitro. III. Inhibition by adherent cells of the T lymphocyte response to Phytohemagglutinin. J. Immunol. 110, 835. HAN T. (1972) Blastogenic response of normal lymphocytes to cultured lymphoid cells and non-lymphoid neoplastic cells. Immunology, 23, 355. HAN T. & MINOWADA J. (1973) A unique leukaemic T lymphoid cell line: Absence of stimulating effect in mixed lymphocyte reaction. Clin. exp. Immunol. 15, 535. HARDY D.A., LING N.R. & KNIGHT S.C. (1969) Exceptional
Human mixed lymphocyte culture lymphocyte stimulating capacity of cells from lymphoid cell lines. Nature (Lond.), 223, 511. HOWE M.L., MANZIELLO B. & GOLDSTEIN A.L. (1972) Proceedings 6th Leucocyte Culture Conference (ed. by M. R. Schwarz) Studies on the nature of the stimulator cell in the mixed lymphocyte interaction. p. 599. Academic Press. New York and London. INSEL R.A., MELEWICZ F.M., LA VIA, M.F. & BALCH C.M. (1975) Morphology, surface markers and in vitro responses of a human leukemic T cell. Clin. Immunol. Immunopathol. 4, 382. KASAKURA S. (1975) MLC stimulatory capacity and production of a blastogenic factor in patients with chronic lymphatic leukemia and Hodgkins disease. Blood, 45, 823. KELLER R. (1975) Major changes in lymphocyte proliferation evoked by activated macrophages. Cellular Immunol. 17, 542. LOHRMANN H.P., NovIKovs L. & GRAW R.G. (1974) Stimulatory capacity of T and B lymphocytes in human mixed leukocyte cultures. Nature (Lond.), 250, 144. LOHRMANN H.P. & WHANG-PENY J. (1974) Human mixed leukocyte culture: Identification of the proliferating lymphocyte subpopulation by sex chromosome markers. J. exp. Med. 140, 54. PAULY J.L., MINOWADA J., HAN T. & MOORE G.E. (1975) Disparity of mixed lymphocyte reactivity to cultured cells of human T and B lymphoid lines. J. nat. Cancer Inst. 54, 557. PLATE J.M.D. & McKENzIE I.F.C. (1973) B cell stimulation
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of allogeneic T cell proliferation in mixed lymphocyte cultures. Nature: New Biology, 245,247. POTTER M.R. & MOORE M. (1975) PHA stimulation of separated human lymphocyte populations. Clin. exp. Immunol. 21, 456. RODE H.N. & GORDON J. (1974) Macrophages in the mixed leucocyte culture reaction (MLC). Cell Immunology, 13, 87. ROYSTON I., GRAZE P.R. & PiTTs R.B. (1974) Failure of cultured human T cell lymphoid lines to stimulate in mixed leukocyte culture. J. natl. Cancer Inst. 53, 361. RUHL H., VOGL W., BOCHERT G., SCHMIDT S., MOELLE R. & SCHAOUA H. (1975) Mixed lymphocyte culture stimulatory and responding capacity of lymphocytes from patients with lymphoproliferative diseases. Clin. exp. Immunol. 19, 55. SHARMA B., TERASAKI P.I. & MICKEY M.R. (1975) Lymphocyte transformation induced by human granulocytes. Transplantation, 20, 499. SMITH M.J., BROWNE E. & SLUNGAARD A. (1973) The impaired responsiveness of chronic lymphatic leukemia lymphocytes to allogeneic lymphocytes. Blood, 41, 505. SONDEL P.M., CHEss L. & SCHLOSSMAN S.F. (1975) Immunologic function of isolated human lymphocyte subpopulations IV. Stimulation of MLC and CML by human T cells. Cell. Immunol. 18, 351. TAKIGUCHI T., ADLER W.H. & SMITH R.T. (1971) Cellular recognition in vitro by mouse lymphocytes. J. exp. Med. 133, 63.
Human mixed lymphocyte culture using separated lymphocyte populations
M. R. POTTER & M. MOORE Immunology Division, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester
Received 5 July 1976; acceptedfor publication
Summary. The ability of human blood lymphocyte populations enriched with T or B cells to act as responder and stimulator populations in the oneway mixed lymphocyte reaction (MLR) was investigated. T- and B-cell-enriched populations were obtained by separation of rosette-forming and non rosette-forming cells and T-cell-enriched populations were also obtained by nylon-fibre column filtration. Using cells prepared by rosette sedimentation, control unseparated and T-cell-enriched populations responded well when stimulated by mitomycin C-treated unseparated cells from a second individual; and stimulation by T- and B-enriched populations generally produced some response, although the magnitude was variable. B-cell-enriched populations gave virtually no response regardless of the composition of the stimulating populations. Nylon-column-enriched T-cell populations responded to stimulation by control unseparated cells but not to T cells purified by the same procedure. T-cell enriched populations prepared by the two methods thus had different activities in the MLR despite containing similar numbers of T cells suggesting that other factors, such as the presence of small numbers of accessory cells, are important in determining the magnitude of the MLR.
5
August 1976
INTRODUCTION Blast transformation in mixed lymphocyte culture (MLC) as first described by Bain, Vas & Lowenstein (1964) is an in vitro expression of an immune reaction against genetically determined incompatibilities. Used in this context lymphocyte suspensions from human peripheral blood or the lymphoid organs of laboratory animals commonly comprise an heterogeneous mixture of lymphocytes and a variable number of non-lymphoid cells. The recognition of two major lymphocyte populations and cells which may be identified and separated on the basis of distinctive cell-surface markers, has prompted several investigators to study their respective roles in MLC. While these studies have unequivocally established that the responding cells belong to the thymus-dependent series (Chess, MacDermott & Schlossman 1974, Von Boehmer, 1974, Takiguchi, Adler & Smith, 1971, Plate & McKenzie, 1973, Lohrmann & Whang-Peny, 1974; Dean, Silva, McCoy, Leonard, Cannon & Herberman, 1974) the nature of the stimulator cells has not been clearly defined. We have previously reported that although certain in vitro properties in a heterogeneous mixture of cell types are frequently attributable to specific cell populations (e.g. phytomitogen responsiveness of T lymphocytes) it need not necessarily follow that the behaviour of such populations will be quantitatively similar in isolation (Potter & Moore, 1975).
Correspondence: Dr M. Moore, Immunology Division, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX.
359
360
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In order to determine the conditions under which optimal stimulation and responsiveness may be achieved in the MLC we have investigated both the stimulatory and responding capacity of human peripheral blood lymphocyte populations enriched with T or B cells by different procedures and in addition the stimulating capacity of a tissue culture cell line expressing B-cell markers (Bri 8), of human derivation.
MATERIALS AND METHODS Blood lymphocytes The initial lymphocyte-rich cell populations used in all experiments were prepared from peripheral blood by centrifugation on Ficoll-Triosil as reported previously (Potter & Moore 1975). Rosette sedimentation and estimation of T and B lymphocytes T- and B-cell enriched preparations were obtained by formation of rosettes with sheep erythrocytes and separation of rosette forming and non-rosette forming cells on Ficoll-Triosil. Using this method it was possible to obtain populations containing 80-95 per cent T or B lymphocytes, as determined by enumeration of the rosette-forming cells and surface immunoglobulin bearing cells in the respective populations (Potter & Moore 1975). By these procedures blood lymphocytes from two individuals were separated into T- and B-cellenriched populations and a control unseparated population. The three populations obtained from one individual were treated with mitomycin C (25 pg/ml for 30 min at 370) and used to stimulate the three populations obtained from a second individual in one-way mixed lymphocyte cultures.
Nylon fibre column separation T-cell enriched lymphocyte populations (85-95 per cent T cells) were also obtained by incubating lymphocytes previously separated on Ficoll-Triosil, on columns packed with nylon fibre and eluting the non-adherent cells as previously described (Potter & Moore 1975). These populations and controls, which had not been so depleted of adherent cells were also used as responding and stimulating cells in one-way MLC.
Depletion ofplastic adherent cells In some experiments Ficoll-Triosil separated blood
lymphocytes were depleted of plastic adherent cells by incubation in tissue culture flasks for 1 h at 370, and the ability of these cells to respond and stimulate in the MLC was also examined. This procedure did not significantly alter the proportion of T and B lymphocytes in the population.
Mixed lymphocyte culture MLC reactions were performed in Falcon microplates using HEPES-buffered Minimum Essential Medium supplemented with 10 per cent human AB serum. The lymphocytes were used at a concentration 2 x 105 responding cells per well together with 4 x 105 stimulating cells in a total volume of 0-2 ml, which in preliminary experiments were found to be the optimum conditions for this system. Control wells containing responding and stimulating cells only were always included and all reactions were performed in triplicate. The plates were incubated for 5 days at 370 in a humid chamber and 1 pCi of [3H]thymidine (Amersham, specific activity 2-5 CfmMol) was added to each well for the final 6 h of incubation. At the end of the culture period the cells were collected on fibre discs using a SAM 2 multiple culture harvester. The discs were dried and transferred to vials containing scintillation fluid (toluene containing PPO and POPOP) for counting. The results were expressed as mean values of triplicate cultures in c.p.m.
Differential cell counts Differential cell counts were performed on lymphocyte preparations using stained cytocentrifuge preparations. Lymphocyte preparations obtained by Ficoll-Triosil centrifugation contained on average 85 per cent lymphocytes; this value was increased to 90 per cent by removal of plastic adherent cells and 95 per cent by nylon-column filtration. RESULTS Rosette sedimentation Blood lymphocyte preparations were separated into a T-cell-enriched rosette-forming population, a Bcell-enriched non-rosette forming population and a control unseparated population. The three populations obtained from one individual were treated with mitomycin C and used to stimulate the three populations obtained from a second individual in a unidirectional MLC.
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Responder cells
Figure 1. MLR between human blood lymphocyte populations separated by rosette sedimentation. Control unseparated, rosette-forming and non-rosette-forming populations from one individual were used as responder populations and the same three populations from a second individual were used as stimulator populations, following treatment with mitomycin C. Cultures were incubated for 5 days and [3H]thymidine incorporation was measured during the final 6 h of culture. Each value represents the mean of triplicate wells in four separate experiments (a, b, c and d). The stimulator populations used were as follows: control unseparated, open columns; rosette forming, hatched columns; non-rosette forming, stippled columns; responder cells only, solid columns.
In three out of four experiments (Fig. la-c) a fairly consistent pattern of response was obtained, but in the fourth experiment there was some variation (Fig. Id). In experiments a, b and c control unseparated cells gave the highest responses when stimulated by mitomycin C-treated control cells, stimulation by the T-cell-enriched populations produced a lower but still substantial reponse while stimulation by the B-cell-enriched populations produced poor stimulation. When the T-cell-enriched populations were used as the responding cells the reactions produced were somewhat higher than those produced in control unseparated cells, but again stimulation by mitomycin C-treated control cells produced the highest responses. The T-cell enriched populations produced a lower level of stimulation and the B-cell enriched populations evoked, at least in two (b and c) of the three tests a feeble reaction. When B-cell-enriched populations were used as the responding cells the reactions were quite different,
0
Control None Control None Non-adherent Non-adherent Responder cells Figure 2. MLR between human blood lymphocyte populations enriched by nylon column filtration (a and b) or by the removal of plastic adherent cells (c and d). Control unseparated and enriched populations from one individual were stimulated by mitomycin C-treated control and enriched populations from a second individual. Cultures were incubated for 5 days and [3H]thymidine incorporation was measured during the final 6 h of culture. Each value represents the mean of triplicate wells in individual experiments. The stimulator populations used were as follows: control unseparated, open columns; depleted (a and b nylon column, c and d plastic adherence), hatched columns; responder cells only, stippled columns.
little or no stimulation was obtained with any of the three stimulating populations. In the fourth experiment (Fig. id) the highest responses were again obtained with the T-cell-enriched responding population; control cells gave a lower response and the B-cell-enriched population a very low response. However the levels of stimulation produced by the different stimulating populations showed one major difference viz, the B-cell-enriched population produced high levels of stimulation while the control and T-cell-enriched populations produced lower but still substantial stimulation. In the case of blood lymphocytes separated by rosette sedimentation therefore the highest responses were consistently observed in the T-cell-enriched populations, unseparated control cells gave a lower response while B-cell-enriched populations were virtually unresponsive. With one exception the most efficient stimulating cells were the control unseparated very
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M. R. Potter & M. Moore
population. By contrast, the stimulation evoked by T-cell-enriched populations was invariably below the level produced by control cells.
(b) 4' n
-=
Nylon-column filtration Blood lymphocyte preparations were separated into A / a T-cell enriched nylon column filtered population and a control unfiltered population. Nylon-column enriched and control cells from one individual were p treated with mitomycin C and used to stimulate the Ui two populations obtained from a second individual in a one-way MLC as previously described. The T-cell-enriched populations obtained by passage through nylon fibre columns gave slightly lower responses than the control populations, but high levels of stimulation of both responding populations were only obtained with mitomycin C-treated -xoz __-x -x control cells. By contrast mitomycin C-treated x x -~~~~~~ T-cell-enriched populations produced poor stimulaI I IJ I 102 WE I tion of control populations and no significant stimu105 2xlO 5x104 0 x04 105 2X105 lation of T-cell-enriched populations (Fig. 2a, b). Stimulating cells (Bri 8) per well Mixed lymphocyte cultures in which both the Figure 3. Stimulation of separated blood lymphocyte responding and stimulating populations were nylon populations by mitomycin C-treated Bri 8 cells. 2 x lO blood column filtered therefore showed virtually no lymphocytes separated by rosette sedimentation (a and b) or response. by nylon column filtration (c and d) were stimulated by 5 x 104, l0s or 2 x lO Bri 8 cells. Cultures were incubated for .
EX in '1--, O
Removal of plastic-adherent cells The effect of depleting adherent cells from FicollTriosil lymphocyte preparations under conditions where the proportion of T and B cells remained unchanged was also investigated. Blood lymphocyte preparations from two individuals were depleted of plastic adherent cells. Control and depleted populations from one individual were treated with mitomycin C and used to stimulate control and depleted populations from a second individual. Both the control and depleted populations responded when stimulated by mitomycin C-treated control cells (Fig. 2c, d) but when the depleted population was used as the stimulator the control population gave a lower response and the depleted population virtually no response at all. Blood lymphocyte response to Bri 8 Mixed lymphocyte cultures were also performed using blood lymphocyte populations separated by rosette sedimentation and nylon-column filtration as the responding population and mitomycin C-
x
x
o
5 days and [3H]thymidine incorporation was measured during the final 6 h of culture. Each value represents the mean of triplicate wells in individual experiments. The following responder populations were used: control rosetted but unseparated (0); rosette forming (A); non-rosette forming (E); control untreated (-); nylon column (A). The background incorporation by Bri 8 cells cultured alone is also given ( x).
treated cells of a human lymphoid cell line (Bri 8) as the stimulating population. In these experiments the T-cell enriched populations obtained by both methods produced responses that were similar to the control unseparated populations (Fig. 3). In the case of T-cell-enriched populations prepared by rosette sedimentation the response produced was equal to or greater than that of control cells and in the case of nylon-column-filtered cells the response was equal to or slightly less than that of control cells. The B-cellenriched populations gave very little or no response to the cell line. The reactions produced by control and T-cell-enriched populations stimulated by Bri 8 were generally quantitatively greater than those produced in response to allogeneic peripheral blood lymphocytes.
Human mixed lymphocyte culture DISCUSSION The failure of B-cell-enriched populations to respond to stimulation by unseparated allogeneic lymphocytes was in general as consistent a feature of this study as the reproducible response of T-cell-enriched populations. These results are therefore in agreement with a number of other studies, using human and animal lymphocytes, indicating that the principal responding cell in the MLC is a thymus dependent lymphocyte (Chess, MacDermott & Schlossman, 1974; Von Boehmer, 1974; Takiguchi et al., 1971; Plate & McKenzie 1973; Lohrmann & Whang-Peny 1974; Dean et al., 1974). The lack of reactivity of separated B-lymphocyte populations does not rule out the possibility that B cells may contribute to the response produced by a population containing T and B cells, for instance, via stimulation by T-cell factors. The behaviour of the various preparations in MLC however revealed other differences which would appear to have implications not only for the nature of the stimulatory, but also for the responding populations of lymphocytes. Thus the responding capacity of T-cell-enriched populations separated by rosette sedimentation was significantly greater than that for T cells prepared by nylon-wool filtration, even though the absolute number of T cells present in each preparation was comparable. Furthermore, stimulatory capacity was not simply related to the proportion of T and B cells in these populations. In the case of cells separated by rosette sedimentation, mitomycin C-treated control cells most frequently induced highest stimulation but neither T- nor B-cellenriched populations were without effect, although the latter were the more variable in this respect. By contrast, nylon fibre column purified T cells evoked a poor response in control populations and none in T-cell-enriched populations. Thus, T-cell-enriched populations prepared by the two methods possessed different stimulating and responding properties, despite the close similarity of T- and B-cell content. This was most evident when both the responding and stimulating populations were T cell enriched; MLCs containing two rosette-cell-enriched populations resulted in substantial blastogenesis while virtually no interaction occurred when both stimulator and responder cells were enriched by nylon column filtration. If extrinsic damage to cells as an artefact of the separation procedures is discounted, these differences may be attributable to variation in the K
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number of residual non-lymphoid cells present in the populations. In that event, the number involved must be very small since rosette-purified T cells contained on average 6 per cent non-lymphoid cells (monocytes and polymorphs) and nylon-column purified T cells only 4 per cent non-lymphoid cells. Control populations that stimulated well contained on average 15 per cent non-lymphoid cells. This interpretation was confirmed by data obtained from MLCs between lymphocyte populations depleted of plastic adherent cells. These were invariably poor stimulators, a fact again indicative of an important accessory role for non-lymphoid cells, since removal of plastic adherent cells did not significantly alter the proportion of T and B lymphocytes in the population. However the experimental design precluded any definitive statement as to which population-stimulator or responder-was primarily influenced by the accessory cells. On the basis of these observations we conclude that both T and B lymphocytes may stimulate in the human MLC but that the magnitude of the response depends not only on the number of T and B cells present but also on the presence of adherent cells. Small numbers of non-lymphoid cells may be envisaged to influence the MLC via a number of possible mechanisms. They may directly stimulate the responding population since it has been shown that monocytes and granulocytes can stimulate allogeneic lymphocytes in some systems (Rode & Gordon, 1974; Sharma, Terasaki & Mickey, 1975). Alternatively the presence of non-lymphoid cells may be necessary to permit stimulation by the lymphoid population either by a co-operative mechanism or by supporting the survival of lymphocytes in culture. In the case of B-cell-enriched populations depletion of a non-lymphoid population is unlikely to be a limiting factor since these populations contained slightly more non-lymphoid cells than the control
populations. Other investigators have reported conflicting results concerning the nature of the stimulating population in the MLC. Using mouse lymphocytes separated by electrophoresis it has been demonstrated that both T- and B-cell enriched populations produce stimulation (Von Boehmer, 1974) and thoracic duct lymphocytes (TDL) from athymic nude mice and heterozygous littermates have comparable stimulatory activity (Cheers & Sprent, 1973). Others have prepared T- and B-cell-enriched human lymphocyte populations by rosette sediment-
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ation (Lohrmann, Novikovs & Graw, 1974) and mouse lymphocytes by the action of cytotoxic antisera and complement (Plate & McKenzie, 1973) and in these experiments B cells provided a better stimulus than T cells. However, in experiments using human lymphocytes separated on immunoabsorbent columns it has been reported that T cells produced greater stimulation than B cells (Chess et al., 1974). Moreover, neonatal thymectomy of mice has been shown to reduce the capacity of spleen cells to respond and stimulate in the MLR (Howe, Manziello & Goldstein, 1972). This wide variation is probably due to some extent to the different separation procedures and lymphocyte preparations used, but again suggests that other factors, in addition to the T-B cell composition, are important in determining the magnitude of the MLR. It is known that small numbers of monocytes or macrophages can increase the response of human and rat lymphocytes in the MLR (Rode & Gordon, 1974; Keller, 1975) and that the addition of adherent cells can greatly increase the reaction between allogeneic T-cell-enriched populations (Sondel, Chess & Schlossman, 1975). However it is apparent that relatively small variations in the number of adherent cells in the different preparations may result in quite different reactions; too few or too many adherent cells may depress the MLR in a similar manner to that of PHA responsiveness (Keller, 1975; Folch, Yoshinaga & Waksman, 1973). The experiments performed using mitomycin Ctreated cells of a human lymphoid cell line to stimulate separated blood lymphocyte populations demonstrated that these cells, which carry B-cell surface markers, produce strong stimulation of T-cell-enriched and control unseparated blood lymphocytes. It is possible that the strong stimulating capacity of the B-cell line compared with B-cellenriched blood lymphocyte populations may be associated with changes that have occurred in culture, for example, the transformation to a rapidly dividing cell or the expression of viral antigens. Other workers have reported strong stimulation of blood lymphocytes by lymphoid cell lines (Hardy, Ling & Knight 1969; Han, 1972) and it has been suggested that this is a property of cell lines carrying B-cell surface markers but not cell lines carrying T-cell surface markers (Han & Minowada 1973, Royston, Graze & Pitts, 1974; Pauly, Minowada, Han & Moore, 1975). Blood lymphocytes from patients with chronic lymphocytic leukaemia (CLL),
which in most cases contain a high proportion of cells with B-lymphocyte surface markers, have also been shown to stimulate in the MLR (Ruhl, Vogl, Bochert, Schmidt, Moelle & Schaoua, 1975; Kasakura, 1975; Smith, Browne & Slungaard, 1973). However, there are also reports of a lymphoid cell line (Callewaert, Kaplan, Peterson & Lightbody, 1975) and CLL cells (Insel, Melewicz, La Via & Balch, 1975) with T-cell surface markers producing stimulation in the MLR, so that there seems to be no absolute correlation between the presence of B-cell surface markers and the stimulating capacity of these cells. ACKNOWLEDGMENTS This work was supported by grants from the Medical Research Council and the Cancer Research Campaign. We are indebted to Miss Wendy White for skilled technical assistance. REFEREN CES BAIN B., VAS M.R. & LOWENSTEIN L. (1964) The development of a large immature mononuclear cells in mixed leukocyte cultures. Blood, 23, 108. VON BOEHMER H. (1974) Separation of T and B lymphocytes and their role in the mixed lymphocyte reaction. J. Imnunol, 112, 70. CALLEWAERT D.M., KAPLAN J., PETERSON W.D. & LIGHTBODY J.J. (1975) Stimulation in the mixed leukocyte culture and generation of effector cells in cell mediated lympholysis by a human T lymphoblast cell line. Cell. Immunol. 19, 276. CHEERS C. & SPRENT J. (1973) B lymphocytes as stimulators of a mixed lymphocyte reaction. Transplantation, 15, 336. CHESS L., MACDERMOTT R.P. & SCHLOSSMAN S.F. (1974) Immunologic functions of isolated human lymphocyte subpopulations. II. Antigen triggering of T and B cells in vitro. J. Immunol. 113, 1122. DEAN J.H., SILVA J.S., McCoy J.L., LEONARD C.M., CANNON G.B. & HERBERMAN R.B. (1975) Functional activities of rosette separated human peripheral blood leukocytes. J. Immunol. 115, 1449. FOLCH H., YOSHINAGA M. & WAKSMAN B.H. (1973) Regulation of lymphocyte responses in vitro. III. Inhibition by adherent cells of the T lymphocyte response to Phytohemagglutinin. J. Immunol. 110, 835. HAN T. (1972) Blastogenic response of normal lymphocytes to cultured lymphoid cells and non-lymphoid neoplastic cells. Immunology, 23, 355. HAN T. & MINOWADA J. (1973) A unique leukaemic T lymphoid cell line: Absence of stimulating effect in mixed lymphocyte reaction. Clin. exp. Immunol. 15, 535. HARDY D.A., LING N.R. & KNIGHT S.C. (1969) Exceptional
Human mixed lymphocyte culture lymphocyte stimulating capacity of cells from lymphoid cell lines. Nature (Lond.), 223, 511. HOWE M.L., MANZIELLO B. & GOLDSTEIN A.L. (1972) Proceedings 6th Leucocyte Culture Conference (ed. by M. R. Schwarz) Studies on the nature of the stimulator cell in the mixed lymphocyte interaction. p. 599. Academic Press. New York and London. INSEL R.A., MELEWICZ F.M., LA VIA, M.F. & BALCH C.M. (1975) Morphology, surface markers and in vitro responses of a human leukemic T cell. Clin. Immunol. Immunopathol. 4, 382. KASAKURA S. (1975) MLC stimulatory capacity and production of a blastogenic factor in patients with chronic lymphatic leukemia and Hodgkins disease. Blood, 45, 823. KELLER R. (1975) Major changes in lymphocyte proliferation evoked by activated macrophages. Cellular Immunol. 17, 542. LOHRMANN H.P., NovIKovs L. & GRAW R.G. (1974) Stimulatory capacity of T and B lymphocytes in human mixed leukocyte cultures. Nature (Lond.), 250, 144. LOHRMANN H.P. & WHANG-PENY J. (1974) Human mixed leukocyte culture: Identification of the proliferating lymphocyte subpopulation by sex chromosome markers. J. exp. Med. 140, 54. PAULY J.L., MINOWADA J., HAN T. & MOORE G.E. (1975) Disparity of mixed lymphocyte reactivity to cultured cells of human T and B lymphoid lines. J. nat. Cancer Inst. 54, 557. PLATE J.M.D. & McKENzIE I.F.C. (1973) B cell stimulation
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of allogeneic T cell proliferation in mixed lymphocyte cultures. Nature: New Biology, 245,247. POTTER M.R. & MOORE M. (1975) PHA stimulation of separated human lymphocyte populations. Clin. exp. Immunol. 21, 456. RODE H.N. & GORDON J. (1974) Macrophages in the mixed leucocyte culture reaction (MLC). Cell Immunology, 13, 87. ROYSTON I., GRAZE P.R. & PiTTs R.B. (1974) Failure of cultured human T cell lymphoid lines to stimulate in mixed leukocyte culture. J. natl. Cancer Inst. 53, 361. RUHL H., VOGL W., BOCHERT G., SCHMIDT S., MOELLE R. & SCHAOUA H. (1975) Mixed lymphocyte culture stimulatory and responding capacity of lymphocytes from patients with lymphoproliferative diseases. Clin. exp. Immunol. 19, 55. SHARMA B., TERASAKI P.I. & MICKEY M.R. (1975) Lymphocyte transformation induced by human granulocytes. Transplantation, 20, 499. SMITH M.J., BROWNE E. & SLUNGAARD A. (1973) The impaired responsiveness of chronic lymphatic leukemia lymphocytes to allogeneic lymphocytes. Blood, 41, 505. SONDEL P.M., CHEss L. & SCHLOSSMAN S.F. (1975) Immunologic function of isolated human lymphocyte subpopulations IV. Stimulation of MLC and CML by human T cells. Cell. Immunol. 18, 351. TAKIGUCHI T., ADLER W.H. & SMITH R.T. (1971) Cellular recognition in vitro by mouse lymphocytes. J. exp. Med. 133, 63.
