Immunology 1978 35 455
Human-mouse mixed lymphocyte cultures
A. W. BOYLSTON & RENE L. ANDERSON Department ofPathology, St Mary's Hospital Medical School, London
Received 24 October 1977; acceptedfor publication 23 January 1978
Summary. Conditions for stimulating human lymphocytes with mouse cells are established. The proliferative and cytotoxic responses generated are shown to be specific for antigens coded for in the major histocompatibility locus.
human and mouse cells may provide a suitable model for immunizing human lymphocytes in vitro (Lindahl & Bach, 1975; Carnaud, Fadai-Ghotbi, Lesavre & Bach, 1977). The experiments reported here were undertaken to explore the conditions required to activate human lymphocytes by mouse cells in vitro and to determine the specificity of the responses produced.
INTRODUCTION Mixed lymphocyte cultures produce complex interactions between different classes of T lymphocytes and the sensitizing cell populations (Cantor & Boyse, 1975). At least two phenomena indicating specific sensitization of the responding cell population can be recognized, a rapid proliferative response to the original stimulating I region antigens and the generation of cytotoxic T cells which recognize the histocompatibility antigens of the stimulator cells (Peck, Alter & Lindahl, 1976). One of the major problems in studying this form of immune responsiveness in man is the necessity to bleed the stimulating cell donor on a number of occasions to provide cells for restimulation and cytotoxicity studies. This problem could be overcome if a convenient laboratory animal could be used as the stimulating antigen. Recent studies suggest that a xenogeneic mixed lymphocyte culture between
MATERIALS AND METHODS Media All cultures were performed in RPMI-1640 made from powdered stock supplied by Flow Laboratories. Stock 1640 adjusted to 0-01 M in HEPES buffer and 5 x 10-5m 2-mercaptoethanol and 20 mg/l gentamycin and 50 mg/l cloxacillin was added. This medium was supplemented with 6 ml of a nutrient cocktail composed of essential and non-essential amino acids, nucleic acid precursors, vitamins, sodium pyruvate and glutamine as described by Click, Benck & Alter (1972). For xenogeneic cultures RPMI-1640 cocktail (1640-C) was supplemented with 5 % decomplemented serum from the responder cell donor
(1640-C-NHS).
Correspondence: Dr A. W. Boylston, Department of Pathology, St Mary's Hospital Medical School, London W2 IPG. 0019-2805/78/0900-0455 $02.00 © 1978 Blackwell Scientific Publications
Animals Mice of BALB/c, Cs7 B16 and their F1 hybrid strains were bred in the animal house at St Mary's Hospital Medical School. 455
456
A. W. Boylston & Rene L. Anderson
Xenogeneic mixed lymphocyte cultures Human lymphocytes were prepared from blood by defibrination with glass beads followed by centrifugation at 200 g for 15 min. The serum was removed, the cells were resuspended in an equal volume of 1640 and layered over Ficoll-Hypaque (Lymphoprep-Nyegaard and Co., Oslo, Norway) and centrifuged at 400 g for 40 min at 20°. The cells at the interface were removed and washed twice in 1640 before culture. Mouse spleen cells were prepared by teasing the spleens into 1640 containing 10% foetal calf serum (FCS), the red cells were lysed by resuspending the cells in 0-17 M NH4Cl, 0-01 M Tris HCl pH 7-2 for 10 min, the cells were washed three times in 1640-FCS and exposed to 3000 rad. Then the mouse cells were washed once in 1640 without serum and resuspended in 1640-C-NHS. Experiments designed to measure proliferative responses were performed in flat-bottomed microtitre plates obtained from NUNC-UK, Hounslow, Middx. (catalogue no. N1480). Varying numbers of mouse stimulating cells were mixed with 2 x 105
human cells per well in a final volume of 0-2 ml 1640-C-NHS. Proliferation was measured by adding 10ul of a 100 pCi/ml solution of 3HTdR (Radiochemical Centre, Amersham, Bucks). Cells were harvested on a Skatron cell harvester, and 3HTdR incorporated measured in a scintillation counter. Large cultures for cytotoxicity studies were performed in Falcon flasks. Volumes from 12-18 ml were cultured in 25 cm2 flasks (no. 3012) lying flat, volumes from 25-50 ml were cultured in 75 cm2 flasks (no. 3024) standing upright, volumes of over 50 ml were cultured in 75 cm2 flasks lying flat. All cultures were performed at an initial concentration of 1 x 106 responder cells/ml, and 3 x 106 stimulating cells/ml in 1640-C-NHS unless otherwise indicated. Cytotoxicity was measured by a 31Cr release method as described by Brunner, Mauel, Rudolf & Chapuis (1970). Preliminary studies showed that the cytotoxicity response was maximal on days 7 to 10 inclusive and the experiments shown here were performed on day 7. All the cytotoxic responses shown were performed at a killer/target cell ratio of 33 : 1 unless otherwise stated. Attempts were made to inhibit cytotoxicity by adding IgG-anti-IgG immune complexes to the reaction. These were prepared by incubating 1 ml of goat-anti-rabbit gamma globulin serum (Calbiochem) containing approximately 1-5 mg of antibody
to rabbit IgG with 10 mg purified rabbit IgG (Pentex) at 370 for 4 h followed by storage overnight at 40. Ten microlitres of these antigen excess com-
plexes were added to killer cells in microtitre plates before the target cells were added. An IgM myeloma serum containing 15 mg/ml IgM was absorbed twice with mouse spleen cells and twice with unstimulated responder lymphocytes prior to addition of 10,ul to killer cells in microtitre plates. Killer cells and IgM-containing serum were incubated at 370 for 1 h before the target cells were added. Mouse tumour cell lines
El-4, a C57BI (H2-B) cell line, WEHI-22, a BALB/c (H2-D) cell line, L1210, a DBA 2 (H2-D, cell line and AKR-A, a H2-K cell line, were maintained in tissue culture as described (Boylston, Watson & Anderson, 1976). P815Y, a DBA-2 (H2-D) tumour, was maintained in the ascites form by serial intraperitoneal passage; Adj-PC-6, a BALB/c (H2-D) tumour, was maintained by serial subcutaneous passage. Where indicated, cultured human lymphocytes or P815Y ascites cells were filtered through nylon wool columns as described by Julius, Simpson & Herzenberg (1973).
RESULTS The ability of different numbers of mouse spleen cells to stimulate 3HTdR incorporation in human peripheral blood cells is shown in Table 1 and the time course of the response is shown in Fig. 1. Since stimulator/responder ratios of 2: 1 and 4: 1 were equivalent, all further studies were performed at a ratio of 3 : 1 and incorporation was measured on day 6 at the peak of the response. The ability of cells from different sources to stimulate human lymphoTable 1. Proliferative response of human lymphocytes to different numbers of mouse spleen cells
Stimulator/responder ratio c.p.m. 3HTdR ± 1 S.D. 8: 1 4: 1 2:1 1:1 0:1
3175 ±1335 8318 ± 1712 8207±69 7215±620 185±20
Human-mouse mixed lymphocyte cultures
457
Although there is a high degree of specificity towards cells bearing the same H2, K and D antigens as the sensitizing strain, in some experiments significant lysis of the control cells was also detected. As can be seen this non-specific lysis is at most one quarter of the specific cytotoxicity. In addition, the effect of different numbers of sensitizing cells on the generation of cytotoxic
20
0
responses is shown in Table 4, where it is seen that a stimulator/responder ratio of 3: 1 is the most
a
if0
3
5
4
6
7
Day Figure 1. Time course of 3HTdR incorporation by human lymphocytes stimulated with mouse spleen cells.
cytes was measured and the results are given in Table 2. Spleen cells produced the most effective stimulation, thymocytes produced weak proliferative responses, and cultured T-cell lines produced no response. Ascites cells from DBA2 mice carrying the P815Y mastocytoma stimulated as well as splenic cells but this response was abolished when the cells were filtered through nylon wool. The ability of human lymphocytes stimulated with BALB/c (H2D), C57B16 (H2B), or their F1 hybrid, CBF1 (H2B/D) spleen cells to kill various H2D (1210, WEHI-22, Adj PC6, P815Y), H2B (El-4), or H2K (AKRA) tumour target cells are shown in Table 3.
effective of those tested. Cultures undertaken at stimulator to responder ratios greater than 5 : 1 show no improvement in cytotoxicity. Attempts were made to ascertain the mechanism of the cytotoxicity generated in these cultures by inhibiting it with IgG-anti-IgG immune complexes, human IgM myeloma serum, or nylon wool filtration of the cultured cells. These experiments were intended to examine whether the cytotoxicity could be attributed to K cell killing, IgM-mediated T-cell cytotoxicity, or by cells adherent to nylon wool. None of these manipulations had any effect on the cytotoxicity developed following xenogeneic stimulation (Table 5). Finally cells stimulated by BALB/c lymphocytes were restimulated after 8 days in culture by spleen cells from different mouse strains. The results of a typical experiment are shown in Fig. 2 where it is seen that the cells from two H2D strains (BALB/c and DBA2) restimulate equally well, and that C57 cells produce no response. The results of restimulation experiments employing cells from five human donors are shown in Table 6. The results are shown as a stimulation index calculated as c.p.m. 3HTdR incorporated by human cells stimulated with
Table 2. Proliferative response of human lymphocytes to stimulation by mouse cells from different sources
Stimulating cell source
BALB/c spleen thymus C57 spleen thymus L1210 cultured EI-4 cultured P815 ascites P815 nylon wool filtered Responder cells alone
c.p.m. 3HTdR ± 1 S.D.
Stimulation index
21,066 ±2406 3505 ±1704 17,594 ± 744 3469 ±951 289 ± 35 289 ± 31 12,099 ±2446 1674 ±285 1401 ± 163
17 3 2-9 14-5 2-8
10-0 1-3
In all cases the c.p.m. incorporation by irradiated stimulator cells was less than 250 ± 46 c.p.m.
A. W. Boylston & Rene L. Anderson
458
Table 3. Cytotoxicity of human lymphocytes stimulated with mouse spleen cells for various mouse tumour cell lines (killer/target ratio 33: 1)
% specific lysis for various target cells* Responding cell donor
Stimulating strain
A
BALB/c
DBA2 C57
1210
P815
W22
El-4
AKR
18-0 8-3 32-5
-
-
40 0-01
-
3.9
0
20-0 20-0 350 18-0 24-0 0
0-2
300 0 0
210 13 -
1-2
-
CBF, B
BALB/c DBA2 BALB/c CBF1 BALB/c BALB/c C57
C
D E F
BALB/c
51*0 31-0 470 -
60-7 17 0 11-2
-
-
-
-
-
24-0 22-0 21-0
12-0
1.0 18 0
1*0
0 0-5 0
6-6 15-8 21-3 25-0 12-6 1*7
0-2 0 05 0 0 0
1-4 -
185 0
-
-
0 21-0
-
0
19 9
-
31-0 2-6
03 -
0005 8-0 9-2
1-3
0-75
0
-
-
* Background 5 Cr release was less than 7 % for all cell lines except P81 5Y which had a spontaneous release of between 10 and 15 % in these experiments. 1
Table 4. Generation of cytotoxic activity at different stimulator/responder cell ratios. (Human-anti-BALB/c culture assayed on 5'Cr L1210 cells)
Stimulator/responder ratio
Killer/target ratio
3:1
100:1 30: 1 10 : 1 100:1 30:1 10:1 100: 1 30:1
1:1 0-5 :1
Percentage lysis 59 45 27 11-7 6-6 3-4 0 0
459
Human-mouse mixed lymphocyte cultures
Table 5. Effect of nylon wool filtration, preincubation with IgG-anti-IgG immune complexes, or preincubation with an IgM myeloma serum on the cytotoxicity generated in human mouse mixed lymphocyte cultures (human-anti-BALB/c cultures assayed on 51'Cr labelled L1210 targets) c.p.m. Cr5 I released ± 1 S.D.
Treatment
Killer/target ratio
Control
Treated
100: 1 30:1 10: 1 100: 1 30:1 10:1 100: 1 30:1 10:1
2954 ±41
3666±400 2207 ±45 1673±6
3340 + 51 2580+87 1321 88 9796 ± 252 6647±89 3370±137 2339 ± 68 1765±3
1193±18
1264±26
Nylon wool filtration
IgG-anti-IgG complexes Preincubated with IgM
2156±104 1251 30 10,377 i 660
7122±258
Table 6. Restimulation of human-anti-mouse mixed lymphocyte culture cells by spleen cells from various mouse strains (stimulator/responder ratio 2: 1)
Stimulation index-restimulating strain Donor
Stimulating strain
A
BALB/c
CBF1 C57 AKR C
BALB/c AKR
D F
G
BALB/c BALB/c C57 BALB/c
B
A
41-0 91
9-6
7-2 12-3 12 4 0-66 05 045 11-8 1-22 21-0 12-5 7-6 0-8 100-0
DBA2
-
90 2-2
3-12 0-7 0-8 3-2
AKR
C57
56
-
09 2-7
0-8
11-2 30
36-0 -
-
CBA
-
-
-
0-8
0-61 -
3-7
07
-
4.5 -
-
-
-
13-2
-
1.1
-
-
-
9*7
11-8
89-0
-
5-77
--
-
1 5 2-3
-
3-8 1 1
-
12-4 -
11 16-0
-
CBF1
-
-
-
A. W. Boylston & Rene L. Anderson
460 7
5 x c
0 0
Ec
cf
3
T7
0
CBA DBA2 BALB C57 Figure 2. Restimulation of human lymphocytes cultured with BALB/c spleen cells by spleen cells from BALB/c, DBA2 (both H2 and D), C57B1 (H2-B) or CBA (H2-K). Results are shown as the stimulation index calculated as c.p.m. stimulated cells c.p.m. unstimulated cells alone
the initial strain spleen cells when restimulated with cells from various restimulating strains/c.p.m. 3HTdR incorporated by unrestimulated cells from the same initial culture. Using this calculation a stimulation index of 1 means there was no difference between cultures containing cells of that mouse strain and human cells alone; indices less than 1 indicate that fewer counts were incorporated in the cultures containing mouse cells than in controls. Although cells bearing the antigens used to stimulate the primary culture always induced the most pronounced restimulation in secondary cultures there was often significant isotope incorporation in cultures exposed to cells from control strains. This was often as high as 25-30% of the specific response and in one experiment amounted to 50 % of the specific stimulation. DISCUSSION The experiments described here confirm the observations of Lindahl & Bach (1975; 1976) and of Carnaud
et al. (1977) that human lymphocytes can respond to the major histocompatibility antigens of mice. Our results differ from those of Carnaud et al. (1977) in that we were unable to stimulate proliferative responses to mouse tumour cell lines. The reason for this discrepancy appears to be the use of ascites tumour cells by Carnaud et al. (1977) which are contaminated with lymphocytes and macrophages from the host. Although P815Y ascites cells produced stimulation in our hands, this was abrogated by absorbing the cells on a nylon wool column, a procedure which removes most of the B lymphocytes and macrophages. Since thymocytes and mouse tumour cell lines known to possess the K and D region antigens, but not the I region antigens of the H2 region do not stimulate human lymphocytes and spleen cell suspensions which contain cells with all these antigens are stimulatory, our results suggest that human cells are indeed stimulated by mouse I region antigens as shown by Peck et al. (1976). Following stimulation, human lymphocytes acquire the ability to kill cells bearing the K and D region antigens of the stimulating strain. This cytotoxic activity appears to be true T-cell cytotoxicity since it cannot be inhibited by human IgM myeloma serum or by IgG-anti-IgG immune complexes. This finding indicates that the observed cytotoxicity is not due to IgM passively adsorbed by T cells either from the donor serum in the culture or produced by B lymphocytes responding in the culture since Fuson & Lamon (1977) showed that IgM induced T-lymphocyte mediated cytotoxicity could be inhibited by incubating T cells with non-specific IgM. Failure of IgG-anti-IgG immune complexes to inhibit indicates that cytotoxicity is not due to a K-cell type mechanism and failure of nylon wool filtration to have an effect indicates that B lymphocytes and macrophages are not involved. The experiments shown here indicate a high degree of strain specificity in both the cytotoxic and proliferative response of human lymphocytes to mouse antigens. However, in some experiments these responses are not absolute and a lesser degree of cytotoxicity or proliferation towards cells bearing antigens other than those of the initial strain can be detected. Non-specific cytotoxicity, which is never more than one quarter of the specific response, might be due to polyclonal activation of human cells similar to that which occurs in mitogen-stimulated cultures so that cells capable of recognizing antigens other than those on the stimulating strain are acti-
Human-mouse mixed lymphocyte cultures vated (Nelson, Bundy, Pitchon, Blaese & Strober, 1976). Alternatively, non-specificity may be due to recognition of species-specific antigens, or possibly even viral antigen since the tumour target cells may all be infected with similar mouse tumour viruses. The non-specific component of the restimulated proliferative response seen in some experiments may have several explanations. It may represent recognition of species-specific non-H2 antigens, species-specific determinants on the H2 antigens themselves, cross reactions between strain-specific antigens detected by human cells, or possibly 'back stimulation' of human cells by products of the irradiated stimulator cells which can recognize, but not proliferate, in response to human cells. Experiments not shown here indicate that the cellular requirements for restimulation are similar to those for initial stimulation and suggest that I region bearing cells may also be important in whichever explanation of non-specific as well as specific H2 restimulation is correct. Xenogeneic mixed lymphocyte cultures may provide a useful model for studying human lymphocyte responses in vitro. Because the response and subsequent development of cytotoxicity are probably the result of complex interactions between subclasses of T lymphocytes this model may prove a useful probe of clinical immunodeficiency states. Furthermore, it opens the prospect of producing long term secondary cultures of specific xenoantigen-selected human lymphocytes for studies of specific membrane properties and biological activities (Hayry & Andersson, 1974; Dennert & Raschke, 1977). Finally these studies raise the intriguing question why and how should human lymphocytes recognize the same specific mouse alloantigens that mice recognize ? ACKNOWLEDGMENTS
This work was supported by a grant from the Medical Research Council. Dr A. W. Boylston is a Wellcome Senior Research Fellow. Dr M. C.
c
461
Berenbaum is thanked for the use of laboratory facilities. REFEREN CES BOYLSTON A.W., WATSON S.R. & ANDERSON R.L. (1976) Mouse T-cell tumour immunoglobulin. I. Antigenic properties and effects on T-cell responses. Immunology, 31, 827. BRUNNER K.T., MAUEL J., RUDOLF H. & CHAPUIs B. (1970) Studies of allograft immunity in mice. I. Induction, development and in vitro assay of cellular immunity. Immunology, 18, 501. CANTOR H. & BOYSE E.A. (1975) Functional subclasses of T lymphocytes bearing different Ly antigens. II. Cooperation between subclasses of Ly+ cells in the generation of killer activity. J. exp. Med. 141, 1390. CARNAUD C., FADAT-GHOTBI M., LESAVRE P. & BACH J.F. (1977) Education of human lymphocytes against mouse cells: specific recognition of H-2 antigens. Eur. J. Immunol. 7, 81. CLICK R.E., BENCK L. & ALTER B.J. (1972) Immune responses in vitro. I. Culture conditions for antibody synthesis. Cellular Immunol. 3, 264. DENNERT G. & RASCHKE W. (1977) Continuously proliferating allospecific T cells, lifespan and antigen receptors. Eur. J. Immunol. 7, 352. FuSON E.W. & LAMON E.W. (1977) IgM-induced cellmediated cytotoxicity with antibody and effector cells of human origin. J. Immunol. 118, 1977. HXYRY P. & ANDERSSON L.C. (1974) Generation of T memory cells in one-way mixed lymphocyte culture. II. Anamnestic responses of 'secondary' lymphocytes. J. Immunol. 3, 823. JULIUS M.H., SIMPSON E. & HERZENBERG L.A. (1973) A rapid method for the isolation of functional thymus derived murine lymphocytes. Eur. J. Immunol. 3, 645. LINDAHL K.F. & BACH F.H. (1975) Human lymphocytes recognize mouse alloantigens. Nature (Lond.), 254, 607. LINDAHL K.F. & BACH F.H. (1976) Genetic and cellular aspects of xenogenic mixed lymphocyte reactions. J. exp. Med. 144, 305. NELSON D.L., BUNDY B.M., PITCHON H.E., BLAESE R.M. & STROBER W. (1976) The effector cells in human peripheral blood mediating mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity. J. Immunol. 117, 1472. PECK A.B., ALTER B.J. & LINDAHL K.F. (1976) Specificity in T cell mediated lympholysis: identical genetic control of the proliferative and effector phases of allogeneic and xenogeneic reactions. Transplant. Rev. 29, 189.
Human-mouse mixed lymphocyte cultures
A. W. BOYLSTON & RENE L. ANDERSON Department ofPathology, St Mary's Hospital Medical School, London
Received 24 October 1977; acceptedfor publication 23 January 1978
Summary. Conditions for stimulating human lymphocytes with mouse cells are established. The proliferative and cytotoxic responses generated are shown to be specific for antigens coded for in the major histocompatibility locus.
human and mouse cells may provide a suitable model for immunizing human lymphocytes in vitro (Lindahl & Bach, 1975; Carnaud, Fadai-Ghotbi, Lesavre & Bach, 1977). The experiments reported here were undertaken to explore the conditions required to activate human lymphocytes by mouse cells in vitro and to determine the specificity of the responses produced.
INTRODUCTION Mixed lymphocyte cultures produce complex interactions between different classes of T lymphocytes and the sensitizing cell populations (Cantor & Boyse, 1975). At least two phenomena indicating specific sensitization of the responding cell population can be recognized, a rapid proliferative response to the original stimulating I region antigens and the generation of cytotoxic T cells which recognize the histocompatibility antigens of the stimulator cells (Peck, Alter & Lindahl, 1976). One of the major problems in studying this form of immune responsiveness in man is the necessity to bleed the stimulating cell donor on a number of occasions to provide cells for restimulation and cytotoxicity studies. This problem could be overcome if a convenient laboratory animal could be used as the stimulating antigen. Recent studies suggest that a xenogeneic mixed lymphocyte culture between
MATERIALS AND METHODS Media All cultures were performed in RPMI-1640 made from powdered stock supplied by Flow Laboratories. Stock 1640 adjusted to 0-01 M in HEPES buffer and 5 x 10-5m 2-mercaptoethanol and 20 mg/l gentamycin and 50 mg/l cloxacillin was added. This medium was supplemented with 6 ml of a nutrient cocktail composed of essential and non-essential amino acids, nucleic acid precursors, vitamins, sodium pyruvate and glutamine as described by Click, Benck & Alter (1972). For xenogeneic cultures RPMI-1640 cocktail (1640-C) was supplemented with 5 % decomplemented serum from the responder cell donor
(1640-C-NHS).
Correspondence: Dr A. W. Boylston, Department of Pathology, St Mary's Hospital Medical School, London W2 IPG. 0019-2805/78/0900-0455 $02.00 © 1978 Blackwell Scientific Publications
Animals Mice of BALB/c, Cs7 B16 and their F1 hybrid strains were bred in the animal house at St Mary's Hospital Medical School. 455
456
A. W. Boylston & Rene L. Anderson
Xenogeneic mixed lymphocyte cultures Human lymphocytes were prepared from blood by defibrination with glass beads followed by centrifugation at 200 g for 15 min. The serum was removed, the cells were resuspended in an equal volume of 1640 and layered over Ficoll-Hypaque (Lymphoprep-Nyegaard and Co., Oslo, Norway) and centrifuged at 400 g for 40 min at 20°. The cells at the interface were removed and washed twice in 1640 before culture. Mouse spleen cells were prepared by teasing the spleens into 1640 containing 10% foetal calf serum (FCS), the red cells were lysed by resuspending the cells in 0-17 M NH4Cl, 0-01 M Tris HCl pH 7-2 for 10 min, the cells were washed three times in 1640-FCS and exposed to 3000 rad. Then the mouse cells were washed once in 1640 without serum and resuspended in 1640-C-NHS. Experiments designed to measure proliferative responses were performed in flat-bottomed microtitre plates obtained from NUNC-UK, Hounslow, Middx. (catalogue no. N1480). Varying numbers of mouse stimulating cells were mixed with 2 x 105
human cells per well in a final volume of 0-2 ml 1640-C-NHS. Proliferation was measured by adding 10ul of a 100 pCi/ml solution of 3HTdR (Radiochemical Centre, Amersham, Bucks). Cells were harvested on a Skatron cell harvester, and 3HTdR incorporated measured in a scintillation counter. Large cultures for cytotoxicity studies were performed in Falcon flasks. Volumes from 12-18 ml were cultured in 25 cm2 flasks (no. 3012) lying flat, volumes from 25-50 ml were cultured in 75 cm2 flasks (no. 3024) standing upright, volumes of over 50 ml were cultured in 75 cm2 flasks lying flat. All cultures were performed at an initial concentration of 1 x 106 responder cells/ml, and 3 x 106 stimulating cells/ml in 1640-C-NHS unless otherwise indicated. Cytotoxicity was measured by a 31Cr release method as described by Brunner, Mauel, Rudolf & Chapuis (1970). Preliminary studies showed that the cytotoxicity response was maximal on days 7 to 10 inclusive and the experiments shown here were performed on day 7. All the cytotoxic responses shown were performed at a killer/target cell ratio of 33 : 1 unless otherwise stated. Attempts were made to inhibit cytotoxicity by adding IgG-anti-IgG immune complexes to the reaction. These were prepared by incubating 1 ml of goat-anti-rabbit gamma globulin serum (Calbiochem) containing approximately 1-5 mg of antibody
to rabbit IgG with 10 mg purified rabbit IgG (Pentex) at 370 for 4 h followed by storage overnight at 40. Ten microlitres of these antigen excess com-
plexes were added to killer cells in microtitre plates before the target cells were added. An IgM myeloma serum containing 15 mg/ml IgM was absorbed twice with mouse spleen cells and twice with unstimulated responder lymphocytes prior to addition of 10,ul to killer cells in microtitre plates. Killer cells and IgM-containing serum were incubated at 370 for 1 h before the target cells were added. Mouse tumour cell lines
El-4, a C57BI (H2-B) cell line, WEHI-22, a BALB/c (H2-D) cell line, L1210, a DBA 2 (H2-D, cell line and AKR-A, a H2-K cell line, were maintained in tissue culture as described (Boylston, Watson & Anderson, 1976). P815Y, a DBA-2 (H2-D) tumour, was maintained in the ascites form by serial intraperitoneal passage; Adj-PC-6, a BALB/c (H2-D) tumour, was maintained by serial subcutaneous passage. Where indicated, cultured human lymphocytes or P815Y ascites cells were filtered through nylon wool columns as described by Julius, Simpson & Herzenberg (1973).
RESULTS The ability of different numbers of mouse spleen cells to stimulate 3HTdR incorporation in human peripheral blood cells is shown in Table 1 and the time course of the response is shown in Fig. 1. Since stimulator/responder ratios of 2: 1 and 4: 1 were equivalent, all further studies were performed at a ratio of 3 : 1 and incorporation was measured on day 6 at the peak of the response. The ability of cells from different sources to stimulate human lymphoTable 1. Proliferative response of human lymphocytes to different numbers of mouse spleen cells
Stimulator/responder ratio c.p.m. 3HTdR ± 1 S.D. 8: 1 4: 1 2:1 1:1 0:1
3175 ±1335 8318 ± 1712 8207±69 7215±620 185±20
Human-mouse mixed lymphocyte cultures
457
Although there is a high degree of specificity towards cells bearing the same H2, K and D antigens as the sensitizing strain, in some experiments significant lysis of the control cells was also detected. As can be seen this non-specific lysis is at most one quarter of the specific cytotoxicity. In addition, the effect of different numbers of sensitizing cells on the generation of cytotoxic
20
0
responses is shown in Table 4, where it is seen that a stimulator/responder ratio of 3: 1 is the most
a
if0
3
5
4
6
7
Day Figure 1. Time course of 3HTdR incorporation by human lymphocytes stimulated with mouse spleen cells.
cytes was measured and the results are given in Table 2. Spleen cells produced the most effective stimulation, thymocytes produced weak proliferative responses, and cultured T-cell lines produced no response. Ascites cells from DBA2 mice carrying the P815Y mastocytoma stimulated as well as splenic cells but this response was abolished when the cells were filtered through nylon wool. The ability of human lymphocytes stimulated with BALB/c (H2D), C57B16 (H2B), or their F1 hybrid, CBF1 (H2B/D) spleen cells to kill various H2D (1210, WEHI-22, Adj PC6, P815Y), H2B (El-4), or H2K (AKRA) tumour target cells are shown in Table 3.
effective of those tested. Cultures undertaken at stimulator to responder ratios greater than 5 : 1 show no improvement in cytotoxicity. Attempts were made to ascertain the mechanism of the cytotoxicity generated in these cultures by inhibiting it with IgG-anti-IgG immune complexes, human IgM myeloma serum, or nylon wool filtration of the cultured cells. These experiments were intended to examine whether the cytotoxicity could be attributed to K cell killing, IgM-mediated T-cell cytotoxicity, or by cells adherent to nylon wool. None of these manipulations had any effect on the cytotoxicity developed following xenogeneic stimulation (Table 5). Finally cells stimulated by BALB/c lymphocytes were restimulated after 8 days in culture by spleen cells from different mouse strains. The results of a typical experiment are shown in Fig. 2 where it is seen that the cells from two H2D strains (BALB/c and DBA2) restimulate equally well, and that C57 cells produce no response. The results of restimulation experiments employing cells from five human donors are shown in Table 6. The results are shown as a stimulation index calculated as c.p.m. 3HTdR incorporated by human cells stimulated with
Table 2. Proliferative response of human lymphocytes to stimulation by mouse cells from different sources
Stimulating cell source
BALB/c spleen thymus C57 spleen thymus L1210 cultured EI-4 cultured P815 ascites P815 nylon wool filtered Responder cells alone
c.p.m. 3HTdR ± 1 S.D.
Stimulation index
21,066 ±2406 3505 ±1704 17,594 ± 744 3469 ±951 289 ± 35 289 ± 31 12,099 ±2446 1674 ±285 1401 ± 163
17 3 2-9 14-5 2-8
10-0 1-3
In all cases the c.p.m. incorporation by irradiated stimulator cells was less than 250 ± 46 c.p.m.
A. W. Boylston & Rene L. Anderson
458
Table 3. Cytotoxicity of human lymphocytes stimulated with mouse spleen cells for various mouse tumour cell lines (killer/target ratio 33: 1)
% specific lysis for various target cells* Responding cell donor
Stimulating strain
A
BALB/c
DBA2 C57
1210
P815
W22
El-4
AKR
18-0 8-3 32-5
-
-
40 0-01
-
3.9
0
20-0 20-0 350 18-0 24-0 0
0-2
300 0 0
210 13 -
1-2
-
CBF, B
BALB/c DBA2 BALB/c CBF1 BALB/c BALB/c C57
C
D E F
BALB/c
51*0 31-0 470 -
60-7 17 0 11-2
-
-
-
-
-
24-0 22-0 21-0
12-0
1.0 18 0
1*0
0 0-5 0
6-6 15-8 21-3 25-0 12-6 1*7
0-2 0 05 0 0 0
1-4 -
185 0
-
-
0 21-0
-
0
19 9
-
31-0 2-6
03 -
0005 8-0 9-2
1-3
0-75
0
-
-
* Background 5 Cr release was less than 7 % for all cell lines except P81 5Y which had a spontaneous release of between 10 and 15 % in these experiments. 1
Table 4. Generation of cytotoxic activity at different stimulator/responder cell ratios. (Human-anti-BALB/c culture assayed on 5'Cr L1210 cells)
Stimulator/responder ratio
Killer/target ratio
3:1
100:1 30: 1 10 : 1 100:1 30:1 10:1 100: 1 30:1
1:1 0-5 :1
Percentage lysis 59 45 27 11-7 6-6 3-4 0 0
459
Human-mouse mixed lymphocyte cultures
Table 5. Effect of nylon wool filtration, preincubation with IgG-anti-IgG immune complexes, or preincubation with an IgM myeloma serum on the cytotoxicity generated in human mouse mixed lymphocyte cultures (human-anti-BALB/c cultures assayed on 51'Cr labelled L1210 targets) c.p.m. Cr5 I released ± 1 S.D.
Treatment
Killer/target ratio
Control
Treated
100: 1 30:1 10: 1 100: 1 30:1 10:1 100: 1 30:1 10:1
2954 ±41
3666±400 2207 ±45 1673±6
3340 + 51 2580+87 1321 88 9796 ± 252 6647±89 3370±137 2339 ± 68 1765±3
1193±18
1264±26
Nylon wool filtration
IgG-anti-IgG complexes Preincubated with IgM
2156±104 1251 30 10,377 i 660
7122±258
Table 6. Restimulation of human-anti-mouse mixed lymphocyte culture cells by spleen cells from various mouse strains (stimulator/responder ratio 2: 1)
Stimulation index-restimulating strain Donor
Stimulating strain
A
BALB/c
CBF1 C57 AKR C
BALB/c AKR
D F
G
BALB/c BALB/c C57 BALB/c
B
A
41-0 91
9-6
7-2 12-3 12 4 0-66 05 045 11-8 1-22 21-0 12-5 7-6 0-8 100-0
DBA2
-
90 2-2
3-12 0-7 0-8 3-2
AKR
C57
56
-
09 2-7
0-8
11-2 30
36-0 -
-
CBA
-
-
-
0-8
0-61 -
3-7
07
-
4.5 -
-
-
-
13-2
-
1.1
-
-
-
9*7
11-8
89-0
-
5-77
--
-
1 5 2-3
-
3-8 1 1
-
12-4 -
11 16-0
-
CBF1
-
-
-
A. W. Boylston & Rene L. Anderson
460 7
5 x c
0 0
Ec
cf
3
T7
0
CBA DBA2 BALB C57 Figure 2. Restimulation of human lymphocytes cultured with BALB/c spleen cells by spleen cells from BALB/c, DBA2 (both H2 and D), C57B1 (H2-B) or CBA (H2-K). Results are shown as the stimulation index calculated as c.p.m. stimulated cells c.p.m. unstimulated cells alone
the initial strain spleen cells when restimulated with cells from various restimulating strains/c.p.m. 3HTdR incorporated by unrestimulated cells from the same initial culture. Using this calculation a stimulation index of 1 means there was no difference between cultures containing cells of that mouse strain and human cells alone; indices less than 1 indicate that fewer counts were incorporated in the cultures containing mouse cells than in controls. Although cells bearing the antigens used to stimulate the primary culture always induced the most pronounced restimulation in secondary cultures there was often significant isotope incorporation in cultures exposed to cells from control strains. This was often as high as 25-30% of the specific response and in one experiment amounted to 50 % of the specific stimulation. DISCUSSION The experiments described here confirm the observations of Lindahl & Bach (1975; 1976) and of Carnaud
et al. (1977) that human lymphocytes can respond to the major histocompatibility antigens of mice. Our results differ from those of Carnaud et al. (1977) in that we were unable to stimulate proliferative responses to mouse tumour cell lines. The reason for this discrepancy appears to be the use of ascites tumour cells by Carnaud et al. (1977) which are contaminated with lymphocytes and macrophages from the host. Although P815Y ascites cells produced stimulation in our hands, this was abrogated by absorbing the cells on a nylon wool column, a procedure which removes most of the B lymphocytes and macrophages. Since thymocytes and mouse tumour cell lines known to possess the K and D region antigens, but not the I region antigens of the H2 region do not stimulate human lymphocytes and spleen cell suspensions which contain cells with all these antigens are stimulatory, our results suggest that human cells are indeed stimulated by mouse I region antigens as shown by Peck et al. (1976). Following stimulation, human lymphocytes acquire the ability to kill cells bearing the K and D region antigens of the stimulating strain. This cytotoxic activity appears to be true T-cell cytotoxicity since it cannot be inhibited by human IgM myeloma serum or by IgG-anti-IgG immune complexes. This finding indicates that the observed cytotoxicity is not due to IgM passively adsorbed by T cells either from the donor serum in the culture or produced by B lymphocytes responding in the culture since Fuson & Lamon (1977) showed that IgM induced T-lymphocyte mediated cytotoxicity could be inhibited by incubating T cells with non-specific IgM. Failure of IgG-anti-IgG immune complexes to inhibit indicates that cytotoxicity is not due to a K-cell type mechanism and failure of nylon wool filtration to have an effect indicates that B lymphocytes and macrophages are not involved. The experiments shown here indicate a high degree of strain specificity in both the cytotoxic and proliferative response of human lymphocytes to mouse antigens. However, in some experiments these responses are not absolute and a lesser degree of cytotoxicity or proliferation towards cells bearing antigens other than those of the initial strain can be detected. Non-specific cytotoxicity, which is never more than one quarter of the specific response, might be due to polyclonal activation of human cells similar to that which occurs in mitogen-stimulated cultures so that cells capable of recognizing antigens other than those on the stimulating strain are acti-
Human-mouse mixed lymphocyte cultures vated (Nelson, Bundy, Pitchon, Blaese & Strober, 1976). Alternatively, non-specificity may be due to recognition of species-specific antigens, or possibly even viral antigen since the tumour target cells may all be infected with similar mouse tumour viruses. The non-specific component of the restimulated proliferative response seen in some experiments may have several explanations. It may represent recognition of species-specific non-H2 antigens, species-specific determinants on the H2 antigens themselves, cross reactions between strain-specific antigens detected by human cells, or possibly 'back stimulation' of human cells by products of the irradiated stimulator cells which can recognize, but not proliferate, in response to human cells. Experiments not shown here indicate that the cellular requirements for restimulation are similar to those for initial stimulation and suggest that I region bearing cells may also be important in whichever explanation of non-specific as well as specific H2 restimulation is correct. Xenogeneic mixed lymphocyte cultures may provide a useful model for studying human lymphocyte responses in vitro. Because the response and subsequent development of cytotoxicity are probably the result of complex interactions between subclasses of T lymphocytes this model may prove a useful probe of clinical immunodeficiency states. Furthermore, it opens the prospect of producing long term secondary cultures of specific xenoantigen-selected human lymphocytes for studies of specific membrane properties and biological activities (Hayry & Andersson, 1974; Dennert & Raschke, 1977). Finally these studies raise the intriguing question why and how should human lymphocytes recognize the same specific mouse alloantigens that mice recognize ? ACKNOWLEDGMENTS
This work was supported by a grant from the Medical Research Council. Dr A. W. Boylston is a Wellcome Senior Research Fellow. Dr M. C.
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Berenbaum is thanked for the use of laboratory facilities. REFEREN CES BOYLSTON A.W., WATSON S.R. & ANDERSON R.L. (1976) Mouse T-cell tumour immunoglobulin. I. Antigenic properties and effects on T-cell responses. Immunology, 31, 827. BRUNNER K.T., MAUEL J., RUDOLF H. & CHAPUIs B. (1970) Studies of allograft immunity in mice. I. Induction, development and in vitro assay of cellular immunity. Immunology, 18, 501. CANTOR H. & BOYSE E.A. (1975) Functional subclasses of T lymphocytes bearing different Ly antigens. II. Cooperation between subclasses of Ly+ cells in the generation of killer activity. J. exp. Med. 141, 1390. CARNAUD C., FADAT-GHOTBI M., LESAVRE P. & BACH J.F. (1977) Education of human lymphocytes against mouse cells: specific recognition of H-2 antigens. Eur. J. Immunol. 7, 81. CLICK R.E., BENCK L. & ALTER B.J. (1972) Immune responses in vitro. I. Culture conditions for antibody synthesis. Cellular Immunol. 3, 264. DENNERT G. & RASCHKE W. (1977) Continuously proliferating allospecific T cells, lifespan and antigen receptors. Eur. J. Immunol. 7, 352. FuSON E.W. & LAMON E.W. (1977) IgM-induced cellmediated cytotoxicity with antibody and effector cells of human origin. J. Immunol. 118, 1977. HXYRY P. & ANDERSSON L.C. (1974) Generation of T memory cells in one-way mixed lymphocyte culture. II. Anamnestic responses of 'secondary' lymphocytes. J. Immunol. 3, 823. JULIUS M.H., SIMPSON E. & HERZENBERG L.A. (1973) A rapid method for the isolation of functional thymus derived murine lymphocytes. Eur. J. Immunol. 3, 645. LINDAHL K.F. & BACH F.H. (1975) Human lymphocytes recognize mouse alloantigens. Nature (Lond.), 254, 607. LINDAHL K.F. & BACH F.H. (1976) Genetic and cellular aspects of xenogenic mixed lymphocyte reactions. J. exp. Med. 144, 305. NELSON D.L., BUNDY B.M., PITCHON H.E., BLAESE R.M. & STROBER W. (1976) The effector cells in human peripheral blood mediating mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity. J. Immunol. 117, 1472. PECK A.B., ALTER B.J. & LINDAHL K.F. (1976) Specificity in T cell mediated lympholysis: identical genetic control of the proliferative and effector phases of allogeneic and xenogeneic reactions. Transplant. Rev. 29, 189.
