Clin. exp. Immunol. (1977) 27, 322-327
Generation of T-cell function in organ culture of foetal mouse thymus II. MIXED LYMPHOCYTE CULTURE REACTIVITY J. H. ROB INSON * & J. J. T. OWEN Department of Anatomy, Medical School, University of Newcastle-upon-Tyne
(Received 23 September 1976) SUMMARY
The generation of MLC reactive cells in organ cultures of embryonic mouse thymus is described. At least 4 days in culture are required before MLC reactive thymocytes are detectable in this system, which is in marked contrast to the short incubation period required for the expression of Thy-1 on T-cell precursors in other systems. Our data demonstrates the generation of T-cell subsets independent of influences from peripheral lymphoid organs, and in the absence of B cells or their products. One very important application of this technique is in the investigation of the mechanism of T-cell tolerance to self.
INTRODUCTION Many important questions concerning the ontogeny of T lymphocytes remain unsolved. Some of these may be defined as follows:-Are the various T-cell subsets, found in peripheral lymphoid organs (Cantor & Weissman, 1976), generated within the thymus itself, or does the full diversification and maturation of T cells depend upon the presence of B cells or their products? Do T cells pass through a stage of maturation when they can readily be rendered tolerant in a comparable manner to that shown recently in maturing B cells (Metcalf & Klinman, 1976). We have argued that a system in which functional T-cell subsets could be generated in vitro within an embryo thymus, which initially contained only immature stem cells, would provide a powerful tool not only for answering questions such as those posed above, but also for studying the factors which regulate thymopoiesis. To study these questions we have used therefore the technique of embryonic thymus organ culture originally introduced by Auerbach to study thymus lymphocyte origins (Auerbach, 1960). In a previous paper we have shown that cells responsive to the polyclonal mitogens PHA, Con A and pokeweed mitogen can be generated in organ cultures of foetal mouse thymus (Robinson & Owen, 1976). Here we show that T cells recognizing and reacting to allogeneic determinants in a mixed lymphocyte culture (MLC) can be generated in embryo thymus organ culture. MATERIALS AND METHODS (1) Mice. C57BL/6 and BALB/c mice were mated in trios and pregnancy was timed from the appearance of vaginal plugs (day 0 gestation). Spleen cell suspensions from CBA/H mice were used in MLC. These three strains of mice are M-locus compatible but differ from each other at K, D and I regions of the MHC. (ii) Organ culture. The organ culture technique has been described in detail (Robinson & Owen, 1976). Briefly thymuses were removed from embryonic mice at 14 or 15 days gestation, and were cultured on Nuclepore filters (type N40, 01300 CPR), floating on RPMI 1640 supplemented with 10% foetal calf serum (RF10), in the centre of Falcon organ culture J. H. Robinson is in receipt of an MRC Student Award. Correspondence: Dr J. H. Robinson, Department of Anatomy, Medical School, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne. *
322
323
In vitro ontogeny of MLC reactivity
dishes (catalogue number 3010). Several explants were placed on one filter and cultures were incubated at 370C in a humid atmosphere of 5% CO2 in air. (iii) MLC. After different periods of organ culture, foetal thymuses were teased apart and the resulting lymphoid cell suspensions were used as the responding population in MLC. One-way mixed lymphocyte cultures were set up in microculture using 2 x 105 responding and stimulating cells in a total of 0-2 ml of RF10 including 5 x 10- 5 M 2-mercaptoethanol. Mitomycin-C-treated spleen cell suspensions (spleenm) from adult BALB/c, C57BL/6, CBA/H mice as well as from the three F1 hybrids between these strains, were used as the stimulating cells. Erythrocytes were removed from spleen cell suspensions by hypotonic shock and cells at 20 x 106/ml were incubated for 30 min at 37°C with 30 pg/ml mitomycin-c (Difco). Treated cells were washed three times in RF10 prior to use. Cultures were set up in duplicate or triplicate in Falcon tissue culture tubes (catalogue number 2054) and incubated for 5 days at 37°C in 5% CO2 in air. DNA synthesis was assayed by the incorporation of 125I-labelled iododeoxyuridine ([125I]lUdR) into DNA as described previously (Robinson & Owen, 1976). Allogeneic mixed lymphocyte reactivity was expressed as a ratio of ct/min. in allogeneic MLC (cultured thymus plus allogeneic adult spleenm) to ct/min in syngeneic MLC (cultured thymus plus syngeneic adult spleenm). The significance (P value) of allogeneic stimulation was assessed using Student's t-test.
RESULTS (i) Cell yields from organ culture We have reported previously (Robinson & Owen, 1976) that in a 7-day culture period, the lymphoid cell yield from 14-day thymuses increases from around 35 x 103 large basophilic cells before culture to 200-500 x 103 viable small and medium lymphoid cells per embryo. The cell yield profile is monophasic and after the peak at 7 days culture, cell yield steadily decreases, although viable cells are still present after 28 days of culture. It has been shown previously that the vast majority of these cells are Thy-i positive (Owen, Raff & Cooper, 1975). TABLE 1. BALB/c cultured thymus responses in MLC to adult BALB/c and CBA/H spleenm
Mean ct/min+ s.e.m.
Culture period* 0 3 5 7
9
11
Syngeneic
Allogeneic
Ratio
271+13 144±136 186+46 415+ 125 406+430 227+45 349+212 1073+88 1035+746 90+79 637+ 99 542+ 106 438+4
469+236 8+ 35 253+ 30 511+235 622+ 180
1-7 0-06 1-4 1-2 1-5 0 75 026 3-1 6-1 10-6 6-7 10-6 8-7
1420+285 13
548+568
45±71 15 Uncultured 19 days gestation
691+ 125 2126+352 442+ 132
171+23t 91+81 3353+162 6285+ 1112 954± 138t 4296+ 507 5758+ 2180 3807+896 13735+ 1226 3234+801 517+ 138t 2335+947 11129+ 1191t 10908+1217
9.7 59 11-5 3-4 5-3 24-7
P value > 0-1 > 0-1
>0.1 >0.1 > 0-1 >0.1 >0-1
Generation of T-cell function in organ culture of foetal mouse thymus II. MIXED LYMPHOCYTE CULTURE REACTIVITY J. H. ROB INSON * & J. J. T. OWEN Department of Anatomy, Medical School, University of Newcastle-upon-Tyne
(Received 23 September 1976) SUMMARY
The generation of MLC reactive cells in organ cultures of embryonic mouse thymus is described. At least 4 days in culture are required before MLC reactive thymocytes are detectable in this system, which is in marked contrast to the short incubation period required for the expression of Thy-1 on T-cell precursors in other systems. Our data demonstrates the generation of T-cell subsets independent of influences from peripheral lymphoid organs, and in the absence of B cells or their products. One very important application of this technique is in the investigation of the mechanism of T-cell tolerance to self.
INTRODUCTION Many important questions concerning the ontogeny of T lymphocytes remain unsolved. Some of these may be defined as follows:-Are the various T-cell subsets, found in peripheral lymphoid organs (Cantor & Weissman, 1976), generated within the thymus itself, or does the full diversification and maturation of T cells depend upon the presence of B cells or their products? Do T cells pass through a stage of maturation when they can readily be rendered tolerant in a comparable manner to that shown recently in maturing B cells (Metcalf & Klinman, 1976). We have argued that a system in which functional T-cell subsets could be generated in vitro within an embryo thymus, which initially contained only immature stem cells, would provide a powerful tool not only for answering questions such as those posed above, but also for studying the factors which regulate thymopoiesis. To study these questions we have used therefore the technique of embryonic thymus organ culture originally introduced by Auerbach to study thymus lymphocyte origins (Auerbach, 1960). In a previous paper we have shown that cells responsive to the polyclonal mitogens PHA, Con A and pokeweed mitogen can be generated in organ cultures of foetal mouse thymus (Robinson & Owen, 1976). Here we show that T cells recognizing and reacting to allogeneic determinants in a mixed lymphocyte culture (MLC) can be generated in embryo thymus organ culture. MATERIALS AND METHODS (1) Mice. C57BL/6 and BALB/c mice were mated in trios and pregnancy was timed from the appearance of vaginal plugs (day 0 gestation). Spleen cell suspensions from CBA/H mice were used in MLC. These three strains of mice are M-locus compatible but differ from each other at K, D and I regions of the MHC. (ii) Organ culture. The organ culture technique has been described in detail (Robinson & Owen, 1976). Briefly thymuses were removed from embryonic mice at 14 or 15 days gestation, and were cultured on Nuclepore filters (type N40, 01300 CPR), floating on RPMI 1640 supplemented with 10% foetal calf serum (RF10), in the centre of Falcon organ culture J. H. Robinson is in receipt of an MRC Student Award. Correspondence: Dr J. H. Robinson, Department of Anatomy, Medical School, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne. *
322
323
In vitro ontogeny of MLC reactivity
dishes (catalogue number 3010). Several explants were placed on one filter and cultures were incubated at 370C in a humid atmosphere of 5% CO2 in air. (iii) MLC. After different periods of organ culture, foetal thymuses were teased apart and the resulting lymphoid cell suspensions were used as the responding population in MLC. One-way mixed lymphocyte cultures were set up in microculture using 2 x 105 responding and stimulating cells in a total of 0-2 ml of RF10 including 5 x 10- 5 M 2-mercaptoethanol. Mitomycin-C-treated spleen cell suspensions (spleenm) from adult BALB/c, C57BL/6, CBA/H mice as well as from the three F1 hybrids between these strains, were used as the stimulating cells. Erythrocytes were removed from spleen cell suspensions by hypotonic shock and cells at 20 x 106/ml were incubated for 30 min at 37°C with 30 pg/ml mitomycin-c (Difco). Treated cells were washed three times in RF10 prior to use. Cultures were set up in duplicate or triplicate in Falcon tissue culture tubes (catalogue number 2054) and incubated for 5 days at 37°C in 5% CO2 in air. DNA synthesis was assayed by the incorporation of 125I-labelled iododeoxyuridine ([125I]lUdR) into DNA as described previously (Robinson & Owen, 1976). Allogeneic mixed lymphocyte reactivity was expressed as a ratio of ct/min. in allogeneic MLC (cultured thymus plus allogeneic adult spleenm) to ct/min in syngeneic MLC (cultured thymus plus syngeneic adult spleenm). The significance (P value) of allogeneic stimulation was assessed using Student's t-test.
RESULTS (i) Cell yields from organ culture We have reported previously (Robinson & Owen, 1976) that in a 7-day culture period, the lymphoid cell yield from 14-day thymuses increases from around 35 x 103 large basophilic cells before culture to 200-500 x 103 viable small and medium lymphoid cells per embryo. The cell yield profile is monophasic and after the peak at 7 days culture, cell yield steadily decreases, although viable cells are still present after 28 days of culture. It has been shown previously that the vast majority of these cells are Thy-i positive (Owen, Raff & Cooper, 1975). TABLE 1. BALB/c cultured thymus responses in MLC to adult BALB/c and CBA/H spleenm
Mean ct/min+ s.e.m.
Culture period* 0 3 5 7
9
11
Syngeneic
Allogeneic
Ratio
271+13 144±136 186+46 415+ 125 406+430 227+45 349+212 1073+88 1035+746 90+79 637+ 99 542+ 106 438+4
469+236 8+ 35 253+ 30 511+235 622+ 180
1-7 0-06 1-4 1-2 1-5 0 75 026 3-1 6-1 10-6 6-7 10-6 8-7
1420+285 13
548+568
45±71 15 Uncultured 19 days gestation
691+ 125 2126+352 442+ 132
171+23t 91+81 3353+162 6285+ 1112 954± 138t 4296+ 507 5758+ 2180 3807+896 13735+ 1226 3234+801 517+ 138t 2335+947 11129+ 1191t 10908+1217
9.7 59 11-5 3-4 5-3 24-7
P value > 0-1 > 0-1
>0.1 >0.1 > 0-1 >0.1 >0-1
