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Biological activities of a murine T-cell clone with reactivity to Mycobacterium leprae
CELLULAR
IMMUNOLOGY
83,2 15-220 (1984)
Biological Activities of a Murine T-Cell Clone with Reactivity to Mycobactefium leprae S. H. E. KAUFMANN Max-Planck-Institut
ftir Immunbiologie,
Stiibeweg 51, D-7800 Freiburg, Federal Republic of Germany
Received June 7, 1983; accepted September 4, 1983
Mice were immunized subcutaneouslywith killed Mycobacterium leprae in incomplete Freund’s adjuvant and draining lymph nodes removed. Lymph node cells were propagated in vitro and cloned at limiting dilution in the presenceof syngeneic accessorycells, antigen, and T-cell growth factor. Cloned T cells were restricted by the H-21-A sublocus. In vitro interaction(s) of cloned T cellswith accessorycells presentingM. lepraederived determinants resultedin T-cell proliferation, interleukin secretion, and macrophage activation. The T cells were stimulated by killed M. leprae and M. bovis (strain BCG), but not Listeria monocytogenes, organisms indicating cross-reactivity between M. leprae and BCG at the clonal level. In vivo, cloned T cells induced protection against the “bystander” bacterium L. monocytogenes. These data suggest that the cloned M. lepraereactive T cells are involved in acquired antimicrobial resistance.
INTRODUCTION Leprosy is a chronic infectious diseasecaused by the intracellular bacterium, Mycobucterium leprue (summarized in (1)). Although it is generally accepted that immunity to M. leprue in principle depends on cooperative events between mononuclear
cells and T lymphocytes (summarized in (1,2)), little is known about cell interactions involved in acquired resistanceto M. leprue, predominantly due to the limited amount of organisms available and to the nonpathogenicity of this bacterium for laboratory animals. However, it has been shown that M. leprue is capable of limited multiplication in the footpad of mice (3, 4) and that T-cell responses can be induced with killed organisms (5, 6). Recent developments in technology have allowed the cloning and in vitro propagation of T cells which maintain their biological activities for long periods of time (summarized in (7)). The aim of the present study was to establish clones of M. leprue-reactive murine T cells and to analyze the cellular immune response to AL leprue using one of these T-cell clones as a probe. MATERIALS
AND METHODS
Mice. Unless otherwise stated male C57B1/6, BlO.MBR, BIO.A (4R), Bl0.A (5R), and BALB/c mice were used when 8 to 12 weeks old. These mice were bred at the Max-Planck-Institute for Immunobiology. Bacteria. Armadillo-derived, cobalt-irradiated (2.5 Mrad), and purified AI. leprue (batch V2) was kindly provided by Dr. R. J. W. Rees, London, U.K., through the World Health Organization Immunology of Leprosy Program. M. leprue was sent 215 0008-8749184$3.00 Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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on dry ice at a concentration of lO’e/ml organisms and kept at -70°C before being diluted. il4. bovis (strain BCG) was kindly provided by Dr. R. North, Trudeau Institute, Saranac Lake, New York. BCG was heat killed by incubation at 60°C (water bath) for 60 min. M. leprae and BCG were suspended at l@/ml organisms in phosphatebuffered saline (PBS) containing 10% fetal calf serum (FCS) and stored at -70°C until used. Listeria monocytogenes (strain EGD) was kept virulent by continuous mouse passage(8). Bacterial cultures were obtained by growing a sample of spleen homogenate in Trypticase soy broth (Oxoid) at 37°C for 16 hr. Heat-killed L. monocytogenes (HIU) were obtained by incubation at 60°C (water bath) for 60 min (9). HKL were suspended in PBS and stored at -70°C at a concentration of 109/ml until used. Establishment of M. leprae-reactive T-cell clones. Irradiated M. leprae organisms (10’) were emulsified in incomplete Freund’s adjuvant (Difco) and injected subcutaneously (SC)into the base of the tail of C57B1/6 mice in a volume of 0.15 ml. After 14 days the immunization regimen was repeated once and another 8 days later inguinal and paraaortic lymph nodes were removed. Lymph node cells (1 X 106/ml) were cultured in 5 ml of Dulbecco’s modification of Eagle’s medium (DMEM), supplemented with 10%selectedFCS (Biolab), 2-mercaptoethanol (Merck), L-asparagine, L-glutamine, penicillin, and streptomycin (Gibco, complete DMEM), together with 106/ml M. leprae organisms in 25-cm2 flasks (Nunc) in an upright position at 37°C in 7% COZ in air. After 5 days, cells were washed and 106/ml cells recultured together with 106/ml irradiated (2200 R) spleen cells and 106/ml M. leprae organisms for 7 days. After centrifugation on a Urovison-Ficoll density gradient (density = 1.077) for 15 min at 7OOg,cells ( 105/ml) were propagated in vitro in the presenceof 5 X 105/ ml irradiated (2200 R) spleen cells, 106/ml M. lepraeorganisms, and 10%semipurihed T-cell growth factor (TCGF) in 5 ml complete DMEM in Costar 3506 trays at 37°C in 7% CO2 in air. TCGF was prepared from concanavalin A-stimulated rat spleen cells as previously described ( 10). Every 3 to 4 days, cells were washed and recultured as described above. Cells were propagated under these conditions for more than 6 weeks. The T-cell line was then cloned at limiting dilution in round-bottom microculture plates (Nunc) containing 2 X 10’ irradiated (2200 R) spleen cells, 2 X lo5 M. leprae organisms, and 5% TCGF in 0.2 ml of complete DMEM. T cells were plated in replicates of 96 wells at threefold dilutions ranging from 1000 to 0.3 cells per well. After 10 days of culture, positive wells of proliferating T cells were identified using an inverted microscope. The frequency of positive wells was l/40. Responding cells at dilutions with frequencies of
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harvest. Supematants were tested for interleukin activity as described (9- 11). In short, 1 X lo6 thymocytes from 3- to 4-week-old BALB/c mice were cultured with 20% supematant in 0.2 ml complete DMEM in round-bottom microculture plates (Nunc) at 37°C in 7% CO2 in air for 3 days, the last 18 hr in the presence of 1 &i [3H]TdR. Mucrophuge activation. Graded amounts of T cells were incubated together with 2 X lo5 peritoneal exudate cells (PEC) and 2 X lo5 killed M. leprae or 2 X lo6 HKL in a total volume of 0.2 ml DMEM at 37°C 7% CO* in air. PEC were obtained from mice injected with 1.5 ml of 10%proteose peptone (Difco) 3 days before harvest. After 24 hr, adherent PEC were washed thoroughly and 2 X lo4 “Cr-labeled EL4 tumor cells added (13). Tumor cells ( lo7 cells/O.2 ml) had been incubated with 0.1 mCi NaZ5’Cr04 (Radiochemical Center Amersham) for 90 min and afterward washed three times, the last time through a layer of FCS. “Cr release was determined after 16 hr. Percentage of specific lysis was calculated according to the equation: % 5’Cr release = 100 X (a - b)/(c - b) (a = “Cr releaseby activated macrophages; b = “Cr releaseby target cells alone; c = 5’Cr release after freeze-thawing). Bystander protection. Graded amounts of cloned T cells were mixed with 2 X lo5 cobalt-irradiated M. leprae and/or 2 X lo5 live L. monocytogerzesorganisms in a total volume of 0.05 ml and injected SCinto one hind footpad of recipient mice (14). After 2 days, feet were removed, rinsed with alcohol (70%), and homogenized with a tissue grinder (Ultra Turrax, IRA). Appropriate dilutions of homogenate were plated out on Trypticase soy agar (Oxoid) and colonies of L. monocytogenes determined after incubation at 37°C for 24 to 36 hr. Data analysis. Data are expressedas means f standard deviation (SD). Student’s t test was used for data analysis. Differences were regarded as significant at P < 0.05. RESULTS AND DISCUSSION After cloning of M. leprae-reactive cells, several clones were obtained. Cloned cells were Thy l+, Ig- (data not shown) and expressedsimilar biological activities. Therefore, representative data of a single M. leprue-reactive T-cell clone are given. The specificity of the cloned T cells was assessedby determination of proliferative responses and interleukin production in cultures of cloned T cells and syngeneic accessorycells in the presence of 2 X 105/0.2 ml M. leprue, 2 X 105/0.2 ml BCG, or 2 X 107/0.2 ml HKL, respectively, as a source of antigen. In earlier experiments it had been found that these antigen concentrations induced maximum responsesin cultures of M. leprae-reactive long-term cultured T-cell lines (M. leprue and BCG) or L. monocytogenes-specific T-cell clones (HKL), respectively (( 10) and unpublished data). As shown in Fig. 1, as few as 3000 cloned T cells were sufficient for significant responses.Cloned T cells were stimulated by M. leprae, as well as by BCG, whereas HKL had no such effect. When a L. monocytogenes-specific T-cell clone (10) was cocultured with M. leprae and accessory cells, neither proliferation nor interleukin production was demonstrable although these T cells were stimulated by HIU (data not shown). Thus, the T-cell clone is specific for a mycobacteriaderived epitope, and any reactivity to armadillo tissue contaminating the M. leprae preparation appears unlikely. The finding that the T-cell clone was not only reactive with M. leprae but also with BCG demonstrates cross-reactivity between these two mycobacteria on the clonal level. This is in agreement with the idea that BCG can stimulate cellular immunity against M. leprue and therefore might facilitate vaccination against leprosy (15).
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-7 4 z; g a 2 7
/ P
3/
2-
/
p
4'
4
l-
4'
-
f&L 01
Number
of T cells
(~10~)
PHOY 0.3 Number
1
3
of Tcells
10 (~10~)
PIG. 1. Proliferation and interleukin induction by cloned M. Zeprue-reactiveT cells. Graded numbers of cloned T cells were cocultured with 2 X 10’ accessory cells in the presence of 2 X lo5 M. teprae(0), 2 X 10’ BCG (A), or 2 X 10’ HRL (0). Left hand panel: proliferative responses;right hand panel: interleukin activity. Means of four determinations + SD.
Macrophage activation by specific T cells is considered a crucial step of acquired resistanceto intracellular bacteria (2). Therefore, it was tested whether the M. lepruereactive T-cell clone was able to activate macrophages for increased tumoricidal activity after antigen specific stimulation in vitro. As indicated by the data in Table 1, in the presence of it4 leprue organisms, as few as 1000 cloned T cells were able to induce significant activation of macrophages. In order to assesswhether the M. leprue-reactive T-cell clone was also active in vivo, graded amounts of T cells, together with M. leprae and/or live L. monucytogenes organisms, were injected SCinto the footpads of normal recipient mice and 2 days later, bacterial numbers determined. In the presence of M. leprue organisms, 30 to 100 X lo3 T cells significantly reduced the numbers of viable L. monocyfogenes organisms (Table 2). Similar effects were observed when BCG was used instead of M. leprue (data not shown). Thus, the cloned hf. leprae-reactive T cells were able to TABLE 1 Macrophage Activation by Cloned M. leprue-reactive T Cells Macrophage activation (‘70specific lysis of S’Cr-labeled EL4 cells)” Number of T cells 0
1x 3x 10 x 30 x
102 lo2 lo2 lo2
+
M. leprae
+HKL
9*1 14 f 28 f 46 f 63 +
13 f 2 7f2 20 + 2 21 +4 16 f 5
2 3 3 4
“Graded amounts of cloned T cells were cocultured with 2 X lo5 PEC in the presence of 2 X lo5
M. leprae or 2 X 10’ HIU. After 24 hr, cells were washedand macrophagesassayedfor tumoricidal activity on 5’Cr-labeled EL4 cells. Means of four determinations f SD. Uncorrected data of “Cr release at 10 X IO2and 30 X lo2 T cells in the presenceof M. Ieprae showed significant differencesagainst “Cr release at 10 X lo2 and 30 X 10’ T cells, respectively, in the presence of HRL and against “Cr release in the presenceof M. leprae but in the absenceof T cells.
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SHORT COMMUNICATIONS TABLE 2 In Vivo Function of Cloned M. leprae-Reactive T Cells
Log protection against L. monocytogenes” Number of T cells
+ M. leprae + L. monocytogenes
1 x IO’
0.12 0.08 0.49 0.76 0.93
3 x 10’
10 x lo3 30 x 10’
100 x 10’
+ + f f +
+ L. monocytogenes
0.13 0.08 0.20 0.17 0.23
0.30 0.24 0.24 0.27 0.31
+- 0.05 -+ 0.09 + 0.10 r 0.08 -t 0.12
a Cloned T cells were injected SCinto one hind footpad of normal recipients together with 2 X 10’ M. leprae organisms and/or 2 X 10’ live L. monocytogenes. Numbers of L. monocytogenes were determined alter 48 hr (14). In the absence of T cells and M. leprae, log viable L. monocytogenes was 7.34 + 0.41, means of five determinations + SD. Uncorrected data of log viable L. momxytogenes at 30 X 10’ and 100 X 10’ T cells in the presence of M. leprae showed significant differences against log viable L. monocytogenes at 30 X 10’ and 100 X 10’ T cells, respectively, in the absenceof M. Zeprue and against log viable L. monocytogenes in the presence of M. leprue but in the absence of T cells.
induce definite though small bystander protection against L. monocytogenes after antigenic stimulation (Table 2). Finally, the H-2 restriction of the biological functions of this T-cell clone was analyzed. Proliferation and interleukin induction were observed when the T cells were cocultured with accessory cells from C57B1/6 and BIO.A (5R) but not with accessorycells from BIO.A (4R) and BlO.MBR mice (Table 3). These data suggest that histocompatibility at the H-21-A sublocus is required and sufficient for effective stimulation of M. leprue-reactive T lymphocytes by antigen-presenting cells. Similarly it has been shown that T-cell clones active in immunity to L. monocytogenes are restricted by the H-21-A sublocus (10). The data presented demonstrate that M. leprae-reactive murine T lymphocytes can be cloned, propagated, and expanded in vitro in the presenceof antigen, accessory cells, and TCGF, and that these T-cell clones retain their biological functions and H-2 restriction during in vitro culture. Cloned &f. leprue-reactive T cells were not TABLE 3 H-2 Restriction of Cloned M. leprae-Reactive T Cells H-2 complex Accessory cell
K
I-A
I-E
D
C57B1/6 BI0.A (4R) BIO.A (5R) BIO.MBR BALBfc
b
b k b
b b k k d
b b d
k b b d
k
d
Proliferative response’ ([‘H]TdR cpm + SD) 7200 f 620
1600 + 190 8300 f 140
1200 + 210 8
960 + 130
Interleukin induction” ([‘H]TdR cpm + SD) 6500 630 5900 980 610
+ 750 + 460 + 680 +- 610 + 300
‘Cloned T cells (10’/0.2 ml) were cocultured with 2 X lo5 M. feprae and 2 X lo5 accessorycells of different haplotype for 5 days, the last 18 hr in the presence 1 &i [“H]TdR. Means of three demrminations f SD.
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only active in vitro but also in vivo mediating protection to a bystander bacterium. Hence, one homogeneous AI. leprue-reactive T-cell population was able to induce different biological functions. Because T-cell clones of the sort described seem to participate in acquired resistance to intracellular pathogens (10, 14), analysis at the single-cell level of both cellular interactions involved in and antigenic determinants relevant to acquired resistance against M. leprue should be facilitated by employing A4. leprae-reactive
T-cell
clones. ACKNOWLEDGMENTS
I thank Dr. R. J. W. Rees for supplying M. leprae organisms (prepared as per protocol l/79, Report to the Enlarged Steering Committee Meeting, 7-8 February 1979, WHO Document TDR, IMMLEP-SWG (S) 80.3). I thank L. J. Wrazel for technical assistanceand H. Kuttler and R. Schneider for typing the manuscript. I would also like to thank Dr. R. J. W. Rees for showing me the techniques concerned with establishing and quantitating infections with M. leprae in mice during a stay at the National Institute of Medical Research,London, England (supported by Stipendium Ka 573/2-l from the Deutsehe Forsehungsgemeinsehaft).
REFERENCES 1. Godal, T., Prog. Allergy 25, 2 1I, 1978. 2. Hahn, H., and Kaufmann, S. H. E., Rev. lnjkcf. Dis. 3, 1221, 1981.
3. Rees, R. J. W., Brit. J. Exp. Pathol. 45, 207, 1964. 4. Shepard, C. C., J. Exp. Med. 112,445, 1960. 5. Patel, P. J., and Lefford, M. J., In&t. Immun. 19, 87, 1978. 6. Patel, P. J., and Lefford, M. J., Infect. Immun. 20, 692, 1978. 7. MiilIer, G. (Ed.), Immunol. Rev. 54, 1, 1981.
8. Kaufmann, S. H. E., Simon, M. M., and Hahn, H., J. Exp. Med. 150, 1033, 1979. 9. Kaufmann, S. H. E., Hahn, H., and Simon, M. M., Scand. J. Immunol. 16, 539, 1982. 10. Kaufmann, S. H. E., and Hahn, H., J. Exp. Med. 155, 1754, 1982. 11. Farr, A. G., Kiely, J. M., and Unanue, E. R., J. Immunol. 122, 2395, 1979. 12. Kaufmann, S. H. E., Simon, M. M., and Hahn, H., Infet. Immun. 38, 907, 1982. 13. Farr, A. G., Weehter, W. J., Kiely, J. M., and Unanue, E. R., J. Immunol. 122, 2405, 1979. 14. Kaufmann, S. H. E., Infect. Immun. 39, 1265, 1983. 15. Brit. Med. J. 1, 24, 1968.
IMMUNOLOGY
83,2 15-220 (1984)
Biological Activities of a Murine T-Cell Clone with Reactivity to Mycobactefium leprae S. H. E. KAUFMANN Max-Planck-Institut
ftir Immunbiologie,
Stiibeweg 51, D-7800 Freiburg, Federal Republic of Germany
Received June 7, 1983; accepted September 4, 1983
Mice were immunized subcutaneouslywith killed Mycobacterium leprae in incomplete Freund’s adjuvant and draining lymph nodes removed. Lymph node cells were propagated in vitro and cloned at limiting dilution in the presenceof syngeneic accessorycells, antigen, and T-cell growth factor. Cloned T cells were restricted by the H-21-A sublocus. In vitro interaction(s) of cloned T cellswith accessorycells presentingM. lepraederived determinants resultedin T-cell proliferation, interleukin secretion, and macrophage activation. The T cells were stimulated by killed M. leprae and M. bovis (strain BCG), but not Listeria monocytogenes, organisms indicating cross-reactivity between M. leprae and BCG at the clonal level. In vivo, cloned T cells induced protection against the “bystander” bacterium L. monocytogenes. These data suggest that the cloned M. lepraereactive T cells are involved in acquired antimicrobial resistance.
INTRODUCTION Leprosy is a chronic infectious diseasecaused by the intracellular bacterium, Mycobucterium leprue (summarized in (1)). Although it is generally accepted that immunity to M. leprue in principle depends on cooperative events between mononuclear
cells and T lymphocytes (summarized in (1,2)), little is known about cell interactions involved in acquired resistanceto M. leprue, predominantly due to the limited amount of organisms available and to the nonpathogenicity of this bacterium for laboratory animals. However, it has been shown that M. leprue is capable of limited multiplication in the footpad of mice (3, 4) and that T-cell responses can be induced with killed organisms (5, 6). Recent developments in technology have allowed the cloning and in vitro propagation of T cells which maintain their biological activities for long periods of time (summarized in (7)). The aim of the present study was to establish clones of M. leprue-reactive murine T cells and to analyze the cellular immune response to AL leprue using one of these T-cell clones as a probe. MATERIALS
AND METHODS
Mice. Unless otherwise stated male C57B1/6, BlO.MBR, BIO.A (4R), Bl0.A (5R), and BALB/c mice were used when 8 to 12 weeks old. These mice were bred at the Max-Planck-Institute for Immunobiology. Bacteria. Armadillo-derived, cobalt-irradiated (2.5 Mrad), and purified AI. leprue (batch V2) was kindly provided by Dr. R. J. W. Rees, London, U.K., through the World Health Organization Immunology of Leprosy Program. M. leprue was sent 215 0008-8749184$3.00 Copyright Q 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
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on dry ice at a concentration of lO’e/ml organisms and kept at -70°C before being diluted. il4. bovis (strain BCG) was kindly provided by Dr. R. North, Trudeau Institute, Saranac Lake, New York. BCG was heat killed by incubation at 60°C (water bath) for 60 min. M. leprae and BCG were suspended at l@/ml organisms in phosphatebuffered saline (PBS) containing 10% fetal calf serum (FCS) and stored at -70°C until used. Listeria monocytogenes (strain EGD) was kept virulent by continuous mouse passage(8). Bacterial cultures were obtained by growing a sample of spleen homogenate in Trypticase soy broth (Oxoid) at 37°C for 16 hr. Heat-killed L. monocytogenes (HIU) were obtained by incubation at 60°C (water bath) for 60 min (9). HKL were suspended in PBS and stored at -70°C at a concentration of 109/ml until used. Establishment of M. leprae-reactive T-cell clones. Irradiated M. leprae organisms (10’) were emulsified in incomplete Freund’s adjuvant (Difco) and injected subcutaneously (SC)into the base of the tail of C57B1/6 mice in a volume of 0.15 ml. After 14 days the immunization regimen was repeated once and another 8 days later inguinal and paraaortic lymph nodes were removed. Lymph node cells (1 X 106/ml) were cultured in 5 ml of Dulbecco’s modification of Eagle’s medium (DMEM), supplemented with 10%selectedFCS (Biolab), 2-mercaptoethanol (Merck), L-asparagine, L-glutamine, penicillin, and streptomycin (Gibco, complete DMEM), together with 106/ml M. leprae organisms in 25-cm2 flasks (Nunc) in an upright position at 37°C in 7% COZ in air. After 5 days, cells were washed and 106/ml cells recultured together with 106/ml irradiated (2200 R) spleen cells and 106/ml M. leprae organisms for 7 days. After centrifugation on a Urovison-Ficoll density gradient (density = 1.077) for 15 min at 7OOg,cells ( 105/ml) were propagated in vitro in the presenceof 5 X 105/ ml irradiated (2200 R) spleen cells, 106/ml M. lepraeorganisms, and 10%semipurihed T-cell growth factor (TCGF) in 5 ml complete DMEM in Costar 3506 trays at 37°C in 7% CO2 in air. TCGF was prepared from concanavalin A-stimulated rat spleen cells as previously described ( 10). Every 3 to 4 days, cells were washed and recultured as described above. Cells were propagated under these conditions for more than 6 weeks. The T-cell line was then cloned at limiting dilution in round-bottom microculture plates (Nunc) containing 2 X 10’ irradiated (2200 R) spleen cells, 2 X lo5 M. leprae organisms, and 5% TCGF in 0.2 ml of complete DMEM. T cells were plated in replicates of 96 wells at threefold dilutions ranging from 1000 to 0.3 cells per well. After 10 days of culture, positive wells of proliferating T cells were identified using an inverted microscope. The frequency of positive wells was l/40. Responding cells at dilutions with frequencies of
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217
harvest. Supematants were tested for interleukin activity as described (9- 11). In short, 1 X lo6 thymocytes from 3- to 4-week-old BALB/c mice were cultured with 20% supematant in 0.2 ml complete DMEM in round-bottom microculture plates (Nunc) at 37°C in 7% CO2 in air for 3 days, the last 18 hr in the presence of 1 &i [3H]TdR. Mucrophuge activation. Graded amounts of T cells were incubated together with 2 X lo5 peritoneal exudate cells (PEC) and 2 X lo5 killed M. leprae or 2 X lo6 HKL in a total volume of 0.2 ml DMEM at 37°C 7% CO* in air. PEC were obtained from mice injected with 1.5 ml of 10%proteose peptone (Difco) 3 days before harvest. After 24 hr, adherent PEC were washed thoroughly and 2 X lo4 “Cr-labeled EL4 tumor cells added (13). Tumor cells ( lo7 cells/O.2 ml) had been incubated with 0.1 mCi NaZ5’Cr04 (Radiochemical Center Amersham) for 90 min and afterward washed three times, the last time through a layer of FCS. “Cr release was determined after 16 hr. Percentage of specific lysis was calculated according to the equation: % 5’Cr release = 100 X (a - b)/(c - b) (a = “Cr releaseby activated macrophages; b = “Cr releaseby target cells alone; c = 5’Cr release after freeze-thawing). Bystander protection. Graded amounts of cloned T cells were mixed with 2 X lo5 cobalt-irradiated M. leprae and/or 2 X lo5 live L. monocytogerzesorganisms in a total volume of 0.05 ml and injected SCinto one hind footpad of recipient mice (14). After 2 days, feet were removed, rinsed with alcohol (70%), and homogenized with a tissue grinder (Ultra Turrax, IRA). Appropriate dilutions of homogenate were plated out on Trypticase soy agar (Oxoid) and colonies of L. monocytogenes determined after incubation at 37°C for 24 to 36 hr. Data analysis. Data are expressedas means f standard deviation (SD). Student’s t test was used for data analysis. Differences were regarded as significant at P < 0.05. RESULTS AND DISCUSSION After cloning of M. leprae-reactive cells, several clones were obtained. Cloned cells were Thy l+, Ig- (data not shown) and expressedsimilar biological activities. Therefore, representative data of a single M. leprue-reactive T-cell clone are given. The specificity of the cloned T cells was assessedby determination of proliferative responses and interleukin production in cultures of cloned T cells and syngeneic accessorycells in the presence of 2 X 105/0.2 ml M. leprue, 2 X 105/0.2 ml BCG, or 2 X 107/0.2 ml HKL, respectively, as a source of antigen. In earlier experiments it had been found that these antigen concentrations induced maximum responsesin cultures of M. leprae-reactive long-term cultured T-cell lines (M. leprue and BCG) or L. monocytogenes-specific T-cell clones (HKL), respectively (( 10) and unpublished data). As shown in Fig. 1, as few as 3000 cloned T cells were sufficient for significant responses.Cloned T cells were stimulated by M. leprae, as well as by BCG, whereas HKL had no such effect. When a L. monocytogenes-specific T-cell clone (10) was cocultured with M. leprae and accessory cells, neither proliferation nor interleukin production was demonstrable although these T cells were stimulated by HIU (data not shown). Thus, the T-cell clone is specific for a mycobacteriaderived epitope, and any reactivity to armadillo tissue contaminating the M. leprae preparation appears unlikely. The finding that the T-cell clone was not only reactive with M. leprae but also with BCG demonstrates cross-reactivity between these two mycobacteria on the clonal level. This is in agreement with the idea that BCG can stimulate cellular immunity against M. leprue and therefore might facilitate vaccination against leprosy (15).
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SHORT COMMUNICATIONS
-7 4 z; g a 2 7
/ P
3/
2-
/
p
4'
4
l-
4'
-
f&L 01
Number
of T cells
(~10~)
PHOY 0.3 Number
1
3
of Tcells
10 (~10~)
PIG. 1. Proliferation and interleukin induction by cloned M. Zeprue-reactiveT cells. Graded numbers of cloned T cells were cocultured with 2 X 10’ accessory cells in the presence of 2 X lo5 M. teprae(0), 2 X 10’ BCG (A), or 2 X 10’ HRL (0). Left hand panel: proliferative responses;right hand panel: interleukin activity. Means of four determinations + SD.
Macrophage activation by specific T cells is considered a crucial step of acquired resistanceto intracellular bacteria (2). Therefore, it was tested whether the M. lepruereactive T-cell clone was able to activate macrophages for increased tumoricidal activity after antigen specific stimulation in vitro. As indicated by the data in Table 1, in the presence of it4 leprue organisms, as few as 1000 cloned T cells were able to induce significant activation of macrophages. In order to assesswhether the M. leprue-reactive T-cell clone was also active in vivo, graded amounts of T cells, together with M. leprae and/or live L. monucytogenes organisms, were injected SCinto the footpads of normal recipient mice and 2 days later, bacterial numbers determined. In the presence of M. leprue organisms, 30 to 100 X lo3 T cells significantly reduced the numbers of viable L. monocyfogenes organisms (Table 2). Similar effects were observed when BCG was used instead of M. leprue (data not shown). Thus, the cloned hf. leprae-reactive T cells were able to TABLE 1 Macrophage Activation by Cloned M. leprue-reactive T Cells Macrophage activation (‘70specific lysis of S’Cr-labeled EL4 cells)” Number of T cells 0
1x 3x 10 x 30 x
102 lo2 lo2 lo2
+
M. leprae
+HKL
9*1 14 f 28 f 46 f 63 +
13 f 2 7f2 20 + 2 21 +4 16 f 5
2 3 3 4
“Graded amounts of cloned T cells were cocultured with 2 X lo5 PEC in the presence of 2 X lo5
M. leprae or 2 X 10’ HIU. After 24 hr, cells were washedand macrophagesassayedfor tumoricidal activity on 5’Cr-labeled EL4 cells. Means of four determinations f SD. Uncorrected data of “Cr release at 10 X IO2and 30 X lo2 T cells in the presenceof M. Ieprae showed significant differencesagainst “Cr release at 10 X lo2 and 30 X 10’ T cells, respectively, in the presence of HRL and against “Cr release in the presenceof M. leprae but in the absenceof T cells.
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SHORT COMMUNICATIONS TABLE 2 In Vivo Function of Cloned M. leprae-Reactive T Cells
Log protection against L. monocytogenes” Number of T cells
+ M. leprae + L. monocytogenes
1 x IO’
0.12 0.08 0.49 0.76 0.93
3 x 10’
10 x lo3 30 x 10’
100 x 10’
+ + f f +
+ L. monocytogenes
0.13 0.08 0.20 0.17 0.23
0.30 0.24 0.24 0.27 0.31
+- 0.05 -+ 0.09 + 0.10 r 0.08 -t 0.12
a Cloned T cells were injected SCinto one hind footpad of normal recipients together with 2 X 10’ M. leprae organisms and/or 2 X 10’ live L. monocytogenes. Numbers of L. monocytogenes were determined alter 48 hr (14). In the absence of T cells and M. leprae, log viable L. monocytogenes was 7.34 + 0.41, means of five determinations + SD. Uncorrected data of log viable L. momxytogenes at 30 X 10’ and 100 X 10’ T cells in the presence of M. leprae showed significant differences against log viable L. monocytogenes at 30 X 10’ and 100 X 10’ T cells, respectively, in the absenceof M. Zeprue and against log viable L. monocytogenes in the presence of M. leprue but in the absence of T cells.
induce definite though small bystander protection against L. monocytogenes after antigenic stimulation (Table 2). Finally, the H-2 restriction of the biological functions of this T-cell clone was analyzed. Proliferation and interleukin induction were observed when the T cells were cocultured with accessory cells from C57B1/6 and BIO.A (5R) but not with accessorycells from BIO.A (4R) and BlO.MBR mice (Table 3). These data suggest that histocompatibility at the H-21-A sublocus is required and sufficient for effective stimulation of M. leprue-reactive T lymphocytes by antigen-presenting cells. Similarly it has been shown that T-cell clones active in immunity to L. monocytogenes are restricted by the H-21-A sublocus (10). The data presented demonstrate that M. leprae-reactive murine T lymphocytes can be cloned, propagated, and expanded in vitro in the presenceof antigen, accessory cells, and TCGF, and that these T-cell clones retain their biological functions and H-2 restriction during in vitro culture. Cloned &f. leprue-reactive T cells were not TABLE 3 H-2 Restriction of Cloned M. leprae-Reactive T Cells H-2 complex Accessory cell
K
I-A
I-E
D
C57B1/6 BI0.A (4R) BIO.A (5R) BIO.MBR BALBfc
b
b k b
b b k k d
b b d
k b b d
k
d
Proliferative response’ ([‘H]TdR cpm + SD) 7200 f 620
1600 + 190 8300 f 140
1200 + 210 8
960 + 130
Interleukin induction” ([‘H]TdR cpm + SD) 6500 630 5900 980 610
+ 750 + 460 + 680 +- 610 + 300
‘Cloned T cells (10’/0.2 ml) were cocultured with 2 X lo5 M. feprae and 2 X lo5 accessorycells of different haplotype for 5 days, the last 18 hr in the presence 1 &i [“H]TdR. Means of three demrminations f SD.
SHORT COMMUNICATIONS
220
only active in vitro but also in vivo mediating protection to a bystander bacterium. Hence, one homogeneous AI. leprue-reactive T-cell population was able to induce different biological functions. Because T-cell clones of the sort described seem to participate in acquired resistance to intracellular pathogens (10, 14), analysis at the single-cell level of both cellular interactions involved in and antigenic determinants relevant to acquired resistance against M. leprue should be facilitated by employing A4. leprae-reactive
T-cell
clones. ACKNOWLEDGMENTS
I thank Dr. R. J. W. Rees for supplying M. leprae organisms (prepared as per protocol l/79, Report to the Enlarged Steering Committee Meeting, 7-8 February 1979, WHO Document TDR, IMMLEP-SWG (S) 80.3). I thank L. J. Wrazel for technical assistanceand H. Kuttler and R. Schneider for typing the manuscript. I would also like to thank Dr. R. J. W. Rees for showing me the techniques concerned with establishing and quantitating infections with M. leprae in mice during a stay at the National Institute of Medical Research,London, England (supported by Stipendium Ka 573/2-l from the Deutsehe Forsehungsgemeinsehaft).
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