INFECTION AND IMMUNITY, Aug. 1979, p. 497-506 0019-9567/79/08-0497/10$02.00/0
Vol. 25, No. 2
Development of Suppressor T-Cells in Mycobacterium habana-Infected Mice SUSAN R. WATSON AND FRANK M. COLLINS* Trudeau Institute, Inc., Saranac Lake, New York 12983
Received for publication 7 May 1979
Mice were infected intravenously with increasing numbers of Mycobacterium habana (simiae serotype II), and the levels of delayed-type hypersensitivity to purified protein derivative and M. habana cytoplasmic protein antigen were determined after 14, 30, and 90 days. A footpad delayed-type hypersensitivity response was seen in 14-day-infected mice and was followed by a persisting anergy. T-cell-enriched suspensions collected 30 and 90 days into the infection (anergic donors) showed depressed transformation indexes after phytohemagglutinin and M. habana cytoplasmic protein antigen treatment in vitro. The corresponding B-cell mitogen (lipopolysaccharide) responses were not affected. Mixing experiments with T-cell-enriched suspensions from day-90 M. habana-infected donors adoptively suppressed lymphocyte transformation by normal and day-14 spleen cells. This effect could be ablated by anti-theta serum and complement treatment of the day-90 cells, indicating that the lack of in vitro responsiveness to cytoplasmic protein antigen was mediated by a population of suppressor T-cells present in the heavily infected spleens. There was no evidence that similar cells were present in the spleens of the 14-day-infected animals. Suppressor T-cells could be induced in vitro by exposure of day-14 spleen cells to concanavalin A or M. habana cytoplasmic protein antigen before they were mixed with normal or day-14 indicator splenic lymphocytes. The timing of the appearance of suppressor T-cells in the infected spleens corresponded to a loss of footpad hypersensitivity by the M. habana-infected animals. Lepromatous leprosy is characterized by the accumulation of large numbers of acid-fast bacilli within tissues which show little evidence of any cell-mediated immunity as the infection progresses (2). The patients also develop a profound state of lepromin anergy which tends to persist even after the mycobacterial load has been reduced by effective chemotherapeutic intervention (1, 11). Unlike tuberculoid leprosy, the peripheral blood lymphocytes from these anergic patients fail to exhibit an enhanced blast transformation in the presence of lepromin (10). A number of atypical mycobacteria are also able to establish persisting systemic infections which are associated with a profound state of skin test energy to the corresponding mycobacterial antigens (7). Heavy BCG infection has been shown to be associated with active suppression of lymphocyte transformation by spleen cells exposed to the nonspecific T-cell mitogen phytohemagglutinin (PHA) (22). At the same time it can be shown that some mycobacterial infections seem to persist virtually indefinitely with no indication of any cellular hypersensitivity or the development of a cell-mediated antibacterial immune response capable of limiting
further growth of the slowly multiplying mycobacterial population within the lymphoreticular organs of the host (14, 26). For instance, mice infected with Mycobacterium simiae or the closely related M. habana (19) develop persisting systemic infections which bear a number of resemblances to M. leprae infections in humans. Such mice develop a persisting anergy to the homologous mycobacterial antigens (26), and their lymphocytes are unable to undergo blast transformation when exposed to M. habana cytoplasmic protein antigens (CPA) in vitro. MATERIALS AND METHODS Animals. Specific pathogen-free C57BL/6 x DBA2 (B6D2) F. hybrid mice were obtained from the Trudeau Animal Breeding Facility. Inbred C57BL/6, DBA-2, and A mice, as well as AB6 and CB6/F, hybrids, were tested. The mice were maintained under barrier conditions on sterile bedding and fed sterile commercial mouse chow and acidified water ad libitum (7, 20). They were 5 weeks old at the time of infection. Organisms. M. bovis (BCG Pasteur TMC 1011) was obtained from the Trudeau Mycobacterial Culture Collection. M. habana T13 was originally obtained from W. B. Schaefer, National Jewish Hospital, Denver, Colo. It was an M. simiae serotype II (K. Mc-
497
498
INFECT. IMMUN.
WATSON AND COLLINS
Clatchy, personal communication). The M. habana was dissociated into translucent (T13Tr) and smoothdomed (T13Smd) colony forms analogous to those described by Schaefer et al. (23) for M. avium. The latter colony type tended to decline at a markedly faster rate in intravenously infected B6D2 mice than either M. simiae or M. habana T13Tr (Fig. 1). The translucent colony variant could be serially transferred on 7H10 agar without reverting to the original mixed colony form. It could even be subcultured in MSTA liquid medium (modified Sauton medium with Tween80 and albumin; 8) for one or two transfers without serious reversion. M. habana T13Tr was transferred from 7H10 agar to MSTA in 500-ml roller tubes and incubated at 370C for 10 days (8). The logarithmicphase culture was dispensed into 1-ml ampoules and stored at -700C (4, 6). The frozen suspension was thawed at 370C, homogenized in an equal volume of fresh medium to break up any clumps, and plated on Middlebrook 7H10 agar. Almost 100%1o of the resulting colonies were of the translucent type. Antigen preparation. The organisms were cultured in MSTA in 1-liter roller bottles for 14 days (8). The cells were centrifuged at 10,000 x g for 30 min at 40C, suspended in 0.05% Tween-saline, and heat inactivated at 1000C for 30 min (26). Sterility tests were carried out by inoculating approximately 108 acid-fast bacilli onto 7H10 agar plates and incubating the cultures at 370C for 6 weeks before examining them for signs of growth. They were uniformly negative. Suspensions to be used for Mitsuda-type footpad tests (14) were standardized to 109 acid-fast bacilli per ml by microscopic counting (24). The mice were injected in a hind footpad with 0.02 ml of the heat-killed suspension (2 x 107 to 5 X 107 acid-fast bacilli). CPA. CPA were prepared by ultrasonic disruption of 5 x 10' acid-fast bacilli per ml at 0°C in 30-s bursts for 5 min (26). The cell breakage rate was followed by the drop in optical density. Residual whole cells and 6
debris were spun down at 10,000 x g for 30 min at 40C, followed by ultracentrifugation at 100,000 x g for 60 min in an International refrigerated ultracentrifuge. The clear supernatant was membrane filtered (Millipore Corp.) and stored at -70°C until required. The protein content was determined by the method of Lowry et al. (17), using a bovine serum albumin standard. The preparation was standardized at 250 ug of protein per ml, and 0.02 ml was injected into the right hind footpad; an equal volume of buffered saline was introduced into the opposite footpad. Swelling was measured with Schnelltaster dial gauge calipers (5) after 3, 6, 24, and 48 h. Some animals were footpad tested by injecting 5 x 107 heat-killed M. habana whole cells in 0.02 ml of 0.05% Tween-saline. Footpad measurements for these mice were also continued twice weekly for up to 6 weeks (26). Normal control mice were injected with an equal amount of each antigen, and the thickness measurements were subtracted from the test values. An increase of 2 U (0.2 mm) or more was significant at the 1% level (4, 5). Enumeration of the in vivo bacterial populations. Groups of five mice were sacrificed, and the lungs, livers, and spleens were removed aseptically and homogenized separately in cold 0.05% Tween-saline (6). Serial saline dilutions were plated on 7H10 agar and incubated in sealed bags at 37°C for 4 weeks before counting. T-lymphocyte purification. Washed nylon wool (Leuko-Pak leukocyte filter; Fenwal Labs, Deerfield, Ill.) was packed into a 60-ml disposable syringe barrel, gas sterilized, and used 3 to 4 days later (15). The column was rinsed with 20 ml of RPMI 1640 medium containing 5% fetal calf serum and left at 37°C to equilibrate for 1 h before it was loaded with cells obtained by expression of cut spleen pieces through a stainless steel grid into cold RMPI 1640 medium. The cells were loaded onto the column at the rate of 109 cells in 20 ml of medium and incubated at 37°C for 45
A
M. SIMIAE
5
4i z 4 cc 0
3
B C,. '5
U
4
Tr
.pL9
__
=-
0 -
3
-.
==-_ Smd _,--a 2 2
6 TIME IN MONTHS
8
10
12
FIG. 1. Growth of M. simiae (A) and M. habana (B) in normal B6D2 mice after intravenous inoculation. The M. habana culture was dissociated into translucent (Tr) and smooth-domed (Smd) colonial types (represented by the open symbols). Symbols: M and 0, spleen (Sp); A and A, lungs (Lg).
VOL. 25, 1979
SUPPRESSOR T-CELLS IN INFECTED MICE
min. The column was kept at 370C, and cells were eluted at a rate of 1 ml/min with warm medium. The eluted (T-cell-enriched) suspension was spun at 250 x g for 15 min, and the cells were resuspended at a standard concentration of 10' lymphocytes per ml, as determined by hemacytometer counts. Viability was checked by dye exclusion and was usually greater than 80%. Lymphocyte transformation. Spleen cells in RPMI 1640 medium containing 5% fetal calf serum were cultured in the presence of the following nonspecific mitogens: PHA (Difco Laboratories, Detroit, Mich.) and lipopolysaccharide (LPS; Escherichia coli 055:BS; Difco). The cells were also exposed to purified protein derivative (PPD; Connaught Laboratories, Toronto, Canada) and to the specific CPA prepared from M. habana (26). Samples (200 pl) of the spleen cell suspension (2 x 106 cells per ml) were dispensed in quadruplicate into flat-bottomed Linbro microtest plates (Flow Laboratories, Irvine, Scotland). The mitogens were added at final concentrations of 0.01, 0.1, 1, and 10 pg/culture well, and the existence of a dose response by the cells was checked each time. However, only the results of the 0.1-ug dose of mitogen are presented below since this seemed to constitute the optimal concentration. The cells were incubated for 48 h at 370C in humidified air containing 5% CO2. Each well received 0.1 uCi of tritiated thymidine (specific activity, 5 Ci/mmol; Radiochemical Centre, Amersham, United Kingdom) given 16 h before harvest with a Mash II sampler (Microbiological Associates, Bethesda, Md.). The radioactivity was measured with a Beckman liquid scintillation counter (20). The incorporation data are expressed as counts per minute (x103; mean ± standard error of the mean) or as the following transformation index: (experimental counts per minute-background counts per minute)/(control counts per minute-control background counts per minute). Anti-theta serum treatment. Lymphocyte suspensions were exposed to 1:80 anti-Thy-i antiserum (Cedarlane Labs, Hicksville, N.Y.) in the presence of 1:10 Low-Tox rabbit complement (21).
RESULTS Growth of M. habana in inbred mouse strains. Groups of 30 ICR, C3H, DBA, C57BL/ 6, A, B6D2, AB6, and CB6 mice were infected intravenously with 1 x 106 to 2 x 106 viable M. habana T13Tr cells. The resulting growth curves are shown in Fig. 2. The M. habana inoculum declined slowly with time in the lungs, livers, and spleens of most of the strains of mice tested, and by 6 months the number of viable organisms recovered represented about 1% of the initial inoculum. However the organisms did multiply slowly in the C57BL/6 mice, some of which died 10 to 12 months later as a result of ongoing lung infections. Delayed-type hypersensitivity to M. habana CPA. B6D2 mice were infected intravenously with 106, 107, or 108 viable M. habana
499
M.HABANA I.V. ICR
7
6
z 4
0 0
g. 2
B6D2
61
4 MA
04 4
0
Sp * Lg * Lr
0
2
4
2 6 0 TIME IN MONTHS
4
6
FIG. 2. Growth curves for M. habana Tr after intravenous (I. V.) inoculation in inbred and hybrid mice. Symbols: U, spleen (Sp); A, lungs (Lg); *, liver
(Lr).
T13Tr cells. Control mice received 106 or 108 viable BCG Pasteur cells. At increasing time intervals after vaccination, groups of mice (including age-matched uninfected controls) were footpad tested with 2.5 ,ug of PPD or 2 and 10 ,g of M. habana CPA (Table 1). By day 21, all three groups of M. habana-infected mice were footpad positive to M. habana CPA but not to PPD. By day 60, all of the animals were anergic to both test antigens. The two groups of BCGvaccinated mice were unresponsive to M. habana CPA. Mice infected with 106 viable BCG developed significant tuberculin sensitivity by day 14, whereas the group infected with 10' BCG remained unresponsive to the PPD injections throughout (Table 1). These latter results are in general agreement with earlier findings (4). Footpad swelling responses to M. habana whole-cell antigen. M. habana-infected mice were footpad tested after 2 months with 5 x 107
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Vol. 25, No. 2
Development of Suppressor T-Cells in Mycobacterium habana-Infected Mice SUSAN R. WATSON AND FRANK M. COLLINS* Trudeau Institute, Inc., Saranac Lake, New York 12983
Received for publication 7 May 1979
Mice were infected intravenously with increasing numbers of Mycobacterium habana (simiae serotype II), and the levels of delayed-type hypersensitivity to purified protein derivative and M. habana cytoplasmic protein antigen were determined after 14, 30, and 90 days. A footpad delayed-type hypersensitivity response was seen in 14-day-infected mice and was followed by a persisting anergy. T-cell-enriched suspensions collected 30 and 90 days into the infection (anergic donors) showed depressed transformation indexes after phytohemagglutinin and M. habana cytoplasmic protein antigen treatment in vitro. The corresponding B-cell mitogen (lipopolysaccharide) responses were not affected. Mixing experiments with T-cell-enriched suspensions from day-90 M. habana-infected donors adoptively suppressed lymphocyte transformation by normal and day-14 spleen cells. This effect could be ablated by anti-theta serum and complement treatment of the day-90 cells, indicating that the lack of in vitro responsiveness to cytoplasmic protein antigen was mediated by a population of suppressor T-cells present in the heavily infected spleens. There was no evidence that similar cells were present in the spleens of the 14-day-infected animals. Suppressor T-cells could be induced in vitro by exposure of day-14 spleen cells to concanavalin A or M. habana cytoplasmic protein antigen before they were mixed with normal or day-14 indicator splenic lymphocytes. The timing of the appearance of suppressor T-cells in the infected spleens corresponded to a loss of footpad hypersensitivity by the M. habana-infected animals. Lepromatous leprosy is characterized by the accumulation of large numbers of acid-fast bacilli within tissues which show little evidence of any cell-mediated immunity as the infection progresses (2). The patients also develop a profound state of lepromin anergy which tends to persist even after the mycobacterial load has been reduced by effective chemotherapeutic intervention (1, 11). Unlike tuberculoid leprosy, the peripheral blood lymphocytes from these anergic patients fail to exhibit an enhanced blast transformation in the presence of lepromin (10). A number of atypical mycobacteria are also able to establish persisting systemic infections which are associated with a profound state of skin test energy to the corresponding mycobacterial antigens (7). Heavy BCG infection has been shown to be associated with active suppression of lymphocyte transformation by spleen cells exposed to the nonspecific T-cell mitogen phytohemagglutinin (PHA) (22). At the same time it can be shown that some mycobacterial infections seem to persist virtually indefinitely with no indication of any cellular hypersensitivity or the development of a cell-mediated antibacterial immune response capable of limiting
further growth of the slowly multiplying mycobacterial population within the lymphoreticular organs of the host (14, 26). For instance, mice infected with Mycobacterium simiae or the closely related M. habana (19) develop persisting systemic infections which bear a number of resemblances to M. leprae infections in humans. Such mice develop a persisting anergy to the homologous mycobacterial antigens (26), and their lymphocytes are unable to undergo blast transformation when exposed to M. habana cytoplasmic protein antigens (CPA) in vitro. MATERIALS AND METHODS Animals. Specific pathogen-free C57BL/6 x DBA2 (B6D2) F. hybrid mice were obtained from the Trudeau Animal Breeding Facility. Inbred C57BL/6, DBA-2, and A mice, as well as AB6 and CB6/F, hybrids, were tested. The mice were maintained under barrier conditions on sterile bedding and fed sterile commercial mouse chow and acidified water ad libitum (7, 20). They were 5 weeks old at the time of infection. Organisms. M. bovis (BCG Pasteur TMC 1011) was obtained from the Trudeau Mycobacterial Culture Collection. M. habana T13 was originally obtained from W. B. Schaefer, National Jewish Hospital, Denver, Colo. It was an M. simiae serotype II (K. Mc-
497
498
INFECT. IMMUN.
WATSON AND COLLINS
Clatchy, personal communication). The M. habana was dissociated into translucent (T13Tr) and smoothdomed (T13Smd) colony forms analogous to those described by Schaefer et al. (23) for M. avium. The latter colony type tended to decline at a markedly faster rate in intravenously infected B6D2 mice than either M. simiae or M. habana T13Tr (Fig. 1). The translucent colony variant could be serially transferred on 7H10 agar without reverting to the original mixed colony form. It could even be subcultured in MSTA liquid medium (modified Sauton medium with Tween80 and albumin; 8) for one or two transfers without serious reversion. M. habana T13Tr was transferred from 7H10 agar to MSTA in 500-ml roller tubes and incubated at 370C for 10 days (8). The logarithmicphase culture was dispensed into 1-ml ampoules and stored at -700C (4, 6). The frozen suspension was thawed at 370C, homogenized in an equal volume of fresh medium to break up any clumps, and plated on Middlebrook 7H10 agar. Almost 100%1o of the resulting colonies were of the translucent type. Antigen preparation. The organisms were cultured in MSTA in 1-liter roller bottles for 14 days (8). The cells were centrifuged at 10,000 x g for 30 min at 40C, suspended in 0.05% Tween-saline, and heat inactivated at 1000C for 30 min (26). Sterility tests were carried out by inoculating approximately 108 acid-fast bacilli onto 7H10 agar plates and incubating the cultures at 370C for 6 weeks before examining them for signs of growth. They were uniformly negative. Suspensions to be used for Mitsuda-type footpad tests (14) were standardized to 109 acid-fast bacilli per ml by microscopic counting (24). The mice were injected in a hind footpad with 0.02 ml of the heat-killed suspension (2 x 107 to 5 X 107 acid-fast bacilli). CPA. CPA were prepared by ultrasonic disruption of 5 x 10' acid-fast bacilli per ml at 0°C in 30-s bursts for 5 min (26). The cell breakage rate was followed by the drop in optical density. Residual whole cells and 6
debris were spun down at 10,000 x g for 30 min at 40C, followed by ultracentrifugation at 100,000 x g for 60 min in an International refrigerated ultracentrifuge. The clear supernatant was membrane filtered (Millipore Corp.) and stored at -70°C until required. The protein content was determined by the method of Lowry et al. (17), using a bovine serum albumin standard. The preparation was standardized at 250 ug of protein per ml, and 0.02 ml was injected into the right hind footpad; an equal volume of buffered saline was introduced into the opposite footpad. Swelling was measured with Schnelltaster dial gauge calipers (5) after 3, 6, 24, and 48 h. Some animals were footpad tested by injecting 5 x 107 heat-killed M. habana whole cells in 0.02 ml of 0.05% Tween-saline. Footpad measurements for these mice were also continued twice weekly for up to 6 weeks (26). Normal control mice were injected with an equal amount of each antigen, and the thickness measurements were subtracted from the test values. An increase of 2 U (0.2 mm) or more was significant at the 1% level (4, 5). Enumeration of the in vivo bacterial populations. Groups of five mice were sacrificed, and the lungs, livers, and spleens were removed aseptically and homogenized separately in cold 0.05% Tween-saline (6). Serial saline dilutions were plated on 7H10 agar and incubated in sealed bags at 37°C for 4 weeks before counting. T-lymphocyte purification. Washed nylon wool (Leuko-Pak leukocyte filter; Fenwal Labs, Deerfield, Ill.) was packed into a 60-ml disposable syringe barrel, gas sterilized, and used 3 to 4 days later (15). The column was rinsed with 20 ml of RPMI 1640 medium containing 5% fetal calf serum and left at 37°C to equilibrate for 1 h before it was loaded with cells obtained by expression of cut spleen pieces through a stainless steel grid into cold RMPI 1640 medium. The cells were loaded onto the column at the rate of 109 cells in 20 ml of medium and incubated at 37°C for 45
A
M. SIMIAE
5
4i z 4 cc 0
3
B C,. '5
U
4
Tr
.pL9
__
=-
0 -
3
-.
==-_ Smd _,--a 2 2
6 TIME IN MONTHS
8
10
12
FIG. 1. Growth of M. simiae (A) and M. habana (B) in normal B6D2 mice after intravenous inoculation. The M. habana culture was dissociated into translucent (Tr) and smooth-domed (Smd) colonial types (represented by the open symbols). Symbols: M and 0, spleen (Sp); A and A, lungs (Lg).
VOL. 25, 1979
SUPPRESSOR T-CELLS IN INFECTED MICE
min. The column was kept at 370C, and cells were eluted at a rate of 1 ml/min with warm medium. The eluted (T-cell-enriched) suspension was spun at 250 x g for 15 min, and the cells were resuspended at a standard concentration of 10' lymphocytes per ml, as determined by hemacytometer counts. Viability was checked by dye exclusion and was usually greater than 80%. Lymphocyte transformation. Spleen cells in RPMI 1640 medium containing 5% fetal calf serum were cultured in the presence of the following nonspecific mitogens: PHA (Difco Laboratories, Detroit, Mich.) and lipopolysaccharide (LPS; Escherichia coli 055:BS; Difco). The cells were also exposed to purified protein derivative (PPD; Connaught Laboratories, Toronto, Canada) and to the specific CPA prepared from M. habana (26). Samples (200 pl) of the spleen cell suspension (2 x 106 cells per ml) were dispensed in quadruplicate into flat-bottomed Linbro microtest plates (Flow Laboratories, Irvine, Scotland). The mitogens were added at final concentrations of 0.01, 0.1, 1, and 10 pg/culture well, and the existence of a dose response by the cells was checked each time. However, only the results of the 0.1-ug dose of mitogen are presented below since this seemed to constitute the optimal concentration. The cells were incubated for 48 h at 370C in humidified air containing 5% CO2. Each well received 0.1 uCi of tritiated thymidine (specific activity, 5 Ci/mmol; Radiochemical Centre, Amersham, United Kingdom) given 16 h before harvest with a Mash II sampler (Microbiological Associates, Bethesda, Md.). The radioactivity was measured with a Beckman liquid scintillation counter (20). The incorporation data are expressed as counts per minute (x103; mean ± standard error of the mean) or as the following transformation index: (experimental counts per minute-background counts per minute)/(control counts per minute-control background counts per minute). Anti-theta serum treatment. Lymphocyte suspensions were exposed to 1:80 anti-Thy-i antiserum (Cedarlane Labs, Hicksville, N.Y.) in the presence of 1:10 Low-Tox rabbit complement (21).
RESULTS Growth of M. habana in inbred mouse strains. Groups of 30 ICR, C3H, DBA, C57BL/ 6, A, B6D2, AB6, and CB6 mice were infected intravenously with 1 x 106 to 2 x 106 viable M. habana T13Tr cells. The resulting growth curves are shown in Fig. 2. The M. habana inoculum declined slowly with time in the lungs, livers, and spleens of most of the strains of mice tested, and by 6 months the number of viable organisms recovered represented about 1% of the initial inoculum. However the organisms did multiply slowly in the C57BL/6 mice, some of which died 10 to 12 months later as a result of ongoing lung infections. Delayed-type hypersensitivity to M. habana CPA. B6D2 mice were infected intravenously with 106, 107, or 108 viable M. habana
499
M.HABANA I.V. ICR
7
6
z 4
0 0
g. 2
B6D2
61
4 MA
04 4
0
Sp * Lg * Lr
0
2
4
2 6 0 TIME IN MONTHS
4
6
FIG. 2. Growth curves for M. habana Tr after intravenous (I. V.) inoculation in inbred and hybrid mice. Symbols: U, spleen (Sp); A, lungs (Lg); *, liver
(Lr).
T13Tr cells. Control mice received 106 or 108 viable BCG Pasteur cells. At increasing time intervals after vaccination, groups of mice (including age-matched uninfected controls) were footpad tested with 2.5 ,ug of PPD or 2 and 10 ,g of M. habana CPA (Table 1). By day 21, all three groups of M. habana-infected mice were footpad positive to M. habana CPA but not to PPD. By day 60, all of the animals were anergic to both test antigens. The two groups of BCGvaccinated mice were unresponsive to M. habana CPA. Mice infected with 106 viable BCG developed significant tuberculin sensitivity by day 14, whereas the group infected with 10' BCG remained unresponsive to the PPD injections throughout (Table 1). These latter results are in general agreement with earlier findings (4). Footpad swelling responses to M. habana whole-cell antigen. M. habana-infected mice were footpad tested after 2 months with 5 x 107
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