CELLULAR
IMMUNOLOGY
22, l-10 (1976)
Stem Cells and T and B Lymphocytes during Tumor Growth R. M. KHAITOV, R. V. PETROV, S. S. GAMBAROV, A. S. NORIMOV, AND V. A. BLINOV Institute
of
Biophysics, Ministry
of
Health, Moscow, USSR
Received April 22,1975 Functional activities of T and B lymphocytes and the kinetics of hematopoietic stem cells were studied in mice with inoculated or spontaneous tumors. The development and growth of the tumor inhibited B cells and helper T cells, while the activity of killer T cells and spleen suppressor cells was markedly enhanced. The processes of stem cell migration from the bone marrow were considerably intensified and altered in tumor-bearing mice. Data were obtained suggesting that helper T cells and killer T cells represent nonidentical compartments within the population of thymus-dependent lymphocytes. Immunosuppression during tumor bearing is probably due to an impairment of T lymphocytes cooperating in immune responses, B-lymphocytes and their precursors.
INTRODUCTION The problem of immunologic reactivity during tumor growth can be approached through experimental studies of the main cellular events responsible for immunogenesis. The existence of two types of immunocompetent cells is well established at present : B cells, thymus-independent precursors of antibody-forming cells (AFC), and T cells, thymus-dependent lymphocytes that regulate the response of B cells to antigens and are active in cell-mediated immune reactions (1, 2). Recent investigations have suggested that the T-cell population is heterogeneous and includes compartments of cells with killer, helper and suppressor effects (3-6). According to current concepts, both T and B cells are derived from hematopoietic multipotential stem cells. The purpose of the present study was to evaluate (i) cooperation of T and B lymphocytes in humoral immune responses ; (ii) inactivation of foreign stem cells by killer T cells in the graft-versus-host (GVH) reaction, (iii) the activity of spleen suppressor cells, and (iv) the kinetics of colony-forming stem cells (or colony-forming units, CFU) during tumor growth in mice. METHODS
Animals. C57B1/6, C3H, A/He, CBA and hybrid (CBA x C57B1/6)F1 mice were used for the experiments. The following groups of animals served as tumor bearers : (i) C57B1/6 and (CBA x C57B1/6) F 1 mice inoculated with carcinoma 755, (Ca-755) which had been induced with methylcholanthrene in C57B1/6
2
KHAITOV
ET AL.
donors ; (ii) CBA mice with transplants of cancer of the cervix (RShM-5) from syngeneic donors ; (ii) C3H and A/He mice with spontaneous mammary cancer. Tumor cells were transplanted by the usual methods (7). Cell suspensions. By use of Medium 199, the bone marrow was washed out of the femoral and tibia1 medullary canals and aspirated repeatedly through a needle to eliminate all coarse particles and ‘threads. Inguinal, mesenteric and axillary lymph nodes and thymuses were minced with ophthalmological scissors in Medium 199 and aspirated repeatedly through a needle. The suspensions obtained were filtered through a stainless-steel sieve. Cooperation of T and B cells. Cooperation in the immune response was assayed by the adoptive transfer technique. Various combinations of marrow cells (B cells) and lymph node or thymus cells (T cells) from tumor bearers or intact mice were injected iv into irradiated recipients 4-6 hr after lethal irradiation with 900 R. In some experiments the spleens of B mice (8) were used as a source of B cells. For antigenic stimulation, 2 x lo8 sheep red blood cells (SRBC) per animal were injected together with the cell suspension. AFC were counted in the spleens of the recipients according to Jerne’s method (9) on the 8th day after cell transfer. The killer effect of T cells. This was measured by the capacity of lymph node cells from C57B1/6 mice (tumor bearing and intact) to inactivate endogenous stem cells in (CBA x C57B1/6) F1 hybrids subjected to sublethal irradiation with 675 R (10,ll). The e#ect of spleen suppressor cells. This effect on the production of AFC to SRBC during the GVH reaction was studied in the model suggested by Miiller (12). Spleen cells (50 x lOa) from intact or tumor-bearing C57B1/6 mice were injected into intact (CBA x C57B1/6)Fl hybrids. Seven days later each of the
AFC 3000
0
Bi+Ti
m
Bit-R
Bt +Ti
2soiY
t
2om
1500 1000 500 0:
Days
after
tumor
inoculation
FIG. 1. Spleen counts of AFC in recipients of T and B cells from intact and tumor-bearing
mice. B,, intact B cells; Bt, B cells from tumor-bearing from tumor-bearing mice.
mice; TI, intact T cells; Tt, T cells
IMMUNOCOMPETENT
CELLS
AND
TUMOR
GROWTH
3
recipients was immunized with 5 x lo* SRBC. Splenic AFC were counted by Jerne’s method (9) on the 5th day after immunization. Assays of CFU. The number of CFU in the marrow was determined according to the exogenous clone method (13). Three or four mice were sampled from each group (control and tumor bearing) and their marrow cells were injected iv into syngeneic recipients which had been irradiated 3-4 hr earlier with a lethal dose of 900 R. Exogenous spleen colonies were counted within 8 days. For assaying endogenous CFU, mice were irradiated with 600 R, and counts of endogenous spleen colonies were done 9 days later (14). Migration of CFU from the marrow was evaluated as described in detail previously ( 15). In brief, during 800-R irradiation the hind extremity of a mouse was protected with a screen down to the midportion of the lower leg. Eight or nine days later the spleen ‘was examined for colonies produced by the CFU that had migrated from the protected segment of the marrow. Irradiation. Irradiation was delivered from a source of gamma rays at a dose rate of 320 or 900 R/min. Statistics. Geometric means and the upper and lower limits of the standard error were calculated, as well as P values. RESULTS Cooperation of T and B Cells As shown in Fig. 1, adoptive transfer of B cells (bone marrow, 1 X 107) from intact mice together with T cells (lymph node cells, 1 X 10e) isolated from tumor bearers 7 days after inoculation of the tumor (Ca-755) resulted in a similar spleen content of AFC in syngeneic C57B1/6 recipients as compared to that in the animals injected with B and T cells from intact mice (control group). However, when normal B cells were transferred with T cells taken from the donors 10 days after tumor inoculation, the numbers of AFC in the spleens of the recipients were 3-3.5 times lower than in control mice. After a combined injection of B cells from intact animals and T cells from mice with a 1Cday period of tumor bearing, the accumulation of AFC was 2.5-3 times less than after the transfer of normal B and T cells. An analysis of the functional activity of B cells showed that the induction of tumor growth affected them earlier than T cells. Thus, by the 7th day after inoculation of Ca-755, cooperation of B cells with intact T cells was considerably reduced (Fig. 1). B cells taken from tumor-bearing animals on the 10th and 14th days were inhibited to a somewhat greater extent. Table 1 illustrates a significant decrease in the cooperative activity of T cells from mice with spontaneous tumors. In a series of double-transfer experiments, B cells were obtained from the spleens of lethally irradiated CBA mice restored with the marrow of intact CBA donors (control B mice) or of tumor bearers 25 days after transplantation of the RShM-5 cancer (experimental B mice). Table 2 shows that following an injection of normal thymocytes together with spleen cells from control B mice the spleen count of AFC in irradiated recipients was seven to eight times higher than after the use of normal bone marrow as a source of B cells. If, however, spleen cells were taken from experimental B mice, the numbers of AFC in the spleens of irradiated secondary recipients decreased sixfold as compared to the use of control B mice.
4
KHAITOV
ET AL.
TABLE
1
NUMBERS OF SPLEEN AFC IN LETHALLY IRRADIATED SYNGENEIC RECIPIENTS INJECTED INTACT B CELLS AND T CELLS FROMMICE BEARING SPONTANEOUSTUMORS Strain of mouse
No. of AFC per spleen (geometric mean f SE)
P
Intact B cells + intact T cells0
Intact B cells + T cells from tumor bearers=
1654 (2032-1329) 1248 (1635- 951)
555 (624494) 369 (430-315)
A/He C3H
WITH
30 10.2 (11.9-8.9) >30
9
14 21
9 10 6
The concentrations and especially the absolute numbers of CFU also fell in the marrow of mice with spontaneous mammary cancer. On the other hand, our assays of endogenous spleen CFU 1421 days after inoculation of Ca-755 showed that sublethally irradiated tumor bearers produced considerably more hematopoietic colonies than control mice (Table 6). Indeed, following irradiation with 600 R the spleen content of endogenous colonies ranged from 6.2 * 0.8 to 10.4 -+ 1.3 in normal animals and was three to five times higher in mice with Ca-755 (about 30-40 colonies which grew confluently). The numbers of exogenous CFU were also increased in the spleens of tumor bearers (Table 7). Special experiments were required in order to explain these findings. The results indicate that the discharge of marrow stem cells into the blood stream was greatly increased in tumor-bearing animals. Thus, in normal mice 9.6 + 1.1 to 10.1 -L 0.7 CFU migrated from the marrow of a hind extremity that had been protected with a screen during lethal irradiation; in mice with Ca-755 tested 14-21 days after tumor grafting more than 30 CFU were discharged from this segment of the marrow (Table 8). Thus, the initial period of tumor bearing in mice with Ca-755 was characterized by intensive proliferation of stem cells and their increased outflow from the bone marrow. Yet at the terminal stage of the disease, the number of stem cells in the marrow was considerably reduced. We observed that in lethally irradiated recipients injected with marrow cells from syngeneic tumor bearers, C57B1/6 or (CBA X C57B1/6)Fr, the average TABLE
7
NUMBERS OF HEMATOPOIETIC STEM CELLS IN THE SPLEENS OF (CBA X C57B1/6)FI MICE WITH Ca-755 TRANSPLANTS Time after Ca-755 inoculation (days)
No. of recipients of spleen cells
-
8
14
9
No. of CFU per lo6 spleen cells (geometric mean f SE)
Total No. of CFU per spleen
17.1 (18.5-15.9) 29.9 (30.5-29.3) P < 0.001
3,480 30,000
8
KHAITOV
ET AL.
TABLE
8
MIGRATION OF HEMATOPOIETIC STEM CELLS FROM THE BONE MARROW OF MICE WITH Ca-755 TRANSPLANTS Time after Ca-755 inoculation (days)
No. of mice
No. of CFU migrating from the marrow segment protected during lethal irradiation (geometric mean f SE)
-
12 16 10 11
10.0 (10.8-9.2) >30 9.4 (10.7-8.2) >30
14 21
weight of the marrow cells was the rule bearing mice
spleen was much higher than in control animals that had received from normal donors (Table 9). Incidentally, extreme splenomegaly in nonirradiated tumor bearers and could be found also in tumorafter sublethal or lethal but nonuniform irradiation. DISCUSSION
The present results show that the ability of marrow B cells and lymph node T cells to cooperate in the humoral immune response to SRBC is reduced during tumor growth, B cells being affected earlier. Since we have found (unpublished data) that tumor growth affects also spleen B cells, this decrease in the cooperative ability of the bone marrow cannot be explained by a loss of its B cells as a result of augmented migration to the spleen. We have also observed that helper T-cells are affected irrespective of their provenance ; i.e., in the spleen and thymus as well as in the lymph nodes of tumor bearers. In adoptive transfer systems, experimental results are undoubtedly influenced by the migration of the cells transferred to the recipients. However, as shown TABLE
9
SPLEEN WEIGHTS IN LETHALLY IRRADIATED RECIPIENTS OF MARROW CELLS FROM INTACT AND TUMOR-BEARING MICE Strain of recipients
Donors
CS7B1/6 CS7B1/6 (CBA (CBA with (CBA with
CS7Bl/6 C57Bl/6
intact with Ca-7.W
X CSfBl/6)Fr X C57B1/6)F1 Ca-7SP x C57Bl/6)F1 Ca-755 b
a Marrow b Marrow
obtained obtained
intact
No. of recipients 9 10
26.8 89.9
(2&l(91.7-
25.5) 88.1)
49.2
(51.4-
47.2)
104.2 (110.2-
98.8)
(CBA
X CS?Bl/6)F1
6
(CBA
X CS7Bl/6)F,
8
(CBA x CS7Bl/6)F,
IO
10 days after Ca-755 inoculation. 20 days after Ca-7.55 inoculation.
Spleen weight, (mg ; geometric mean f SE)
142.7 (146.9-138.6)
P
IMMUNOLOGY
22, l-10 (1976)
Stem Cells and T and B Lymphocytes during Tumor Growth R. M. KHAITOV, R. V. PETROV, S. S. GAMBAROV, A. S. NORIMOV, AND V. A. BLINOV Institute
of
Biophysics, Ministry
of
Health, Moscow, USSR
Received April 22,1975 Functional activities of T and B lymphocytes and the kinetics of hematopoietic stem cells were studied in mice with inoculated or spontaneous tumors. The development and growth of the tumor inhibited B cells and helper T cells, while the activity of killer T cells and spleen suppressor cells was markedly enhanced. The processes of stem cell migration from the bone marrow were considerably intensified and altered in tumor-bearing mice. Data were obtained suggesting that helper T cells and killer T cells represent nonidentical compartments within the population of thymus-dependent lymphocytes. Immunosuppression during tumor bearing is probably due to an impairment of T lymphocytes cooperating in immune responses, B-lymphocytes and their precursors.
INTRODUCTION The problem of immunologic reactivity during tumor growth can be approached through experimental studies of the main cellular events responsible for immunogenesis. The existence of two types of immunocompetent cells is well established at present : B cells, thymus-independent precursors of antibody-forming cells (AFC), and T cells, thymus-dependent lymphocytes that regulate the response of B cells to antigens and are active in cell-mediated immune reactions (1, 2). Recent investigations have suggested that the T-cell population is heterogeneous and includes compartments of cells with killer, helper and suppressor effects (3-6). According to current concepts, both T and B cells are derived from hematopoietic multipotential stem cells. The purpose of the present study was to evaluate (i) cooperation of T and B lymphocytes in humoral immune responses ; (ii) inactivation of foreign stem cells by killer T cells in the graft-versus-host (GVH) reaction, (iii) the activity of spleen suppressor cells, and (iv) the kinetics of colony-forming stem cells (or colony-forming units, CFU) during tumor growth in mice. METHODS
Animals. C57B1/6, C3H, A/He, CBA and hybrid (CBA x C57B1/6)F1 mice were used for the experiments. The following groups of animals served as tumor bearers : (i) C57B1/6 and (CBA x C57B1/6) F 1 mice inoculated with carcinoma 755, (Ca-755) which had been induced with methylcholanthrene in C57B1/6
2
KHAITOV
ET AL.
donors ; (ii) CBA mice with transplants of cancer of the cervix (RShM-5) from syngeneic donors ; (ii) C3H and A/He mice with spontaneous mammary cancer. Tumor cells were transplanted by the usual methods (7). Cell suspensions. By use of Medium 199, the bone marrow was washed out of the femoral and tibia1 medullary canals and aspirated repeatedly through a needle to eliminate all coarse particles and ‘threads. Inguinal, mesenteric and axillary lymph nodes and thymuses were minced with ophthalmological scissors in Medium 199 and aspirated repeatedly through a needle. The suspensions obtained were filtered through a stainless-steel sieve. Cooperation of T and B cells. Cooperation in the immune response was assayed by the adoptive transfer technique. Various combinations of marrow cells (B cells) and lymph node or thymus cells (T cells) from tumor bearers or intact mice were injected iv into irradiated recipients 4-6 hr after lethal irradiation with 900 R. In some experiments the spleens of B mice (8) were used as a source of B cells. For antigenic stimulation, 2 x lo8 sheep red blood cells (SRBC) per animal were injected together with the cell suspension. AFC were counted in the spleens of the recipients according to Jerne’s method (9) on the 8th day after cell transfer. The killer effect of T cells. This was measured by the capacity of lymph node cells from C57B1/6 mice (tumor bearing and intact) to inactivate endogenous stem cells in (CBA x C57B1/6) F1 hybrids subjected to sublethal irradiation with 675 R (10,ll). The e#ect of spleen suppressor cells. This effect on the production of AFC to SRBC during the GVH reaction was studied in the model suggested by Miiller (12). Spleen cells (50 x lOa) from intact or tumor-bearing C57B1/6 mice were injected into intact (CBA x C57B1/6)Fl hybrids. Seven days later each of the
AFC 3000
0
Bi+Ti
m
Bit-R
Bt +Ti
2soiY
t
2om
1500 1000 500 0:
Days
after
tumor
inoculation
FIG. 1. Spleen counts of AFC in recipients of T and B cells from intact and tumor-bearing
mice. B,, intact B cells; Bt, B cells from tumor-bearing from tumor-bearing mice.
mice; TI, intact T cells; Tt, T cells
IMMUNOCOMPETENT
CELLS
AND
TUMOR
GROWTH
3
recipients was immunized with 5 x lo* SRBC. Splenic AFC were counted by Jerne’s method (9) on the 5th day after immunization. Assays of CFU. The number of CFU in the marrow was determined according to the exogenous clone method (13). Three or four mice were sampled from each group (control and tumor bearing) and their marrow cells were injected iv into syngeneic recipients which had been irradiated 3-4 hr earlier with a lethal dose of 900 R. Exogenous spleen colonies were counted within 8 days. For assaying endogenous CFU, mice were irradiated with 600 R, and counts of endogenous spleen colonies were done 9 days later (14). Migration of CFU from the marrow was evaluated as described in detail previously ( 15). In brief, during 800-R irradiation the hind extremity of a mouse was protected with a screen down to the midportion of the lower leg. Eight or nine days later the spleen ‘was examined for colonies produced by the CFU that had migrated from the protected segment of the marrow. Irradiation. Irradiation was delivered from a source of gamma rays at a dose rate of 320 or 900 R/min. Statistics. Geometric means and the upper and lower limits of the standard error were calculated, as well as P values. RESULTS Cooperation of T and B Cells As shown in Fig. 1, adoptive transfer of B cells (bone marrow, 1 X 107) from intact mice together with T cells (lymph node cells, 1 X 10e) isolated from tumor bearers 7 days after inoculation of the tumor (Ca-755) resulted in a similar spleen content of AFC in syngeneic C57B1/6 recipients as compared to that in the animals injected with B and T cells from intact mice (control group). However, when normal B cells were transferred with T cells taken from the donors 10 days after tumor inoculation, the numbers of AFC in the spleens of the recipients were 3-3.5 times lower than in control mice. After a combined injection of B cells from intact animals and T cells from mice with a 1Cday period of tumor bearing, the accumulation of AFC was 2.5-3 times less than after the transfer of normal B and T cells. An analysis of the functional activity of B cells showed that the induction of tumor growth affected them earlier than T cells. Thus, by the 7th day after inoculation of Ca-755, cooperation of B cells with intact T cells was considerably reduced (Fig. 1). B cells taken from tumor-bearing animals on the 10th and 14th days were inhibited to a somewhat greater extent. Table 1 illustrates a significant decrease in the cooperative activity of T cells from mice with spontaneous tumors. In a series of double-transfer experiments, B cells were obtained from the spleens of lethally irradiated CBA mice restored with the marrow of intact CBA donors (control B mice) or of tumor bearers 25 days after transplantation of the RShM-5 cancer (experimental B mice). Table 2 shows that following an injection of normal thymocytes together with spleen cells from control B mice the spleen count of AFC in irradiated recipients was seven to eight times higher than after the use of normal bone marrow as a source of B cells. If, however, spleen cells were taken from experimental B mice, the numbers of AFC in the spleens of irradiated secondary recipients decreased sixfold as compared to the use of control B mice.
4
KHAITOV
ET AL.
TABLE
1
NUMBERS OF SPLEEN AFC IN LETHALLY IRRADIATED SYNGENEIC RECIPIENTS INJECTED INTACT B CELLS AND T CELLS FROMMICE BEARING SPONTANEOUSTUMORS Strain of mouse
No. of AFC per spleen (geometric mean f SE)
P
Intact B cells + intact T cells0
Intact B cells + T cells from tumor bearers=
1654 (2032-1329) 1248 (1635- 951)
555 (624494) 369 (430-315)
A/He C3H
WITH
30 10.2 (11.9-8.9) >30
9
14 21
9 10 6
The concentrations and especially the absolute numbers of CFU also fell in the marrow of mice with spontaneous mammary cancer. On the other hand, our assays of endogenous spleen CFU 1421 days after inoculation of Ca-755 showed that sublethally irradiated tumor bearers produced considerably more hematopoietic colonies than control mice (Table 6). Indeed, following irradiation with 600 R the spleen content of endogenous colonies ranged from 6.2 * 0.8 to 10.4 -+ 1.3 in normal animals and was three to five times higher in mice with Ca-755 (about 30-40 colonies which grew confluently). The numbers of exogenous CFU were also increased in the spleens of tumor bearers (Table 7). Special experiments were required in order to explain these findings. The results indicate that the discharge of marrow stem cells into the blood stream was greatly increased in tumor-bearing animals. Thus, in normal mice 9.6 + 1.1 to 10.1 -L 0.7 CFU migrated from the marrow of a hind extremity that had been protected with a screen during lethal irradiation; in mice with Ca-755 tested 14-21 days after tumor grafting more than 30 CFU were discharged from this segment of the marrow (Table 8). Thus, the initial period of tumor bearing in mice with Ca-755 was characterized by intensive proliferation of stem cells and their increased outflow from the bone marrow. Yet at the terminal stage of the disease, the number of stem cells in the marrow was considerably reduced. We observed that in lethally irradiated recipients injected with marrow cells from syngeneic tumor bearers, C57B1/6 or (CBA X C57B1/6)Fr, the average TABLE
7
NUMBERS OF HEMATOPOIETIC STEM CELLS IN THE SPLEENS OF (CBA X C57B1/6)FI MICE WITH Ca-755 TRANSPLANTS Time after Ca-755 inoculation (days)
No. of recipients of spleen cells
-
8
14
9
No. of CFU per lo6 spleen cells (geometric mean f SE)
Total No. of CFU per spleen
17.1 (18.5-15.9) 29.9 (30.5-29.3) P < 0.001
3,480 30,000
8
KHAITOV
ET AL.
TABLE
8
MIGRATION OF HEMATOPOIETIC STEM CELLS FROM THE BONE MARROW OF MICE WITH Ca-755 TRANSPLANTS Time after Ca-755 inoculation (days)
No. of mice
No. of CFU migrating from the marrow segment protected during lethal irradiation (geometric mean f SE)
-
12 16 10 11
10.0 (10.8-9.2) >30 9.4 (10.7-8.2) >30
14 21
weight of the marrow cells was the rule bearing mice
spleen was much higher than in control animals that had received from normal donors (Table 9). Incidentally, extreme splenomegaly in nonirradiated tumor bearers and could be found also in tumorafter sublethal or lethal but nonuniform irradiation. DISCUSSION
The present results show that the ability of marrow B cells and lymph node T cells to cooperate in the humoral immune response to SRBC is reduced during tumor growth, B cells being affected earlier. Since we have found (unpublished data) that tumor growth affects also spleen B cells, this decrease in the cooperative ability of the bone marrow cannot be explained by a loss of its B cells as a result of augmented migration to the spleen. We have also observed that helper T-cells are affected irrespective of their provenance ; i.e., in the spleen and thymus as well as in the lymph nodes of tumor bearers. In adoptive transfer systems, experimental results are undoubtedly influenced by the migration of the cells transferred to the recipients. However, as shown TABLE
9
SPLEEN WEIGHTS IN LETHALLY IRRADIATED RECIPIENTS OF MARROW CELLS FROM INTACT AND TUMOR-BEARING MICE Strain of recipients
Donors
CS7B1/6 CS7B1/6 (CBA (CBA with (CBA with
CS7Bl/6 C57Bl/6
intact with Ca-7.W
X CSfBl/6)Fr X C57B1/6)F1 Ca-7SP x C57Bl/6)F1 Ca-755 b
a Marrow b Marrow
obtained obtained
intact
No. of recipients 9 10
26.8 89.9
(2&l(91.7-
25.5) 88.1)
49.2
(51.4-
47.2)
104.2 (110.2-
98.8)
(CBA
X CS?Bl/6)F1
6
(CBA
X CS7Bl/6)F,
8
(CBA x CS7Bl/6)F,
IO
10 days after Ca-755 inoculation. 20 days after Ca-7.55 inoculation.
Spleen weight, (mg ; geometric mean f SE)
142.7 (146.9-138.6)
P
