Further characterization of the suppressor cells, activated by goat anti-Th-B antibody reagent and responsible for enhanced growth of sarcoma 180 in AKR mice

CELLULARIMMUNoLoGY 37, 61-76 (1978)

Further Characterization of the Suppressor Cells, Activated Goat Anti-Th-B Antibody Reagent and Responsible for Enhanced Growth of Sarcoma 180 in AKR Mice KIICHI

KAKIMOTO, Department

HIROSHI

FUJI,

ALLAN

by

L. GROSSBERG, AXD DAVID PRESSMAN

of Immmwlogy

Research, Roswell Park Memorial Institute,l Buffalo, New York 14263

Received

September

28,1977

In our previous study, thymus cells were shown to be responsible for enhancing the growth of the allogeneic sarcoma 180 (S180) in AKR mice that had been injected with goat anti-Th-B antibody reagent (antiserum raised in goats against Balb/c myeloma MOPC 104E cells and purified). We suggested that the cells producing enhancement are suppressor T cells. We now show that the cells responsible for tumor enhancement are indeed T cells, since they carry the Thy-l antigen on their surface. Treatment of the cells in vitro with anti-Thy-l plus complement completely eliminates their ability to enhance tumor growth. The thymocytes responsible for tumor enhancement do not carry the Th-B determinant. Treating thymocytes in. vitro with goat antiTh-B antibody reagent plus complement does not abrogate their tumor-enhancing activity. This suggests that the suppressor T cells involved in tumor enhancement are generated by the interaction of anti-Th-B antibodies with precursor suppressor cells which do carry Th-B. Once generated, the active suppressor cells lose the Th-B antigen. This suggestion is supported by our finding that the thymic precursors of Con A-inducible suppressor cells bear Th-B, since they are killed by anti-Th-B plus complement, whereas active suppressor cells induced by Con A do not carry Th-B, since they are not killed by anti-Th-B plus complement. Neither splenic precursors of Con A-inducible suppressor cells nor the active suppressor cells thus induced carry Th-B since neither is killed by anti-Th-B plus complement. We have also found that there are apparently nonthymic suppressor cell precursors which can also be activated by anti-Th-B, since spleen cells from thymectomized mice bearing S180 and treated with anti-Th-B can transfer the tumor-enhancing effect. We conclude that precursors of suppressor cells carry the Th-B determinant. These precursors differentiate to active suppressor cells when stimulated by anti-Th-B antibodies. This process can take place either outside the thymus or in the thymus. Once differentiated, the mature suppressor cells no longer bear the Th-B marker and migrate from their sites of induction. Such cells can suppress immune mechanisms responsible for allogeneic tumor graft rejection and thus cause tumor enhancement.

INTRODUCTION Recently, the existence of suppressor cells in cell-mediated immunity has been demonstrated by several investigators (l-4). Furthermore, evidence suggesting that suppressor cells are responsible for the enhanced growth of tumor cells has f A unit of the New York State Department of Health.

0008-8749/78/0371-0061$02.00/0 I

Copyright0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

62

KAKIMOTO

ET

AL.

also been presented by Fujimoto et al. (5, 6)) Treves et al. (7), Small (S), and Kilburn et al. (9). In our preceding paper (lo), we demonstrated that the injection of goat anti-Th-B antibody reagent (prepared from antiserum raised against Balb/c myeloma MOPC 104E cells and purified as described by Yutoku et al. (11) ) into AKR mice resulted in significant enhanced growth of the allogeneic sarcoma 180. This effect on tumor growth is transferable most effectively by the injection of thymus cells and less so by spleen cells derived from anti-Th-Btreated, tumor-bearing animals into tumor recipients. It appears that a precursor of suppressor cells carries the Th-B determinant and that anti-Th-B antibodies stimulate these precursor cells into mature active suppressor cells instead of killing them. Then, these mature active suppressor cells apparently interfere with the immune mechanism responsible for the rejection of sarcoma 180 in AKR mice. In the present study we have further characterized these suppressor cells and obtained evidence that they are not macrophages but are thymocytes carrying the Thy-l antigen, In addition, we now show that the tumor-enhancing effect of antiTh-B induced thymocytes is not abrograted by treating these cells with anti-Th-B antibody plus complement. Therefore it appears that on being generated by antiTh-B antibody reagent, mature suppressor cells lose the Th-B determinant since they are no longer sensitive to anti-Th-B antibody. It was also shown that thymectomy does not abrogate the tumor-enhancing effect of injected goat anti-Th-B antibody, so that it appears that these are also nonthymic precursors of the suppressor cells and these also carry the Th-B determinant. MATERIALS

AND

METHODS

Mice. Balb/c Cr, DBA/2 Cr, and AKR/SN female mice and Ha/ ICR Swiss male mice, 6 to 8 weeks old, were supplied by the West Seneca Breeding Center of RPMI. Inbred Swiss mice were made available by the Grace Cancer Drug Center of this Institute. Tumors. Sarcoma 180 ( SlSO) was obtained from the Grace Cancer Drug Center and maintained in our laboratory by serial transplantation in both female and male inbred Swiss mice. Tumor transplantation was carried out subcutaneously by standard trocar techniques. Sarcoma 180 is a “nonspecific” tumor of unknown host origin and grows initially after subcutaneous implantation but finally regresses in almost 100% of AKR Mice. Ehrlich ascites tumor was propagated in Ha/ICR Swiss males. P815 mastocytoma ascites tumor was propagated in syngeneic DBA/Z females. Tumor growth measurements. The longest diameter and that perpendicular to it were measured for each tumor and the average of these values was taken as the measure of size. Statistical analysis of the significance of differences in tumor size was done using Student’s t test. Preparation of goat antibody reagent against MOPC 104E cells. The goat antisera raised against MOPC 104E cells were either the same serum described previously (11) or additional bleedings from the same goat. Not all goats injected with MOPC 104E cells yielded antibodies of this specificity. Only one out of eight goats gave high-titer anti-Th-B antibody. In vivo absorption of antiserum was done as described previously (1 l), by passage through Balb/c mice. In &JO purified antiserum was inactivated at 56°C for 30 min and stored frozen and is termed the goat anti-Th-B antibody reagent (goat anti-Th-B). A goat normal serum control

SUPPRESSOR

CELLS

IN

TUMOR

ENHANCEMENT

63

reagent (goat NS) was prepared in the same manner using normal goat serum ilJ place of the antiserum. Cell suspensions. Preparation of single cell suspensions of each tissue was done as described previously (10). Total cells were counted following staining with 0.1% crystal violet in 0.1 M citric acid, and the viability of cells was checked by trypan blue dye exclusion test. Spleen and thymus cell transfers. Spleen and thymus were obtained after eight injections of goat anti-Th-B or goat NS into SlSO-bearing AKR mice. Single cell suspensions of spleen cells and thymocytes which were carefully separated from parathymic lymph nodes were prepared in balanced salt solution, and 5 X lo7 cells in 0.5 ml were injected iv into AKR mice which had been transplanted with S180 on the previous day. Adult thymectomy (ATx). AKR mice were thymectomized at 6 or 10 weeks of age and used 4 weeks later. Control mice were sham-thymectomized and used in the same manner. Thymectomized mice were checked after the experiment, and when remnants of the thymus were found, those mice were removed from the experimental group. Alloantiserum against Thy- 1.1 antigen was preAnti-Thy-l .1 alloantiserum. pared by a method described by Reif and Allen (12). Briefly, C3H/HeHa mice were injected ip six times at weekly intervals with 1 to 2 x lo7 AKR/SN thymus cells. The antiserum obtained, at a dilution of 1 to 512, killed 100% of the AKR/SN thymus cells in the presence of rabbit complement. The antiserum (or normal C3H serum as a control), each diluted to a l/4 concentration, were used for treatment of AKR thymus cells. Cytotoxicity test with antibody and complement. This was carried out by the method described by Gorer and O’Gorman (13) and modified by Boyse et al. ( 14). For details, see Ref. ( 11) . Con A-induced suppressor activity in cell-mediated cytotoxic responses against allogeneic cells. This was done essentially using a method described by Jandinski etal. (4). (a) Treatment of spleen cells or thymocytes before Con A activation. C57BL/6 spleen cells or thymocytes (5 x 106/ml), as precursors of regulator cells, were incubated with equal volumes of goat anti-Th-B antibody reagent or goat NS control reagent, each diluted fourfold, together with fourfold-diluted rabbit complement, at 37°C for 45 min. DNase (0.04%) was present in the medium to prevent cells from aggregating. After washing, 1.5 X lo7 viable cells were cultured alone with 1.5 pg/ml of Con A for 48 hr. (b) Treatment of spleen cells and thymocytes after Con A activadion. C57BL/6 spleen cells or thymocytes (1.5 x 107) were cultured alone or with 1.5 pg/ml of Con A for 48 hr. After harvesting and washing them, viable cells (5 x lOe/ml) were incubated with equal volumes of goat anti-Th-B or goat NS control reagent, each diluted fourfold, together with fourfold-diluted rabbit complement at 37°C for 45 min. After either procedures a or b above, harvested cells were washed, and graded numbers of viable cells were added as regulator cells to a mixture of 1.5 x 105 C57BL/6 spleen cells (H-Zb) and 5 X lo6 mitomycin C-treated Balb/c spleen cells ( H-2d) and cultured for 5 days. The cytotoxic activity of viable cells in these cultures, obtained by either pro-

64

KAKIMOTO

ET

AL.

cedure a or b, was determined against P815 DBA/2 mastocytoma (H-2d) cells by a 51Cr-release assay. Assay of cell-mediated cytotoxicity by 51C~ release. This was carried out essentially as described by Cerottini et al. (15). Results are expressed as the percentage of 51Cr release, as determined by the following formula : y0 specific Wr release (51Cr release by cytotoxic

lymphocytes)

= pc r re 1ease by freeze-thawed

- (spontaneous 51Crrelease)

target cells) - (spontaneous 51Crrelease)

x 100,

where spontaneous Yr release is 51Cr released from the target cells incubated in culture medium only. Percentage suppression by regulator cells was calculated from the percentage specific release values for cultures containing Con A-treated (Con A +) and cultures containing untreated (Con A -) regulator cells by the formula : y. suppression

=

ye specific release by (Con A-) - y0 specific release by (Con A+) y. specific release by (Con A-)

x 100.

RESULTS The effect of anti-Thy-l.1 and comjlement on thywms cells activated by goat anti-Th-B reagent. We found that the transferred cells responsible for tumor growth enhancement carry the Thy-l, 1 determinant, The tumor growth-enhancing activity of cells transferred from mice treated with goat anti-Th-B had been found to be much greater with thymus cells than with spleen cells (10). Therefore, the present in v&o experiments were carried out only with thymus cells. AKR mice transplanted with the allogeneic tumor sarcoma 180 on Day 0 were injected ip with 0.25 ml of goat anti-Th-B reagent or goat NS (as control) on Day 1 and then on alternate days for a total of eight injections as described previously (10). Three days after the last injection, thymus cells were collected, and after washing, suspensions of 5 x lo6 cells/ml were treated with the same volume (at a l/4 concentration) of either a mixture of C3H anti-Thy-l.1 serum plus rabbit complement or a mixture of normal C3H serum plus rabbit complement at 37°C for 45 min. Over 95% of thymus cells were killed by anti-Thy-l.1 as determined by trypan blue dye exclusion. After washing, treated thymus cells, 5 X lo7 (viable cell number before treatment), were injected i.v. into AKR mice which had been implanted with S180 on the previous day. As shown in Fig. lA, Group I, the thymus cells derived from mice treated with goat anti-Th-B, when treated in &fro with normal C3H serum plus complement, retained their ability to produce tumor enhancement. Thymus cells derived from mice treated with goat anti-Th-B when treated in vitro with anti-Thy-l.1 serum plus complement (Fig. lA, Group II), lost their ability to produce tumor enhancement, since tumor growth was reduced to the level of control groups (Fig. lA, Groups III, IV, and V).

SUPPRESSOR

0

CELLS

5

IN

TUMOR

D

ENHANCEMENT

15

65

20

Days after tronsplontotion

FIG. 1A. Sarcoma 180 tumor growth curves in AKR mice (10 mice per group) given thymus cells derived from SlWbearing AKR mice injected with either goat anti-Th-B reagent (I and II) or goat norrflal serum control reagent (III and IV). Thymus cells before injection were treated in vitro with complement plus either C3H anti-Thy-l.1 serum (II and IV) or normal C3H serum (I and III). Thymus cells were injected on Day 1 following tumor transplantation on Day 0. See the text for additional details of the source of thymus cells and the in vitro treatment of thymus cells. An additional group of tumor-bearing mice (V) was not injected with thymus cells. Note that treatment of the anti-Th-B-activated thymus cells with anti-Thy-l.1 serum plus complement abrogates their tumor-enhancing ability (compare Group I with Group II). The statistical significance of the difference in tumor size between Group I and the other groups is given in Table 1A.

These findings indicate that the suppressor cells involved in this system carry the Thy-l.1 marker on their surface. Thus, they are thymocytes rather than macrophages, since the latter do not carry the Thy-l.1 marker. Macrophages have been reported to act as suppressor cells in some tumor systems (16, 17). The effect of goat anti-Th-B reagent and coulzplement on thymus cells activated by goat anti-Th-B reagent. We have now found that the transferred cells responsible for tumor growth enhancement do not carry the Th-B determinant. Thymus cells harvested from the mice injected with goat anti-Th-B were treated with anti-Th-B reagent plus complement. The same treatment was given thymus cells obtained from mice injected with goat NS. By this treatment, 60 to 70% of the thymus cells were killed, as previously shown (11). The control in z&o treatment of both kinds of thymus cells was with goat NS plus complement. After washing, 5 x lo7 cells (viable cell number before treatment) were transferred iv into AKK mice implanted with SlSO on the previous day. As seen in Fig. 1B (Group VI), treatment of goat anti-Th-B-activated thymus cells with goat anti-Th-B reagent plus complement did not abrogate their tumor enhancing activity since their enhancing activity was as great as that of cells treated with goat NS plus complement (Group I). It appears to us that cells responsible for tumor growth enhancement in this system are generated precursor cells which have the Th-B determinant but lose it once they have been stimulated to become active suppressor cells by goat antiTh-B treatment. Efect of goat anti-Th-B reagent and comfllewcnt on Con A-gelzerated suppressor cell activity. We now show that goat anti-Th-B antibody plus complement does not kill Con A-activated suppressor cells, indicating that such cells lack the Th-R determinant. On the other hand, anti-Th-B antibody plus complement does kill the

66

KAKIMOTO

ET

AL.

Precursors of Con A-activated thymus suppressor cells, indicating that the precursors carry Th-B. It is well known that an appropriate concentration of Con A nonspecifically generates suppressor cells which are suppressive in cell-mediated cytotoxicity as well as in PFC response (18-20). Therefore, we were interested to see the effect of the treatment of Con A-activated suppressor cells with goat anti-Th-B on their suppressive activity. Although nonspecific activation of suppressor cells by Con A usually has been reported with spleen cells, we found that Con A can induce suppressive activity in thymus cells, though less effectively than in spleen cells. After in vitro culture of C57BL/6 (H-Zb) spleen cells or thymocytes with 1.5 pg/ml of Con A for 2 days, the cells were harvested, washed, and incubated with goat antiTh-B (or goat NS) pl us rabbit complement at 37°C for 45 min. Then, graded numbers of viable cells were added as the regulator cells to a culture containing C57BL/6 (H-Zb) spleen cells as the responder cells and Mitomycin C-treated Balb/c (H-Zd) spleen cells as the sensitizing cells. After 5 days in culture, the cytotoxic activity of C57BL/6 spleen cells against H-Zd alloantigqs was determined by the 61Cr-release assay. As presented in Table 4, Con A-stimulated spleen or thymus cells did suppress the cell-mediated cytotoxic response of C57BL/6 spleen cells against H-Zd alloantigens. The treatment with goat anti-Th-B and complement of Con A-generated suppressor thymus cells or suppressor spleen cells resulted in TABLE The Statistical

Significance Group Tumor

Day

1A

of the Difference in Tumor I and Other Groups5

P

size (mm) II

I 13 15

17

13.6 f 1.4 12.4 f 1.0 11.6 z!c 1.0

7.8 f 6.9 f 5.7 f

17 19

13.6 12.4 11.6 10.6

1.5 17 19

12.4 f 11.6 f 10.6 f

1.5

i zk zk f

0.1 0.01 0.05 0.05

0.01 < P < 0.05 0.01 < P < 0.05 0.01 < P < 0.05

V 0.9 0.6

8.6 f 0.8 8.7 zk 0.6 8.4 7.5 7.1 6.4 7.9

13.6 i

1.4 1.0

11.6 f 1.0 10.6 f 0.6 0.9

0 Refer also to Fig. 1A.

f f f f IV

12.4 f 12.2 f

0.05 < P < 0.001 < P < 0.01 < P < 0.01 < P <

7.3 * 2.2 6.7 f 2.3 5.8 * 2.0

1.5

17 19

2.0 1.3 1.4 1.7

1.0 1.0 0.9

13

11

< < <

9.0 7.6 6.4 5.5

I 11.9 f 12.0 i

0.01 0.01 0.01

1.4 1.0 1.0 0.9

I

7 9

P < 0.05 P < 0.05 P < 0.05

1.8 2.1 2.0

III

I 13

Size between

f * zk fzk

0.9 0.7 0.6 0.8 0.9 I

0.001

<

P < 0.01

SUPPRESSOR

2

CELLS

IN

TUMOR

67

ENHANCEMENT

IO

f L 8 i7j

5 m sm+NSnqmt PI XE

0

Goat Anti-Th-BW WNSmpnt

GmNsmg8rd Soat mti-Th-Enogmt Soaf anti-Th-8-t

10

5

15

20

Days after transplantation FIG. 1B. Sarcoma 180 growth curves, as in Fig. lA, for mice given thymus cells treated as indicated. Note that treatment of thymus cells with anti-Th-B plus complement does not abrogate their tumor-enhancing effect, i.e., there is no difference between Groups I and VI. The statistical significance of the difference in tumor size between groups is given in Table 1B.

no change in their suppressive activity, indicating the absence of the Th-B determinant on their surface. In the next experiment, lymphoid cells were treated with goat anti-Th-B or goat

NS plus complement before Con A stimulation. C57BL/6 spleen cells or thymocytes were treated with goat anti-Th-B (or goat NS) plus rabbit complement in a manner similar to the treatment after Con A stimulation. After washing, 1.5 X lo7 viable cells were cultured with 1.5 pg/ml of Con A for 48 hr. Then, the cells were harvested and washed, and graded numbers of viable cells were added as the regulator cells to cultures of C57BL/6 spleen cells and Mitomycin C-treated Balb/c spleen cells, in a manner similar to the experiment described above. Cytotoxicity was determined after 5 days of culture by the 51Cr-release assay. As shown in Table 5, the pretreatment with goat anti-Th-B and complement caused a negligible effect on the induction of splenic suppressor cells by Con A. On the other hand, the induction of thymus suppressor cells by Con A was almost completely abolished by the pretreatment with goat anti-Th-B plus complement, while no effect was obtained with goat NS treatment plus complemnt. This suggests that the Th-B determinant reactive with goat anti-Th-B exists on the precursors of thymus suppressor cells but not on the precursors of spleen suppressor cells. The fact that precursors of Con A-stimulated thymus suppressor cells are sensitive to anti-Th-B but not the active suppressor cells is consistent with the in viva results shown in Fig. 1B. The lack of effect of adult thyunectomy on tumor growth enhancement by goat anti-Th-B. Since we showed that thymocytes were mainly responsible for tumor enhancement caused by anti-Th-B injection, we attempted to abrogate the enhancement

by thymectomizing

adult

mice.

However,

as shown

in Figs.

2A

and 2B,

adult thymectomy had no abrogating effect but if anything further increased the enhancement caused by goat anti-Th-B as compared to enhancement in shamthymectomized AKR mice. This result was reproducible in two experiments in which adult thymectomy was carried out on mice at age 6 weeks and at age 10 weeks. Thus, it appears that thymectomy does not inhibit the ability of mice to show tumor growth enhancement when injected with goat anti-Th-B reagent.

68

KAKIMOTO

ET

AL.

Enhancewent by spleen cells transferred from thymectomized mice treated with goat anti-Th-B. To determine if spleen cells from thymectomized mice injected with anti-Th-B could transfer the tumor-enhancing effect, we injected goat antiTh-B or goat NS into thymectomized or sham-thymectomized AKR mice which had been transplanted with S180 cells, as described above (Fig. 2B). The mice were sacrificed on Day 18, and their spleen cells, as well as the thymus cells of the sham-thymectomized mice, were injected into AKR mice that were transplanted with S180 on the previous day. As shown in Fig. 3, spleen cells from thymectomized mice that had been injected with S180 and goat anti-Th-B had as much enhancing effect on tumor growth as the thymus cells from sham-thymectomized mice injected with S180 and goat anti-Th-B. On the other hand, spleen cells from sham-thymectomized mice injected with S180 and goat anti-Th-B had essentially no enhancing effect on S180 growth as observed previously for intact mice (10). Thus, it appears that in thymectomized mice suppressor cells may be activated outside of the thymus by anti-Th-B reagent treatment and home in on the spleen, whereas in sham-thymectomized mice (or intact mice) they are activated either within the thymus or home in on the thymus rather than the spleen. Probable absence of enhancing antibodies. To determine if the tumor-enhancing effect of spleen cells from thymectomized mice treated with anti-Th-B reagent was mediated by the production of enhancing antibody rather than by suppressor cells, we injected mice with SE30 plus serum from thymectomized mice carrying enhanced tumors caused by anti-Th-B injection. Such serum caused no tumor enhancement in the tumor-bearing recipients when compared with serum from thymectomized mice injected with control serum reagent. Thus, the enhancing effect on TABLE The

1B

Statistical Significance of the Difference in Tumor Size between Groups” Tumor size (mm)

Day I 11 13 15 17 19 22

12.2 13.6 12.4 11.6 10.6 9.1

f f f f f *

15 17 19 22

7.6 6.4 5.5 4.2

13 1.5 17 19 22

13.7 13.1 12.3 11.3 10.4

P VII

0.6 1.4 1.0 1.0 0.9 0.8

8.3 8.6 7.7 7.0 6.5 5.6

1.3 1.4 1.7 2.6

13.1 12.3 11.3 10.4

2.0 1.4 1.4 1.3 0.8

8.6 7.7 7.0 6.5 5.6

III f f * f

* Refer also to Fig. 1B.

1.5 1.4 1.2 1.1 1.0 0.7

0.01 0.01 0.001 0.01 0.01 0.001

< < < < < <

P P P P P P

< < < < < <

0.05 0.05 0.01 0.05 0.05 0.01

1.4 1.4 1.3 0.8

0.001 0.001 0.001 0.01

< < < <

P P P P

< < < <

0.01 0.01 0.05 0.05

1.4 1.2 1.1 1.0 0.7

0.01 0.001 0.001 0.001 0.001

< < < < <

P P P P P

< < < < <

0.05 0.01 0.01 0.01 0.01

VI f & zk f

VII

VI f f f * zk

f f f f f f

f f f f f

SUPPRESSOR

CELLS

IN

TUMOR

I

b 0

5

lo

60

ENHANCEMENT

1

15

20

Days after transpbntdion

A

B

Doys after tronsplontation

FIG. 2. The effect of adult thymectomy on tumor growth enhancement caused by goat antiTh-B antibody reagent. Adult thymectomy was carried out in the AKR/SN mice at age of 6 weeks and 10 weeks. TX mice were used 4 weeks later. Injection of 0.25 ml of either antiTh-B reagent or control normal (NS) reagent was done on alternate days as indicated by the arrows. Note that TX does not change the tumor-enhancing effect of anti-Th-B injection when compared to the sham TX controls or causes even greater tumor enhancement by antiTh-B injection when compared to the sham TX control. The statistical significance of the difference in tumor size between goat anti-Th-B-treated groups and goat NS-treated groups is given in Tables 2A and 2B.

S180 growth of transferred spleen cells from goat anti-Th-B-treated thymectomized mice bearing S180 probably cannot be attributed to enhancing antibodies against S180 produced by the spleen cells. DISCUSSION In a previous paper, we showed that the enhancing effect on S180 growth in AKR mice injected with goat anti-Th-B could be attributed to lymphoid cells, especially to the thymocytes of these animals, since this effect was transferable quite effectively with thymus cells and less effectively with spleen cells derived from goat anti-Th-B-treated mice bearing S180 (10). It appeared that goat anti-Th-B caused this effect on S180 by stimulation or activation of suppressor ‘1‘ cells, possibly due to the presence of the Th-11 tleterniinant on the sup~m5wr cell surface.

70

KAKIMOTO

ET

TABLE The Statistical

Tumor Anti-Th-B 13.8 14.4 13.6 12.0 10.1

group f f i f i

12.6 11.8 9.5 9.6

f f zt f

P

size (mm)

(TX)

1.2 1.0 1.3 1.2 1.1

NS group 4.4 4.0 8.5 7.3 6.3

Anti-Th-B group (sham-TX) 1.5 17 19 21

2A

Significance of the Difference in Tumor Size between Goat Anti-Th-B-Treated Groups and Goat NS-Treated Groups : 6-Week-Old Mice”

Day

11 13 15 17 19

AL.

0.5 1.1 0.6 0.6

NS group 4.4 8.0 6.8 6.0

(TX)

* 1.1 f 1.1 f 1.6 f 1.2 f 1.2

0.01 0.001 0.01 0.01 0.01

< < < < <

P P P P P

< < < < <

0.05 0.01 0.05 0.05 0.05

(sham-TX) f 0.7 f 0.8 f 0.6 f 0.5

0.01 < P < 0.01 0.01 < P < 0.05 0.001 < P < 0.01 P < 0.001

5 Refer also to Fig. 2A.

Since the possible interactions of anti-Th-B with cells in viva are extremely complex, we have concentrated our attention in the present study on the characteristics of the thymus cells which are responsible for transferring the observed enhancing effect. Thus, the work reported here further characterizes the putative suppressor cells that appear to be involved in this system. We show that such cells carry the Thy-l antigen, since anti-Thy-l. 1 antiserum plus complement eliminates the enhancing effect on tumor growth of transferred thymus cells, TABLE The Statistical

Significance of the Difference in Tumor Size between Groups and Goat NS-Treated Groups: lo-Week-Old Tumor

Day Anti-Th-B

group

11 13

15.8 * 1.4 16.0 i 0.9

1.5

16.1 f 1.0

18

15.3 i

(TX)

14.4 13.5 12.7 11.2

Anti-Th-B 15

18

f f & f

16.1 i 15.3 f

a Refer also to Fig. 2B.

1.0 1.1

NS group

8.3 i

0.8 0.9 1.0 1.1

group

P

10.7 *

1.1

NS group 10.7 10.4 9.6 7.4

(TX)

Goat Anti-Th-B-Treated Mice’

size (mm) (TX) 1.1

10.5 dz 1.1 10.0 zk 1.0

Anti-Th-B group (sham-TX) 11 13 1.5 18

2B

1.2

0.01

< P < 0.05 P < 0.001 P < 0.001 P < 0.001

(sham-TX) f

1.2

f 1.1 f f

1.1 0.9

0.01 0.01 0.01 0.001

< < < <

P P P P

< < < <

0.05 0.05 0.05 0.01

Anti-Th-B group (sham-TX) 12.7 & 1.0 11.2 f 1.1

0.01 < P < 0.05 0.001 < P < 0.01

SUPPRESSOR

0

CELLS

5

IN

TUMOR

lo

ENHANCEMENT

15

71

20

Days after transplantation FIG. 3. Sarcoma 180 growth curves in AKR mice given spleen cells from thymectomized (TX) mice or given either spleen cells or thymus cells from sham-TX mice. All donor mice had been inoculated with S180 and were treated with goat anti-Th-B antibody reagent or goat normal serum control reagent as described in the text and in Fig. 2. Group 1: mice given spleen cells derived from thymectomized mice given goat anti-Th-B antibody reagent. Group 2: mice given thymus cells derived from sham-thymectomized mice given goat anti-ThB antibody reagent. Group 3: mice given spleen cells from sham-thymectomized mice given goat anti-Th-B antibody reagent. Group 4: mice given spleen cells from thymectomized mice given normal goat serum control reagent. Group 5: mice given spleen cells from shamthymectomized mice given normal goat serum control reagent. Group 6: mice given thymus cells from sham-thymectomized mice given normal goat serum control reagent. Note that spleen cells from TX mice given goat anti-Th-B (curve 1) cause as much or more tumor enhancement as thymus cells from sham TX mice given goat anti-Th-B (curve 2). In contrast, spleen cells from sham TX mice given goat anti-Th-B cause no tumor enhancement (curve 3). The statistical significance of the difference in tumor size between Group I and Groups 2, 3, and 4 is given in Table. 3.

indicating that they are, in fact, thymocytes. They are not macrophages since they lack Thy-l. Macrophages have been reported to function as suppressor cells in several tumor systems (16, 17). More interestingly, if the thymocytes are treated in vitro with goat anti-Th-B plus complement, they still retain suppressor activity in viva when transferred, in contrast to their loss of activity by anti-Thy-l.1 treatment. A probable explanation is that precursors of the suppressor cells express Th-B antigens and, therefore, are stimulated by the in tivo treatment with anti-Th-B antibodies. The mature suppressor cells thus generated have lost the Th-B determinant from their surface and are then no longer sensitive to anti-Th-B plus complement. This explanation is consistent with the results of in vitro experiments on the effects of goat antiTh-B and complement on Con A-generated suppressor cells. After Con A induction of suppressor cells from spleen cells or thymocytes, goat anti-Th-B plus complement has no effect on their suppressor activity. However, when the treatment is carried out before Con A stimulation, the thymocytes lose their suppressive activity completely, probably because the precursor cells have been killed. Thus, the Th-B determinant seems to be expressed on the precursors of suppressor thymocytes inducible by either anti-Th-B or Con A stimulation. Once generated, the suppressor cells are not sensitive to the treatment with anti-Th-B plus complement. In contrast, the precursors of Con A-inducible suppressor cells in the spleen

72

KAKIMOTO

ET

AL,

apparently do not carry the Th-B determinant, because the pretreatment of spleen cells by anti-Th-B and complement did not abrogate Con A induction of splenic suppressor activity. These results are consistent with our previous conclusion that the Th-B expression in the T-cell lineage is limited to immature thymocytes (11). Further study is needed, however, to determine whether Th-B+ precursor suppressor cells in the thymus and Th-B- precursor suppressor cells in the spleen are the cells at different stages of maturation or are cells belonging to different subpopulations of T cells. Unexpectedly, adult thymectomy did not abrogate the tumor-enhancing effect of goat anti-Th-B but rather further enhanced it. In addition, spleen cells from anti-Th-B-treated thymectomized mice bearing tumors did transfer the tumor growth-enhancing activity as effectively as did the thymocytes from goat anti-Th-Btreated sham-thymectomized mice bearing tumors. The sera from the donors of the spleen cells or the thymus cells did not have the tumor-enhancing activity. These results indicate that the tumor-enhancing activity of the spleen cells from the thymectomized mice is cell-mediated and that the precursors of anti-Th-B-inducible suppressor cells may be present not only in the thymus but also outside of the thymus, probably in the bone marrow. The latter cells may migrate to the spleen in thymectomized mice and develop to suppressor cells when stimulated by antiTh-B. It is yet to be established, however, that the splenic suppressor cells responsible for tumor enhancement in this case were also T cells. TABLE

3

The Statistical Significance of the Difference in Tumor Size between Group 1 and Groups 2, 3, and 4a

P

Tumor size (mm)

Day

Group 1

Group 2

17 19

16.2 f 0.9 15.6 f 0.9

13.1 zlz 1.0 11.1 f 1.1

Group 1

Group 3

4 6 8 10 12 14 17 19

10.4 f 13.4 f 16.4 f 17.8 f 16.8 f 16.0 f 16.2 f 15.6 f

4 6 8 10 12 14 17 19

10.4 f 13.4 f 16.4 f 17.8 f 16.8 f 16.0 f 16.2 f 15.6 f

1.4 1.1 0.8 0.8 1.2 1.2 0.9 0.9

Group 1

n Refer also to Fig. 3.

1.4 1.1 0.8 0.8 1.2 1.2 0.9 0.9

5.5 f 8.1 f 9.1 f 12.0 f 12.1 f 12.2 f 10.8 f 7.9 i

0.8 1.8 1.7 0.7 0.9 1.0 0.9 0.9

0.01 < P < 0.05 0.001 < P < 0.01

0.001 0.01 0.001 P 0.001 0.01 P P

< < < < < < < <

P < 0.01 P < 0.05 P < 0.01 0.001 P < 0.01 P < 0.05 0.001 0.001

0.01 P P P 0.01 0.001 0.001 P

< < < < < < < <

P < 0.05 0.001 0.001 0.001 P < 0.05 P < 0.01 P < 0.01 0.001

Group 4 5.4 f 7.2 f 9.8 f 10.8 f 10.5 f 9.9 f 10.4 f 9.4 f

1.3 1.1 1.1 1.3 1.4 1.4 1.6 1.2

SUPPRESSOR

CELLS

IN

TUMOR

TABLE Goat Anti-Th-B

Treatment of regulator CdS

Number of regulator cells

4

Keayent Plus Complement Does Not Kill Suppressor Cells from Spleen or Thymus”

Experiment

Con A present

(‘OH A-Induced

Experiment

1

Percentage specific releases in the presence of regulator cells cultured with:

7.1

ENHANCEMENT

Suppression* (%)

Treatment of regulator Cl418

Number of regulator cells

2

ConA present

Con A absent

Suppressionh (%I

Percentage specific r&as@ in the presence of regulator cells cultured with : Con A absent

Spleen cells a8 regulator cells

Goat Anti-Th-B

Goat NS

10’ 106 10’

72 f 56 fl

2c

17fl

70 f 1’ 66 f0

49f2

104 10’ 10%

73 f3 64h3

76 f3 66f2

1.5 f 2

52 zt 1

10’ 106 108

68 f2 44&O 23zt2

63 f2 57z!c2 45f2

10’

59 f 53 f2 39f3

1 59 f 0

-2rk

4c

15f

2

65z!z

9

3*x 4f 71f

7 7

Goat Anti-Th-B

Goat NS

;t 1~ 57 f 1~ 66zk2 58 f 1 28 *o 76&O 14&O

104 106 10’ 5 x 10s

60

10’ 106 10” 5 x 106

59 2~0 65 It 1 24 f3 20 * 1

104 106 106 5 x 108

64 59

104 106 10% 5 x 10”

65fl

56fl

f 75 f 77 f 83 *

-6

f2J

15 f4 53 *2

82 f 0

1 1 1 1

69 f6 76 zt 2

1 78 f 2 1 79 It 1 64&O 69&O

25 * 2 43 f2 70 f2

68

13 f2

l.i f2

Thymus cells as regulator cells Goat Anti-Th-B

Goat NS

106 106

62 f4 63fl

-a* 22f 49

6 4

f

10

032 fll

1

15 38f

Regulator cells absent: 70 f 4% specific release Spontaneous release = 11% of WZr Release by freezing and thawing = 81 f 3X of Wr

Goat Anti-Th-B

Goat NS 7

f f

37fl 21&l 69 f 26&O 14fO

67f2 1 74 zk 0 57fl 69&I

18 *4

3 * 3 7f2 55 f.2 80 f 2

Regulator cells absent: 70 f 1% specific release Spontaneous release = 10% of WIRelease by freezing and thawing = 84 k 1% of ZlCr

~1Con A-induced (or untreated) regulator spleen cells were treated with either goat anti-Th-B or goat NS. as described under Materials and Methods. After washing. graded numbers of regulator cells were added to mixtures of C57BL/6 (H-2b) spleen cells and Mitomycin-treated Balb/c (H-2”) spleen cells and cultured for 5 days as described. The cytotoxic activity of the cultured spleen cells was determined by a Wr-release assay with Wr-labeled P815 T)BA i2 (H-2’) maatocytoma cells as target. b The values are calculated as described under Materials and Methods. c The values are averages of duplicates f deviation from the average. d The average f one-half of the range.

It must be noted that we have not provided evidence that anti-Th-B can induce suppressor T cells either in viva or in vitro by a direct action on thymocytes carrying the Th-B determinant, although we propose this as a likely possibility. It may be that anti-Th-B acts in a more indirect manner, in viva. Thus, since antiTh-B can also interact with the Th-B determinant on B cells, .another possibility is that a product of this interaction is responsible for activation of suppressor T cells. Additional mechanisms may exist, and it will require further work to sort out the possibilities. Recently, Feldmann et al. (21) reported that the cooperation between long-lived Tz cells resistant to adult thymectomy (ATx) and short-lived T1 cells resistant to anti-lymphocyte serum (ALS) is necessary for the full expression of the suppressor function of T lymphocytes in the in vitro humoral response. Their results showed that ATx-resistant cells are suppressor precursors and ALS-resistant cells are suppressor cell amplifiers. In our study, it might be assumed, therefore, that anti-Th-B reacts with amplifier cells which may be Th-B+, and the amplifier cells

74

KAKIMOTO

ET

AL.

then stimulate Th-B- precursors of suppressor cells to become Th-Bmature suppressor cells. This mechanism is a possible one in our experiments. Although we used ATx mice 4 weeks after operation, at which time they are substantially depleted of Tr cells (22), nonetheless some T1 cells may still have remained (23). Thus, the spleen cells from ATx mice treated with anti-Th-B which caused tumor enhancement may have been suppressor cells induced by amplifier T1 cells stimulated by anti-Th-B. Murphy et al. (24) have described the I-J-defined determinant on suppressor T cells. Frelinger et al. (25) have shown that anti-I-J alloantiserum plus complement can kill Con A-reactive T cells. It is unlikely, however, that the Th-B determinant is the same as the determinant coded by the I-J subregion, since Th-B is present on B cells as well as on immature T cells, whereas the I-J-defined determinant is absent from B cells. On the other hand, a possibility still exists that goat anti-Th-B antibody may recognize common determinants of antigens coded by various I subregions. We suppose that very immature cells express the Th-B determinant while in the bone marrow or during differentiation and migration to the thymus. In the thymus they lose the determinant as they mature (11). Mature T cells that have TABLE

5

Goat Anti-Th-B Reagent Plus Complement Kills Precursors of Con A-Induced Suppressor Cells from Thymus but Not from Spleen’ Experiment Treatment of precursors of regulator

Number of regulator cells

CdIS

Percentage specific reIeaseb in the presence or regulator cells cultured with:

Goat NS

Anti-Th-B

Suppressic& (%)

Treatment of precursors of regulator cells

Number of regulater C&S

2

Percentage specific release” in the presence of regulator cells cultured with:

Suppressiona (%)

ConA present

ConA absent

106

Spleen cells as precursors of regulator cells 104 -3f3d 40 f 1c 39 l 1c 105 11 f6 Anti-Th-B 37 zti 42 fl 106 31 f6 29 &l 42 fl

59 f20 44A4 35f4

53 *2= 63fl 74fl

10” 10’ 100

37 fl 28 *1 33 l o

106 106

62 d2 65 f 1 50 l 1 73 l 1 39 f 1 75 *2

5*3 32 f4 48 A.5

104 106

40 f 1 31fl 32 fl

Thymus cells as precursors of regulator cells 104 5f3 42 zk 1 10’ Anti-Th-B 32fl 3h6 108 Oh3 32 3~0

60 f 1 56 ;tO .54*0 54fO 55 fl 56 f 1

-7f2

ConA present

Anti-Th-B

Experiment

1

104 10’

106

ConA absent

41 f 1 33 f0 50 fl

9f3 17 f3 35 l 4

lf6 46 zt2 46 f 1 2f2 so*0 Slfl 106 Goat NS 37 f3 106 28 f 1 45 f 0 Regulator cells absent: 55 f 1% specific release Spontaneous release = 12% of Wr Release by freezing and thawing = 92% of Wr 104

104

Goat NS

-10 f 6d 31 f9 52 f7

1 f0

2f3

51f4 60&O 15 f 7 49&l 58&l 16 f4 Goat NS 108 32 *O 59 4~1 45 *2 Regulator cells absent: 57 f 0% specific release Spontaneous release = 10% of Wr Release by freezing and thawing = 72 5~ 1% of Wr 101 106

a Precursors of regulator spleen cells were treated first with either goat anti-Th-B or with goat NS and then cultured in the presence or absence of Con A as described under Materials and Methods. Graded numbers of viable r%UlatOr cells were added to mixtures of C57BL/6 (H-Zh) spleen cells and Mitomycin-treated Balb/c (H-2d) Spleen Cells and cultured for 5 days as described. The cytotoxicity of the cultured spleen cells was determined bY a 6’Cr-release assay with Wr-labeled P815 DBA/2 (H-2d) mastocytoma cells as target. b The values are calculated as described under Materials and Methods. c The values are averages of duplicates f deviation from the average. d The average k one-half of the range.

SUPPRESSOR

CELLS

IN

TUMOR

ENHANCEMENT

7s

lost the Th-B determinant migrate to the periphery and participate in cellular and humoral immune reactions as peripheral T cells. Goat anti-Th-B may react with the Th-B determinant expressed on the cell surface at certain periods during differentiation and stimulate cells, thereby causing acceleration of the maturation of a certain subset of the T-cell population. Probably the rate at which suppressor subpopulations mature in the thymus exceeds the rate of migration of those cells into the periphery, which would explain why a more significant effect on tumor growth enhancement is seen with thymus cells than with spleen cells. It might be that after adult thymectomy, which eliminates the site of removal of the Th-B antigen from the cell surface (11)) the number of peripheral T cells carrying the Th-B determinant is increased over that of normal mice. Thus, thymectomized mice would contain cells sensitive to goat anti-Th-B, thereby producing the observed enhanced growth of S180 in thymectomized mice treated with goat antiTh-B. Recently, Small (8) reported that immature thymocytes are responsible for T-cell-mediated enhanced growth of syngeneic Lewis lung carcinoma in C57BL,% mice. The responsible cells were found to be rapidly dividing and to disappear quickly from the thymus 24 hr after cortisone administration. These results are compatible with our results, although our system is an allogeneic tumor system, whereas theirs was a syngeneic one. Although our evidence shows that the thymocytes from S180-bearing mice treated with anti-Th-B antibody reagent have tumor-enhancing activity, we have found (unpublished data) that thymocytes from normal mice treated with anti-ThB antibody reagent do not. Therefore, it appears that the induction of suppressor cells in our system requires the presence of the tumor cell antigen. Further studies are needed to determine whether or not the suppressor cell is antigen-specific. ACKNOWLEDGMENT The authors wish to thank Miss Maryann Geleta, Mrs. Kimberly Jankowski, Mr. Arthur J. Trott, Mr. Mike Palcan, Mrs. Aino Simm, and Mrs. Cheryl Zuber for skillful technical assistance. This investigation was supported by Grant No. SP30CA17609 and Grant No. C.414562, awarded by the National Cancer Institute, DHEW.

REFERENCES 1. Rich, S. S., and Rich, R. R., J. Exp. Med. 140, 1588, 1974. 2. Zembala, M., and Asherson, G. L., Nature (London) 244, 277, 1973. 3. Ha, T-Y., and Waksman, B. H., J. Zmntlcnol. 110, 1290, 1973. 4. Jandinski, J., Cantor, H., Tadakuma, T., Peavy, D. L., and Pierce, C. W., J. E.rj. Med. 143, 1382, 1976. 5. Fujimoto, S., Greene, M. I., and Sehon, A. H., J. Zmmunol. 116, 791, 1976. 6. Fujimoto, S., Greene, M. I., and Sehon, A. H., .Z. Zmmunol. 116, 800, 1976. 7. Treves, A. J., Carnaud, C., Trainin, N., Feldman, M., and Cohen, I. R., Z%r. J. Z~~~rrmtol. 4, 722, 1974. 8. Small, M., J. Zmmunol. 118, 1517, 1977. 9. Kilburn, D. G., Smith, J. B., and Gorczinski, P. M., Eur. J. Zmmunol. 4, 784, 1974. 10. Kakimoto, K., Fuji, H., Grossberg, A. L., and Pressman, D., Cancer Res. 37, 3145, 1977. Il. Yutoku, M., Grossberg, A. L., Stout, R., Herzenberg, L. A., and Pressman, D., Cell. Zmmunol. 23, 140, 1976. 12. Reif, A. E., and Allen, J. M. W., J. Exp. Med. 120, 413, 1964. 13. Gorer, P. A., and O’Gorman, P., Transplant. Bull. 3, 142, 1956. 14. Boyse, E. A., Old, L. J., and Stockert, E., Anti. N.Y. Acad. Sci. 99, 574, 1962.

76

KAKIMOTO

ET

AL.

15. Cerottini, J-C., and Brunner, K. T., In “In vitro Methods in Cell-Mediated Immunity” (B. R. Bloom and P. R. Glade, Eds.), p. 369. Academic Press, New York, 1971. 16. Kirchner, H., Chused, T. M., Herberman, R. B., Holden, H. T., and Larvin, D. H., J. Exp. Med. 139, 1473, 1974. 17. Pope, B. L., Whitney, R. B., Levy, J. G., and Kolbum, D. G., J. Zmmunol. 116, 1342, 1976. 18. Peavy, D. L., and Pierce, C. W., J. Exp. Med. 140, 356, 1974. 19. Dutton, R. W., J. Exp. Med. 136, 144.5,1972. 20. Rich, R. R., and Pierce, C. W., J. Exp. Med. 137, 694, 1973. 21. Feldmann, M., Beverley, P. C. L., Woody, J., and McKenzie, I. F. C., J. Exp. Med. 145, 793, 1977. 22. Simpson, E., and Cantor, H., Eur. J. Zmmunol. 5, 337, 1975. 23. Kappler, J. W., Hunter, P. C., Jacobs, D., and Lord, E., J. Znmwnol. 113, 27, 1974. 24. Murphy, D. B., Herzenberg, L. A., Okumura, K., Herzenberg, L. A., and McDevitt, H. O., J. Exp. Med. 144, 699, 1976. 25. Frelinger, J. A., Niederhuber, J. E., and Schreffler, D. C., J. Exp. Med. 144, 1141, 1976.