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Antigen-specific augmentation of delayed-type hypersensitivity by immune serum factor in mice: Augmentation of anti-tumor cytostatic activity
CELLULAR
IMMUNOLOGY
103,3 1l-325 (1986)
Antigen-Specific Augmentation of Delayed-Type Hypersensitivity by Immune Serum Factor in Mice: Augmentation of Anti-Tumor Cytostatic Activity’ SEIJI NAKAMURA,* KUNISUKE HIMENO, MASAMITSU MITANI, AND KIKUO
AKIRA YAMADA, NOMOTO
Department of Immunology, Medical Institute of Bioregulation and Second Department of Oral Surgery, Faculty ofDentistry, Kyushu University, 3-l-l Maidashi. Higashi-ku. Fukuoka 812, Japan Received January 7.1986; acceptedJuly 14, 1986 A humoral factor capable of augmenting delayed-type hypersensitivity antigen specificity (DAF) is present in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR). In the present study, a similar factor was identified when xenogeneic tumor cells were used as antigens. This factor also could augment the in vitro anti-tumor cytostatic activity against homologous tumor cells, which correlated with in vivo DIR to the same tumor cells. The cytostatic activity augmented by the transfer of this factor had the following characteristics: (i) The activity appeared in the whole peritoneal exudate cells (PEC) from serum recipients at 4 days after the antigenic challenge. (ii) Such an activity was revealed in the collaboration of plastic dish-nonadherent and -adherent PEC as the primary and final effecters, respectively. (iii) The appearance of primary effector cells for such an activity was also accelerated in spleen and lymph node cells. (iv) However, a sufficient number of macrophages were always required as the final effecters in their functional expression. (v) These primary effecters were sensitized T lymphocytes which produced lymphokine(s) such as macrophage-activating factor(s) and which contributed to this augmented cytostatic activity through the activation of macrophages. Thus, this immune serum factor seemsto exert functional expression by accelerating the generation of lymphokine-producing delayed-type T lymphocytes, which is also responQ 1986 Academic Fms, IOC. sible for CytOStatiC anti-tumor immUUity.
INTRODUCTION Delayed-type hypersensitivity (DTH)3 was proposed to be one of the main effector mechanisms in the anti-tumor immunity, in syngeneic or allogeneic tumor systems ’ This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan. * To whom correspondence should be addressed at the Department of Immunology, Medical Institute of Bioregulation, Kyushu University 69,3-l- 1 Maidashi, Higashi-ku, Fukuoka 8 12, Japan. 3Abbreviations used: CFA, complete Freund’s adjuvant; CRBC, chicken erythrocytes; CY, cyclophosphamide; DFR, delayed footpad reaction; DTH, delayed-type hypersensitivity; HBSS, Hanks’ balanced salt solution; Ig, immunoglobulin; ip, intraperitoneally; iv, intravenously; LN, inguinal and popliteal lymph nodes; MAF, macrophage-activating factor(s); MMC, mitomycin C; MMC-AH 130, MMC-treated AH130; PBS, phosphate-buffered saline; PEC, peritoneal exudate cells; sc, subcutaneously; 5’Cr, Na2%Zr04; [‘*‘I]UdR, [‘2SI]5-iodo-2’-deoxyuridine. 311 OOO8-8749186 $3.00 Cowri&t 0 1986 by Academic F’ms, Inc. Au rights of reproduction in any form resmed.
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(l-4), in the acquired resistance against some kinds of infection with intracellular parasites such as Mycobacteria and Listeriu (5-g), and in the transplantation immunity to allografi ( 10) and xenograft (11). In some transplanted tumor systems, antitumor cytostatic mechanisms produced by lymphokine-producing lymphocytes and macrophages were assumed to play a main role in the rejection (3,4, 11, 12). The in vitro cytostatic activity has been demonstrated to correlate well with in vivo DTH reactions (3,4). Thus, lymphokine-producing lymphocytes responsible for cytostatic activity and those for DTH reaction may be assignedto the same population. It has been established that DTH as well as antibody production and cytotoxic T cells were regulated by helper and suppressor T cells. The primary in vitro induction of DTH to influenza virus was shown to be enhanced by antigen-specific helper T cells (13). In the induction of DTH to heterologous erythrocytes, helper T cells may be required for both in vitro (14) and in vivo (15) induction. In some cases,such collaboration is mediated through antigen-specific T-cell factors. Ptak et al. (16) and Askenase et al. ( 17) demonstrated the role of T-cell factors which acted at the expression phase, while Colizzi et al. (18) reported the T-helper factor which acted at the induction phase. Recently, we found a humoral factor capable of augmenting DTH antigen specificity in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DIR) (19,20), or in the culture supematant of their immune spleen cells (21). As shown below, a similar factor was also found in the serum of mice sensitized with rat AH 130 tumor cells to induce DFR. In the present study, we investigated whether this factor could augment in vitro anti-tumor cytostatic activity as well as DFR. We obtained evidence that this augmentation factor exerted its effect in the induction phase of cytostatic activity. MATERIALS AND METHODS
Mice. Female C57BL/6 and (C57BL/6 X DBA/2)Fl (BDFl) and male DBA/2 mice were obtained from the Shizuoka Laboratory Animal Center (Hamamatsu, Japan). Male Donryu rats were obtained from the Rat Japan, Inc. (Urawa, Japan). All mice were used for experiments at 8 to 12 weeks of age and all rats were used at 4 to 5 weeks of age. Tumors. AH 130, a rat ascitic-form hepatoma (22), was maintained in vivoby serial intraperitoneal (ip) passagein Donryu rats. P8 15, a mastocytoma of DBA/2 origin, was maintained in vivoby serial ip passagein syngeneic DBA/2 mice. Antigens. Chicken erythrocytes (CRBC) obtained from normal hens by cardiac puncture were washed three times with phosphate-buffered saline (PBS) before use. Treatment with mitomycin C. AH 130 tumor cells were suspended in Hanks’ balanced salt solution (HBSS) at a concentration of 2 X 10’ cells/ml. HBSS was supplemented with 100 &ml mitomycin C (MMC; Kyowa Hakko Co., Ltd., Tokyo, Japan). After the incubation at 37°C for 60 min, the cells were washed three times with HBSS and used for experiments. Preparation of immune seru. Mice were immunized with 1 X 10’ MMC-treated AH1 30 (MMC-AH130) tumor cells or 1 X 10’ CRBC emulsified with complete Freund’s adjuvant (CFA; Difco, Detroit, Mich.) supplemented with 5 mg/ml of heatkilled Mycobacterium tuberculosisAoyama B (donor immunization). Emulsion (0.2 ml) was subcutaneously (SC)injected into a hind thigh. In some experiments, mice
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313
were inoculated SCwith 1 X lo7 viable AH 130 tumor cells in 0.2 ml PBS into a hind thigh (donor immunization). Cyclophosphamide (CY; Endoxan, Shionogi & Co., Osaka, Japan) was dissolved in sterile distilled water and administered ip in a dose of 100 mg/kg, 2 days before the immunization. At 7 days after the immunization, 1 x lo7 MMC-AH 130 tumor cells or 2.5 X lo7 CRBC in 0.05 ml PBS were injected into the right footpad of each mouse to elicit DFR. The same antigen was used for the immunization and elicitation of serum donors. Twenty-four hours later, blood samples were collected and the sera were prepared. In some experiments, samples were obtained 8 days after the immunization, without elicitation of serum donors. When donor mice were inoculated with viable AH130 tumor cells, samples were obtained not only 8 days but also 5 and 15 days after the inoculation, without elicitation of serum donors. Assayfor the activity of augmentationfactor by DFR. One milliliter of the immune serum was transferred intravenously (iv) via the tail vein into recipient mice pretreated ip with 100 mg/kg of CY 4 days before. Three to six hours later, mice were immunized with 1 X lo7 MMC-AH130 tumor cells or 2.5 X lo6 CRBC in 0.05 ml PBS into the right hind footpad (recipient immunization). Three to four days after the immunization, DFR was elicited by injection of 1 X lo6 MMC-AH 130 tumor cells or 2.5 X lo7 CRBC in 0.05 ml PBS into the left hind footpad. The degree of swelling was measured 24 hr later with a dial thickness gauge. Reactions were expressed as the difference in thickness between the left and right footpad. The results are shown as the mean f the standard error of the mean (SEM) of five to eight animals. Tissueculture medium. RPM1 1640 medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% NU-SERUM (Collaborative Research, Inc., Lexington, Mass.), 20 mM Hepes, 100 units/ml penicillin, and 100 pg/ml streptomycin was used for in vitro experiments. Efictor cellsfor cell-mediated cytostatic (CMS) assays.Peritoneal exudate cells (PEC) were used as effector cells in most experiments. The PEC were induced by injecting ip with 1 ml of 2% boiled and autoclaved starch (Wako Pure Chemical Industries, Ltd., Osaka, Japan) 2 days before the assay. In some experiments, the whole PEC were fractionated into adherent cells and nonadherent cells. The whole PEC were suspended in the RPM1 culture medium at a concentration of 1 X lo6 cells/ml and incubated in plastic dishes (Falcon 300 1, Becton-Dickinson, Oxnard, Calif.) for 60 min at 37°C. The nonadherent cells were put into other plastic dishes for the second incubation, and the adherent cells were washed three times with warm HBSS (37°C) to remove the residual nonadherent cells. The adherent cells were incubated in PBS containing 0.6 mM EDTA on ice for 15 min and harvested by gently pipetting. The nonadherent cells were harvested at the end of the second incubation at 37°C for 60 min. For reconstitution experiments, the adherent cells were added to the nonadherent cells at the ratio of 1:1. In some experiments, spleen cells and inguinal and popliteal lymph nodes (LN) cells were used as the effector cells for the CMS assay. Assaysfor the activity of augmentationfactor by cytostaticactivity. The CMS assay was carried out as described (3, 4, 11, 12) with slight modifications. AH 130 tumor cells were incubated at 37°C for 1 hr in plastic dishes (Falcon 300 1) at a concentration of 1 X 1O6cells/ml to remove macrophages of host origin and used as target cells. Cell suspensions containing 2 X 1O4unlabeled target cells and 5 or 10 X 1O4effector cells
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(effector:target ratio of 2.5: 1 or 5: 1) in 0.2-ml aliquots of the RPM1 culture medium in flat-bottom microculture plates (Cell Wells 25860, Corning Glass Works, Corning, N.Y.) were cultured in a humidified atmosphere of 5% CO2 at 37°C for 24 hr. DNA synthesis was assessedby adding 0.4 &i [‘251]5-iodo-2’deoxyuridine ([iZ51]UdR; Amersham International plc, Amersham, England) per well 9 hr before the end of the culture (23). The cultures were harvested using a Labo Mash Cell Harvester (Lab0 Science Co., Ltd., Tokyo, Japan) and the radioactivity was measured with a well-type gamma-counter (Shimadzu, Tokyo, Japan). Percentage of cytostasis (% CTS) was calculated, using the following formula: %CTS = 100 ’
[i2?]UdR uptake by target cells with immune effector cells ’ - [i2’I]UdR uptake by target cells with nonimmune effector cells *
Assay for the activity of augmentation factor by macrophage-activating factor(s) (MAF)-production by spleen and LN cells. Spleen and LN cells from serum recipients immunized according to the same protocol as that for DFR were cultured at 5 X lo6 cells/ml for 24 hr at 37°C under 5% C02, with or without 5 X lo6 MMC-AH130 tumor cells/ml. MAF activity in these supematants was evaluated as the ability to induce nonspecific cytolytic activity in peptone-induced BDFl macrophages toward P8 15 tumor cells, according to Pace and Russel(24) and Schreiber et al. (25), with some modifications. P8 15 tumor cells were suspended at a concentration of 1 X 10’ cells/ml and incubated at 37°C for 1 hr with 50 &i of Na25’CrG4(“Cr; Japan Atomic Energy Research Institute, Tokyo, Japan). Thereafter, these cells were washed three times with HBSS and used as “0-labeled target cells. The peptone-induced PEC were obtained 3 days after injecting ip with 1 ml of 10% proteose peptone (Difco), and washed three times with HBSS. The PEC were adjusted to a concentration of 2 X lo6 cells/ml in the RPM1 culture medium and the suspension was put into flatbottom microculture plates (Cell Wells 25860) at a volume of 0.1 ml per well. After culturing for 3 hr, the nonadherent cells were aspirated, and the adherent monolayers were washed twice with the RPM1 culture medium. Such monolayers were composed of more than 98% of monocyte-macrophage series, determined morphologically after staining with Giemsa. After the final wash and aspiration, serial dilutions of 0.2ml test media were added and the monolayers were cultured for 12 hr at 37°C. The monolayers were then washed and 2 X lo4 5’Cr-labeled P8 15 target cells suspended in 0.2-ml aliquots of the RPM1 culture medium supplemented with 10 rig/ml LPS (Escherichia coli strain 0 111:B4, Difco) were added to each well. The cell mixtures were cultured for 18 hr and “Cr releasewas determined in 0.1 ml of the supematant. The radioactivity was measured with a well-type gamma counter. Percentagespecific lysis was calculated, using the following formula: % specific lysis =
experimental release - control release x 100. maximum release - control release
Control releasewas the amount released from labeled target cells cultured with nonactivated macrophages treated only with the RPM1 culture medium. Maximum release was the amount released from labeled target cells when incubated with 0.1 ml of 10% Triton X-100 (Wako Pure Chemical Industries, Ltd.). The data shown were calculated from six replicate determinations.
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315
Treatment of spleenand LN cells with nylon wool column. Nylon wool nonadherent T-cell-enriched fraction was prepared by a modification of the method of Julius et al. (26). After double passageover a nylon wool column, from 10 to 15% of the cells were recovered in the eluate, of which more than 90% were Thy- 1.2 positive, as determined by fluorescence staining and flow cytometric analysis. Anti- Thy-l .2 antibody plus complementtreatment of spleenand LN cells.Separate aliquots of cells ( 1 X 1O7cells/ml) were incubated in a 1:2000 dilution of monoclonal anti-Thy- 1.2 antibody (clone F7D5, Olac 1976 Ltd., Oxan, U.K.) at 4°C for 40 min. After washing, these cells were incubated further for 60 min at 37°C with 1:10 diluted Low-Tox rabbit complement (Cedarlane Laboratories, Ltd., Homby, Ontario, Canada). Thereafter, the cells were washed three times with HBSS, resuspended in the RPM1 culture medium, and then used as T-cell-depleted spleen and LN cells. Absorption of immune serum by anti-mouseIg-conjugated Sepharose.Anti-mouse Ig-conjugated Sepharose was prepared as described (19). Absorption of immune serum by anti-mouse Ig-conjugated Sepharosewas performed at 4°C for 12 hr. Statistics.The statistical significance of the data was determined by Student’s t test. A P value of lessthan 0.05 was taken as significant. RESULTS
Antigen-specific augmentation of DFR in mice transferred with immune serum. When recipients are transferred with the serum from donors immunized and elicited with heterologous erythrocytes and immunized with the same antigen, DFR became positive 3 days later. At 3 days after the immunization, however, DFR was not positive in mice without such a transfer, as reported (19,20). Accordingly, a similar transfer experiment was carried out to determine whether the immune serum possessed the ability to augment DFR, when xenogeneic tumor cells were used as the antigen. Immune sera were obtained as described under Materials and Methods. As shown in Table 1, the transfer of immune serum from AH 130-immunized donors significantly augmented AH 130-specific DFR but did not augment CRBC-specific DFR in transferred recipients. Inversely, the transfer of immune serum from CRBC-immunized donors could augment only CRBC-specific DFR. Thus, the serum from donors immunized and elicited with AH 130 tumor cells contained the augmentation factor capable of augmenting DFR antigen specificity in transferred recipients. Experiments with the same protocol were performed three times and similar results were obtained. In further experiments, data were repeatedly confirmed at least twice. Augmentation of anti-tumor cytostatic activity in PEC from mice transferred with immune serum. The CMS assaywas carried out by using PEC from mice transferred with the immune serum and immunized with MMC-AH130 tumor cells 5 days before. The immune serum used in the assay for augmentation of DFR was also used in this experiment. The PEC were obtained by injecting ip with 1 ml of 2% starch 2 days before the assay.As shown in Table 2, the immune serum augmented cytostatic activity in PEC, as well as DFR, compared to that in PEC from mice immunized without such a transfer. Cytostatic activity was not detected in PEC from mice transferred with the immune serum, without the immunization of recipients. Cytostatic activity in PEC was always higher in mice transferred with the immune serum and immunized than that in mice immunized without such a transfer at 4 to 6 days after the antigenic challenge. These results suggestedthat the immune serum possessedthe
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TABLE 1 Antigen-Specific Augmentation of DFR in Mice Transferred with Immune Serum Transferred serum”
Antigen b
DFR’ (X 0.1 mm)
Nontransferred controls Nonimmunized donors AH I30-immunized CRBC-immunized
AH130 AH130 AH130 AH130
0.4 + 0.3 0.2 + 0.2d 3.9 -t 0.3’ 0.5 * 0.2d
Nontransferred controls Nonimmunized donors AH 130-immunized CRBC-immunized
CRBC CRBC CRBC CRBC
0.4+0.1 0.2 zk0.2d 0.4 r 0.2d 3.5 * 0.4e
’ CY-pretreated C57BL/6 mice were immunized SCwith 1 X 10’ MMC-AH130 tumor cells or 1 X 10s CRBC emulsified with CFA as serum donors. At 7 days after the immunization, these mice were injected with the same antigen without CFA, i.e., 1 X lo6 MMC-AH130 tumor cells or 2.5 X 10’ CRBC, into a hind footpad for elicitation of DFR. One day later, sera were obtained from these mice. ’ One milliliter of immune serum was transferred iv to CY-pretreated C57BL/6 mice. Three to six hours later, the recipients were immunized with 1 X 10’ MMC-AH130 tumor ceils or 2.5 X lo6 CRBC. Three days after the immunization, these mice were injected with the same antigen, i.e., 1 X lo6 MMC-AH130 tumor cells or 2.5 X 10’ CRBC into a hind footpad for elicitation of DFR. ’ DFR was expressed as footpad swelling 24 hr after the elicitation. Each value represents the mean f SEM of footpad swelling in five mice. d Not significant. eValues that differ significantly from nontransferred controls, P < 0.00 1.
ability to augment cytostatic activity, and that the expression of augmentation effect in this system required the antigenic challenge in transferred recipients. When effector cells were cultured with target cells at the effector:target ratio of 5: 1, the baseline of [‘*‘I]UdR uptake by target cells was decreased,as compared with that TABLE 2 Augmentation of Cytostatic Activity in PEC from Mice Transferred with Immune Serum Transfer of immune serum’
Immunization of recipient b
-
-
+ +
+ +
[i2SI]UdR uptake (% CTS) ET, 5:l
E:T, 2.5: 1
9842 + 392’ 9216 zk237d (6.3) 4008+211’ (59.3) 1680 -+ 1lO”‘(82.9)
14442 f 758 14992 + 621 d (-3.8) 13002 + 327d (10.0) 6722 + 625
a Seefootnote a of Table 1. The immune serum from AH 130-immunized donors was used in this experiment. b One milliliter of immune serum was transferred iv to CY-pretreated C57BL/6 mice. Three to six hours later, the recipients were immunized with 1 X 10’ MMC-AH 130 tumor cells. Starch-induced PEC were harvested at 5 days after the immunization and were used as effector cells. Each group included five mice. ’ Each value represents the mean of sii replicates + SEM. d Values that are not significant, compared with a nontransferred and nonimmunized control group. pValues that differ significantly from a nontransferred and nonimmunized control group, P < 0.00 1. ‘Values that differ significantly from a nontransferred, but immunized group, P < 0.001.
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TABLE 3 Absorption of Immune Serum with Rabbit Anti-Mouse Ig-Conjugated Sepharose4B
Transferred serum’
Immunization of recipientsb
-
-
Control serum Native immune serum Rabbit anti-mouse IgSepharoseabsorbed
+ + + +
[1251]UdRuptake (% CTS) E:T, 5: 1
7960 2 399’ 8016 + 671 (-0.6) 7234 -t 444d (9.2) 5263 f 325e(33.9) 4135 *218”(48.1)
’ Immune serum was obtained as described in footnote a of Table 2. This immune serum was absorbed with rabbit anti-mouse Ig-conjugated Sepharose 4B at 4’C for 12 hr. Control serum was obtained from normal mice. bSee footnote b of Table 2. cEach value represents the mean of six replicates k SEM. d Values that are not significant, compared with a nontransferred but immunized group. eValues that differ significantly from a nontransferred hut immunized group, P < 0.005.
at the effector:target ratio of 2.5: 1. This decrease may be caused by changing the culture conditions or by natural cytotoxicity of macrophages, since the total number of cells in each well increases approximately twice, under the assay condition at the effector:target ratio of 5: 1.
Absorption of immune serum with rabbit anti-mouseIg-conjugated Sepharose4B. The immune serum containing the augmentation factor for cytostatic activity was absorbed with rabbit anti-mouse IgG (K, X, y specific)-conjugated Sepharose 4B at 4°C for 12 hr, and the activity was assessed.As shown in Table 3, the augmentation activity was not absorbed with rabbit anti-mouse Ig-Sepharose. These results suggest that the augmentation factor differed from conventional immunoglobulins.
Augmentation activity of various kinds of immune serumfor cytostatic activity in PEC. Experiments were done to determine the augmentation activity of various kinds of immune serum (Table 4). The immune serum from donor mice immunized with MMC-AH 130 tumor cells in CFA and elicited 24 hr before the serum harvest possesseda high degree of augmentation activity, as described above. On the other hand, such an activity was not seenwhen such donors were not injected with eliciting antigens 1 day before the serum harvest. Accordingly, the elicitation with antigen is necessaryfor the production of augmentation factor, when donors were immunized with MMC-AH 130 tumor cells in CFA. However, such an activity was evident in the serum from donors without the elicitation with antigen, when such donors were inoculated with viable AH 130 tumor cells, although such an activity was somewhat weak compared with that in the serum from antigen-elicited donors. In mice inoculated SCwith 1 X 10’ viable AH1 30 tumor cells, tumors grew for 7 days after the inoculation, thereafter rapidly regressed,and were completely rejected at 15 days after the inoculation. The augmentation activity was evident in the serum from donors inoculated 5 or 8 days previously; however, it was not detected
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TABLE 4 Augmentation Effect of Various Rinds of Immune Serum on Cytostatic Activity in PEC Treatment of serum donors Expt. I
Immunization
Elicitation’
Nontransferred Nontransferred MMC-AH 130 in CFAd
Nontransferred Nontransferred MMC-AH 130 MMC-AH 130 Nontransferred Nontransferred MMC-AH 130 Nontransferred Nontransferred -
-
II
III
MMC-AH 130 in CFA Nontransferred Nontransferred Viable AH 1308 Viable AH 130 Nontransferred Nontransferred Viable AH 130 (5 days) Viable AH 130 (8 days) Viable AH 130 ( 15 days)
-
Immunization of recipient@
[‘*‘I]UdR uptake (% CTS) ET, 5: I
+ + + + + + + + + + + +
106.52r 897” 7348 r 698 (30.2) 7432 r 525’ (30.2) 6982 + 595’ (34.5) 3453 f 623’(67.6) 10022 + 466 81402454 (18.8) 8334 + 442’ (16.8) 5846 f 254’(41.7) 4147 k 281/(58.6) 12045 f 735 9574 z!z675 (20.5) 4185 +- 379’(65.3) 4356 +- 562’(63.8) 9648 5 957’ (19.9)
’ In Experiments I and II, AH 130-immunized serum donors were injected with 1 X 10’ MMC-AH130 tumor cells into a hind footpad at 7 days after the immunization for elicitation of DIR. One day later, sera were obtained from these mice. Sera from nonelicited mice were obtained at 8 days after the immunization. ’ Seefootnote b of Table 2. ’ Each value represents the mean of six replicates +- SEM. d C57BL/6 mice were immunized sc with 1 X 10’ MMC-AH130 tumor cells emulsified with CFA as serum donors. Two days before the immunization, these mice were pretreated with CY. ’ Values that are not significant, compared with a nontransferred but immunized group. ‘Values that differ significantly from a nontransferred but immunized group, P c 0.005. g C57BL/6 mice were inoculated scwith 1 X 10’ viable AH 130 tumor cells. Two days before the inoculation, these mice were pretreated with CY. In Experiment III, sera from nonelicited mice were obtained at 5,8, and 15 days after the inoculation.
in the serum from donors inoculated 15 days previously without the elicitation with antigen.
Cell fraction analysis of augmentedcytostatic activity in PEC induced by transfer of immune serum. When whole PEC were separated into plastic dish-adherent and -nonadherent cells, cytostatic activity was not detected in each fraction of adherent and nonadherent cells (Table 5). However, when adherent cells of PEC from normal mice were added at the ratio of 1:1 to nonadherent cells from mice transferred with the immune serum and immunized with MMC-AH 130 tumor cells, cytostatic activity was restored and the augmenting effect by the transfer of immune serum also became detectable. When adherent cells of PEC from mice transferred with the immune serum and immunized or mice immunized without such a transfer were added to these nonadherent cells, almost the same results were obtained, compared to that in adherent cells of PEC from normal mice. Taken together, the expression of cytostatic activity in our system required the collaboration of adherent and nonadherent cells, and the adherent cells of PEC, even from normal mice, were available as the source of adherent cells for the restoration of cytostatic activity in the presence of immune nonadherent cells.
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TABLE 5 Cell Fraction Analysis of Augmented Cytostatic Activity in PEC Induced by Transfer of Immune Serum Transfer of immune serum’
Immunization of recipientsb
Cell fraction of PEC’
-
-
Whole Adherent Nonadherent Nonadherent + normal adherent Whole Adherent Nonadherent Nonadherent + normal adherent Whole Adherent Nonadherent Nonadherent + normal adherent
-
+
[ ‘ZSI]UdR uptake (96CTS)d E:T, 5: 1 12921 -t 456’ 12102 + 478 16952 + 548 13301+- 819 10897 + 465 (15.7) 11749 + 754/(2.9) 17405 + 715’(-2.7) 10335 + 230 (22.3) 8041 +318x(37.8) 11355 2 657’(6.2) 16868 2 985/(0.5) 6410+ 34jg(51.8)
DSee footnote a of Table 2. b Seefootnote b of Table 2. Each group in this experiment included 20 mice. c Whole PEC of each group were fractionated and nonadherent cells were reconstituted with adherent cells of starched-induced PEC from normal mice, as described under Materials and Methods. d Each % CTS was calculated, compared with the same fraction of a nonimmunized control group. ’ Each value represents the mean of six replicates -t SEM. ‘Values that are not significant, compared with the same fraction of a nonimmunized control group. gValues that differ significantly from the same fraction of a nontransferred but immunized group, P
Augmentation of cytostatic activity in spleen and LN cellsfrom mice transferred with immune serum. Cytostatic activity could not be detected in spleen and LN cells from mice transferred with the immune serum and immunized with MMC-AH 130 tumor cells or mice immunized without such a transfer (Table 6). However, when the adherent cells of PEC from normal mice were added to the spleen and LN cells at the ratio of 1:1, cytostatic activity could be detected, and the activity of such cells from mice transferred with the immune serum and immunized was higher than that of cells from mice immunized without such a transfer. Thus, the spleen and LN cells from immune mice included primary effector cells for cytostatic activity, but a sufficient number of adherent cells was always required for functional expression. Furthermore, these results suggestthat the appearance of primary effector cells for cytostatic activity is also accelerated in spleen and LN cells by the transfer of immune serum. When the immune spleen and LN cells were treated with monoclonal anti-Thy1.2 and complement, such cells could not render the cytostatic activity to the adherent cells of PEC from normal mice (Table 7), suggesting that primary effector cells for cytostatic activity are Thy- 1-positive T cells or cytostatic activity is T-cell dependent. For further confirmation, nylon wool nonadherent T-cell-enriched fractions of spleen and LN cells were used as effector cells. When the adherent cells of PEC from normal mice were added to such cells, cytostatic activity was detected. These results
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TABLE 6 Augmentation ofcytostatic Activiy in Spleen and LN Cells from Mice Transferred with Immune Serum Transfer of immune serum0
-
-
+
Immunization of recipientsb
Effector cells’
-
PEC Spleen + LN Spleen + LN + normal adherent PEC Spleen + LN Spleen + LN + normal adherent PEC Spleen + LN Spleen + LN + normal adherent
[rZ51]UdRuptake (% CTS)d ET, 5:l 10139 + 571e 151422401 10965 + 581 6895 rfr508 (32.0) 16215 + 323’(-7.1) 8538 + 453 (22.1) 2399 + 1428(76.3) 15669 + 403/(-3.5) 4210 + 304#(40.9)
a Seefootnote a of Table 2. b Seefootnote b of Table 2. ‘Whole PEC and spleen and LN cells were used as effector cells. Spleen and LN cells were added to adherent cells of starch-induced PEC from normal mice, at the ratio of 1:1. d Each % CTS was calculated, compared with the same kind of effector cells of a nonimmunized control group. eEach value represents the mean of six replicates + SEM. lValues that are not significant, compared with the same kind of effector cells of a nonimmunized control group. 8 Values that differ from the same kind of effector cells of a nontransferred but immunized group, P
indicate that lymphokines such as MAF released from T cells contribute to the expression of cytostatic activity by activating macrophages, and that the transfer of immune serum acceleratesthe generation of such T cells. Augmentation of MAF production by spleen and LN cells from mice transferred with immune serum. To determine that MAF production was also augmented by the transfer of immune serum, the following experiments were carried out. MAF activity could be assessedby its ability to induce nonspecific anti-tumor cytotoxic (cytostatic and/or cytolytic) potentials in macrophages. In this study, MAF activity was assessed by both cytostatic and cytolytic assay and similar results were obtained. Only data obtained with the cytolytic assay, a more popular assay system (24, 29, are shown. Immune spleen and LN cells were cultured with MMC-AH 130 tumor cells and MAF activity in these cultured-supematants was assessed(Fig. 1). Such an activity was detected in l/2 and l/4 dilutions of the supematant from the immune spleen and LN cells, when these cells were harvested from mice transferred with the immune serum and immunized with MMCAH130 tumor cells at 4 days after the antigenic challenge (Fig. 1A). On the other hand, when the immune cells were harvested from mice immunized without such a transfer, a low degree of MAF activity was detected only in a l/2 dilution of the supematant. When the immune cells were harvested from mice transferred with the immune serum and immunized with MMC-AH 130 tumor cells at 5 days after their antigenic
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TABLE 1 Expression of Augmented Cytostatic Activity Requiring Sensitized T Lymphocytes Transfer of immune serum’
Immunization of recipientsb
-
-
-
+
+
+
-
-
-
+
+
+
Treatment of spleen and LN cells’ Untreated Untreated C alone Anti-Thy- 1 + C Untreated C alone Anti-Thy- 1 + C Unfractionated Nylon-nonadherent Unfractionated Nylon-nonadherent Unfractionated Nylon-nonadherent
[“‘I]UdR
uptake (% CTS)d E:T, 5: 1
10034 f 457e 703 1 + 378 (29.9) 6742 f 375 (32.8) 10127 f 468/(-0.9) 2642 + 172g(73.7) 2930 f 1638(70.8) 9821 k 389’-(2.1) 10632 f 597 10323 IL 699 9418+842 (11.4) 8487 f 7 12 (20.2) 3514 f 124g(66.7) 2889 + 1048(72.8)
DSeefootnote a of Table 2. b See footnote b of Table 2. ’ Spleen and LN cells of each group were treated with anti-Thy- 1 and complement or complement alone (the first experiment), or with nylon wool column (the second experiment), then added to adherent cells of starch-induced PEC from normal mice, at the ratio of l:l, and used as effector cells. Untreated or unfractionated spleen and LN cells were used as positive control. dEach % CTS was calculated, compared with untreated or unfractionated spleen and LN cells of a nonimmunized control group. ’ Each value represents the mean of six replicates + SEM. /Values that are not significant, compared with untreated spleen and LN cells from a nonimmunized control group. 8 Values that differ significantly from spleen and LN cells received the same treatment from a nontransferred but immunized group, P < 0.00 1.
challenge (Fig. 1B), a high degreeof MAF activity could be detected in a 1/ 16 dilution of supematant from these immune cells. In contrast, when the immune cells were harvested from mice immunized without such a transfer, MAF activity was detected in l/2 and l/4 dilutions of the supematant, although not in a l/8 dilution of the supematant. Therefore, the former supematant may have approximately two- to fourfold MAF activity, compared with the latter. MAF production was also dependent on Thy- 1-positive T cells (Figs. 2A, B), aswell asprimary effector cells for cytostatic activity. These results suggestthat the cytostatic activity in this study is compatible with MAF activity which can be assessedby “Cr-release assay and that the transfer of immune serum accelerates the generation of lymphokine-producing T cells responsible for cytostatic activity and MAF production. DISCUSSION We detected a factor(s) in the serum of mice sensitized with xenogeneic tumor cells to induce DFR, which was capable of augmenting DFR antigen specificity in transferred recipients (Table l), as similarly shown in the experiments with heterolo-
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0 2
4
I
13
REClPROCll
32
2
4
OF SUPERNATANT
5
13
32
DllUTlON
FIG. 1. Augmentation of MAF production by spleen and LN cells from mice transferred with immune serum. Transfer system and transferred immune serum were the same as in Table 2. Spleen and LN cells were harvested at 4 days (A) or 5 days (B) after the immunization of recipients, cultured with MMC-AH 130 tumor cells, and MAF activity of these supematants was evaluated as described under Materials and Methods. Spleen and LN cells from mice transferred with the immune serum and immunized with MMCAH 130 tumor cells (Cl), mice immunized without such a transfer (A), and nonimmunized mice (0) were used. Closed symbols represent MAF activity of the supematant from each group of spleen and LN cells cultured without antigens. The data are expressed as the means of six replicates + SD.
B
2
4
8
RECIPIIOCAL
13
32 OF SUPERNATANT
OlLUTlON
FIG. 2. Effect of anti-Thy- 1.2 plus complement on MAF production by spleen and LN cells. Transfer system and transferred immune serum were the same as in Table 3. Spleen and LN cells were harvested at 5 days after the immunization from the recipients transferred with the immune serum and immunized with MMC-AH130 tumor cells (A) or mice immunized without such a transfer (B). These cells treated or untreated (-) were cultured with anti-Thy- 1.2 and complement (. . . ), complement alone (-.-), with MMC-AH 130 tumor cells, and MAF activity of these supematants was evaluated as described under Materials and Methods. The data are expressed as the means of six replicates + SD.
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323
gous erythrocytes (19,20). This factor could also augment in vitro anti-tumor cytostatic activity. This augmented cytostatic activity appeared at 4 days after the antigenie challenge of serum recipients, in the whole PEC (Table 2, Fig. 1). The activity was attenuated or abolished when plastic dish-adherent or nonadherent PEC alone were used as effector cells, and was restored when adherent PEC from normal mice were added to the immune nonadherent PEC (Table 5). The appearance of primary effector cells responsible for cytostatic activity was also accelerated in spleen and LN cells from mice transferred with the immune serum before the antigenic challenge (Table 6). However, sufficient numbers of adherent PEC, in either caseof nonadherent PEC and spleen and LN cells, were always necessary as the direct effecters for functional expression and adherent PEC even from normal mice were available as the source of adherent cells. These primary effecters were T lymphocytes (Table 7). Lymphokine(s) such as MAF releasedfrom such sensitized T lymphocytes contribute to the expression of cytostatic activity by activating adherent cells (macrophages) (Figs. 2,3). Thus, the augmentation factor may exert its effect in the induction phase to raise sensitized T lymphocytes, and may accelerate the generation of lymphokineproducing T lymphocytes responsible for cytostatic activity. The contribution of lymphotoxin, cytotoxic T cells, natural killer (NK) cells, arming macrophages, or antibody-dependent cell-mediated cytotoxicity (ADCC) may be negligible, since this augmented cytostatic activity requires the collaboration of both antigen-specific sensitized T lymphocytes and macrophages. Furthermore, cytotoxic activity assessedby a 4-hr “Cr release from labeled AH 130 target cells was hardly detectable in PEC and spleen and LN cells from these immunized mice (data not shown), as reported earlier ( 11). NK activity in PEC and spleen cells assessedby a 4-hr 5‘Cr releasefrom labeled YAC-1 target cells was not augmented by the transfer of immune serum (data not shown). Cytotoxic antibodies could not be detected in assay systems for ADCC and for complement-dependent cytotoxic antibodies, in the immune serum we used in the present study (data not shown). The proposed participation of cytostatic effector cells in the functional expression of DTH may be supported by our previous studies (3,4, 11, 12), and in vitro cytostatic activity may be an useful in vitro assay system for in vivo DTH reaction. In that system which involves use of a murine allotransplantable tumor, sarcoma 180, the inhibition pattern of in vivo tumor growth correlated closely with the in vitro cytostatic activity and in vivo DFR in mice with an acquired anti-tumor resistance against such a tumor. The cytostatic activity was also seenin macrophages activated by sensitized lymphocytes, and cytotoxic lymphocytes were not detected (3). The same result was obtained even in syngeneic tumor systemswith Meth A tumor of BALB/c origin and others (4, 12). We observed the augmentation activity of our factor in a transfer system, as described above, but have not yet determined its role in the immunized hosts (donors of the augmentation factor). When serum donors were immunized with MMC-AH 130 tumor cells in CFA, the elicitation with antigen was essentially required for production of the augmentation factor in the serum donors (Table 4), as required in systems with heterologous erythrocytes (19,20). In contrast, when serum donors were inoculated with viable AH 130 tumor cells 5 or 8 days previously, a sufficient degree of augmentation activity was detected, without elicitation of serum donors. However, when serum donors were inoculated 15 days previously, such an activity was not detected without elicitation of serum donors. In serum donors inoculated with viable
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AH130 tumor cells 5 or 8 days previously, tumor masseswere palpable, but not in mice inoculated 15 days previously. At 5 or 8 days after the inoculation, sensitized T lymphocytes may be restimulated at the site of direct elimination of tumor cells and produce the augmentation factor. These findings suggestthat the augmentation factor may exert its effect on the induction phase, in the immunized hosts. Lawrence reported that a low-molecular-weight substance from leucocyte lysate was able to transfer DTH (termed transfer factor) (27). Such a transfer factor can introduce DTH antigen specificity in recipients without additional antigenic challenge. On the other hand, our augmentation factor required the antigenic challenge of recipients for the expression of activity. Our factor exerts its effect on the induction phase of DTH, while “transfer factor” is effective on the expression phase. Ptak and co-workers reported that an antigen-binding factor produced by T cells was able to transfer an antigen-specific immediate hypersensitivity-like skin reaction in mice (16). Their factor leads to mast cell activation and the release of vasoactive amines, however, has no effect on the induction phase and does not accelerate the induction of sensitized lymphocytes after immunization. Antigen-specific factors released in the serum of donors immunized and elicited in the same manner as described in the present paper may participate in the acceleration and augmentation of DTH and acquired resistance against various kinds of particulate antigens. In our studies, the presence of such a factor was suggestedin experimental systems with Listeriu monocytogenes to which acquired resistance can be attributed to the cooperation of lymphokine-producing sensitized T lymphocytes and macrophages (Yamada et al., manuscript in preparation). Furthermore, there is the possibility that the same type of antigen-specific factor contributes to the resistance to some kinds of syngeneic tumors, since lymphokine-induced cytostatic macrophageswere confirmed to be responsible for the elimination of syngeneic tumor cells such as Meth A (4, 12,28). The biological meaning of this factor in the host resistance will be defined by further studies. ACKNOWLEDGMENTS We thank Shigeko Ueda, Chikako Ishikawa, Izumi Yoshimatsu, and Atsuko Yamada for technical assistance and secretarial services and Mariko Ohara for comments on the manuscript.
REFERENCES 1. Paranjpe, M. S., and Boone, C. W., Znt. J. Cancer 13, 179, 1974. 2. Macek, C. M., Kahn, B. D., and Pellis, N. R., J. Zmmunol. 125, 1639,198O. 3. Tomita, Y., Himeno, K., Fujiki, M., Nomoto, K., and Hirohata, T., Oncology 38,30 1, 1981. 4. Matsumoto, T., Himeno, K., Mitani, M., Mori, K., Miake, S., and Nomoto, K., J. C/in. Lab. Zmmunol. 19,83, 1986. 5. Mackaness,G.B., J. Exp. Med. 116,381, 1962. 6. Miki, K., and Mackaness, G. B., J. Exp. Med. 120,93,1964. 7. Lane, F. C., and Unanue, E. R., J. Exp. Med. 1351104, 1972. 8. Mitsuyama, M., Nomoto, K., Akeda, H., and Takeya, K., Znfect.Zmmun. 3&l, 1980. 9. Mitsuyama, M., Nomoto, K., and Takeya, K., Infect. Zmmun. 36,72, 1982. 10. Loveland, B. E., Hogarth, P. M., Ceredig, Rh., and McKenzie, I. F. C., J. Exp. Med. 153, 1044, 1981. 11. Nomoto, K., Terasaka, R., Himeno, K., and Shiraishi, M., Japan. J. Exp. Med. 51,8 1, 1981. 12. Mitani, M., Matsumoto, T., Mori, K., Miake, S., Himeno, K., and Nomoto, K., J. Clin. Lab. Zmmunol. 18,97, 1985. 13. Leung, K. N.,andAda, G. L.,J. Exp. Med. 153,1029,1981. 14. Tucker, M. J., and Bretscher, P. A., J. Exp. Med. 155, 1037, 1982.
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15. Wright, K., and Ramshaw, I. A., J. Immunol. 130,1596, 1983. 16. Ptak, W., Askenase, P. W., Rosenstein, R. W. and Gershon, R. K., Proc. Natl. Acad. Sci. USA 79, 1969,1982. 1I. Askenase, P. W., Rosenstein, R. W., and Ptak, W., J. Exp. Med. 157,862, 1983. 18. Colizzi, V., Asherson, G. L., James, B. M. B., and Malkovsky, M., Zmmunology52,261, 1984. 19. Yamada, A., Himeno, K., Miyata, H., Kumazawa, Y ., and Nomoto, K., Cell. Zmmunol. 88,184,1984. 20. Himeno, K., Yamada, A., Miyata, H., Nanishi, F., and Nomoto, K., CelI. Zmmuno/. 95,35, 1985. 2 1. Yamada, A., Himeno, K., Kumazawa, Y., and Nomoto, K., Cell. Zmmunol. 84,206, 1984. 22. Shigeyoshi, O., Natl. Cancer Inst. Monogr. 16,5 I, 1967. 23. le Mevel, B. P., Oldham, R. K., Wells, S. A., and Herberman, R. B., J. Natl. Cancer Inst. 51, 1551, 1973. 24. Pace, J. L., and Russell, S. W., J. Zmmunol. 126, 1863, 1981. 25. Schreiber, R. D., Altman, A., and Katz, D. H., J. Exp. Med. 156,677, 1982. 26. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Zmmunol. 3,645, 1973. 21. Lawrence, H. S., J. Clin. Invest. 34,3 19, 1955. 28. Mitani, M., Mori, K., Himeno, K., Matsumoto, T., Taniguchi, K., and Nomoto, K., Cell. Zmmunol. 92,22, 1985.
IMMUNOLOGY
103,3 1l-325 (1986)
Antigen-Specific Augmentation of Delayed-Type Hypersensitivity by Immune Serum Factor in Mice: Augmentation of Anti-Tumor Cytostatic Activity’ SEIJI NAKAMURA,* KUNISUKE HIMENO, MASAMITSU MITANI, AND KIKUO
AKIRA YAMADA, NOMOTO
Department of Immunology, Medical Institute of Bioregulation and Second Department of Oral Surgery, Faculty ofDentistry, Kyushu University, 3-l-l Maidashi. Higashi-ku. Fukuoka 812, Japan Received January 7.1986; acceptedJuly 14, 1986 A humoral factor capable of augmenting delayed-type hypersensitivity antigen specificity (DAF) is present in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR). In the present study, a similar factor was identified when xenogeneic tumor cells were used as antigens. This factor also could augment the in vitro anti-tumor cytostatic activity against homologous tumor cells, which correlated with in vivo DIR to the same tumor cells. The cytostatic activity augmented by the transfer of this factor had the following characteristics: (i) The activity appeared in the whole peritoneal exudate cells (PEC) from serum recipients at 4 days after the antigenic challenge. (ii) Such an activity was revealed in the collaboration of plastic dish-nonadherent and -adherent PEC as the primary and final effecters, respectively. (iii) The appearance of primary effector cells for such an activity was also accelerated in spleen and lymph node cells. (iv) However, a sufficient number of macrophages were always required as the final effecters in their functional expression. (v) These primary effecters were sensitized T lymphocytes which produced lymphokine(s) such as macrophage-activating factor(s) and which contributed to this augmented cytostatic activity through the activation of macrophages. Thus, this immune serum factor seemsto exert functional expression by accelerating the generation of lymphokine-producing delayed-type T lymphocytes, which is also responQ 1986 Academic Fms, IOC. sible for CytOStatiC anti-tumor immUUity.
INTRODUCTION Delayed-type hypersensitivity (DTH)3 was proposed to be one of the main effector mechanisms in the anti-tumor immunity, in syngeneic or allogeneic tumor systems ’ This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan. * To whom correspondence should be addressed at the Department of Immunology, Medical Institute of Bioregulation, Kyushu University 69,3-l- 1 Maidashi, Higashi-ku, Fukuoka 8 12, Japan. 3Abbreviations used: CFA, complete Freund’s adjuvant; CRBC, chicken erythrocytes; CY, cyclophosphamide; DFR, delayed footpad reaction; DTH, delayed-type hypersensitivity; HBSS, Hanks’ balanced salt solution; Ig, immunoglobulin; ip, intraperitoneally; iv, intravenously; LN, inguinal and popliteal lymph nodes; MAF, macrophage-activating factor(s); MMC, mitomycin C; MMC-AH 130, MMC-treated AH130; PBS, phosphate-buffered saline; PEC, peritoneal exudate cells; sc, subcutaneously; 5’Cr, Na2%Zr04; [‘*‘I]UdR, [‘2SI]5-iodo-2’-deoxyuridine. 311 OOO8-8749186 $3.00 Cowri&t 0 1986 by Academic F’ms, Inc. Au rights of reproduction in any form resmed.
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(l-4), in the acquired resistance against some kinds of infection with intracellular parasites such as Mycobacteria and Listeriu (5-g), and in the transplantation immunity to allografi ( 10) and xenograft (11). In some transplanted tumor systems, antitumor cytostatic mechanisms produced by lymphokine-producing lymphocytes and macrophages were assumed to play a main role in the rejection (3,4, 11, 12). The in vitro cytostatic activity has been demonstrated to correlate well with in vivo DTH reactions (3,4). Thus, lymphokine-producing lymphocytes responsible for cytostatic activity and those for DTH reaction may be assignedto the same population. It has been established that DTH as well as antibody production and cytotoxic T cells were regulated by helper and suppressor T cells. The primary in vitro induction of DTH to influenza virus was shown to be enhanced by antigen-specific helper T cells (13). In the induction of DTH to heterologous erythrocytes, helper T cells may be required for both in vitro (14) and in vivo (15) induction. In some cases,such collaboration is mediated through antigen-specific T-cell factors. Ptak et al. (16) and Askenase et al. ( 17) demonstrated the role of T-cell factors which acted at the expression phase, while Colizzi et al. (18) reported the T-helper factor which acted at the induction phase. Recently, we found a humoral factor capable of augmenting DTH antigen specificity in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DIR) (19,20), or in the culture supematant of their immune spleen cells (21). As shown below, a similar factor was also found in the serum of mice sensitized with rat AH 130 tumor cells to induce DFR. In the present study, we investigated whether this factor could augment in vitro anti-tumor cytostatic activity as well as DFR. We obtained evidence that this augmentation factor exerted its effect in the induction phase of cytostatic activity. MATERIALS AND METHODS
Mice. Female C57BL/6 and (C57BL/6 X DBA/2)Fl (BDFl) and male DBA/2 mice were obtained from the Shizuoka Laboratory Animal Center (Hamamatsu, Japan). Male Donryu rats were obtained from the Rat Japan, Inc. (Urawa, Japan). All mice were used for experiments at 8 to 12 weeks of age and all rats were used at 4 to 5 weeks of age. Tumors. AH 130, a rat ascitic-form hepatoma (22), was maintained in vivoby serial intraperitoneal (ip) passagein Donryu rats. P8 15, a mastocytoma of DBA/2 origin, was maintained in vivoby serial ip passagein syngeneic DBA/2 mice. Antigens. Chicken erythrocytes (CRBC) obtained from normal hens by cardiac puncture were washed three times with phosphate-buffered saline (PBS) before use. Treatment with mitomycin C. AH 130 tumor cells were suspended in Hanks’ balanced salt solution (HBSS) at a concentration of 2 X 10’ cells/ml. HBSS was supplemented with 100 &ml mitomycin C (MMC; Kyowa Hakko Co., Ltd., Tokyo, Japan). After the incubation at 37°C for 60 min, the cells were washed three times with HBSS and used for experiments. Preparation of immune seru. Mice were immunized with 1 X 10’ MMC-treated AH1 30 (MMC-AH130) tumor cells or 1 X 10’ CRBC emulsified with complete Freund’s adjuvant (CFA; Difco, Detroit, Mich.) supplemented with 5 mg/ml of heatkilled Mycobacterium tuberculosisAoyama B (donor immunization). Emulsion (0.2 ml) was subcutaneously (SC)injected into a hind thigh. In some experiments, mice
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were inoculated SCwith 1 X lo7 viable AH 130 tumor cells in 0.2 ml PBS into a hind thigh (donor immunization). Cyclophosphamide (CY; Endoxan, Shionogi & Co., Osaka, Japan) was dissolved in sterile distilled water and administered ip in a dose of 100 mg/kg, 2 days before the immunization. At 7 days after the immunization, 1 x lo7 MMC-AH 130 tumor cells or 2.5 X lo7 CRBC in 0.05 ml PBS were injected into the right footpad of each mouse to elicit DFR. The same antigen was used for the immunization and elicitation of serum donors. Twenty-four hours later, blood samples were collected and the sera were prepared. In some experiments, samples were obtained 8 days after the immunization, without elicitation of serum donors. When donor mice were inoculated with viable AH130 tumor cells, samples were obtained not only 8 days but also 5 and 15 days after the inoculation, without elicitation of serum donors. Assayfor the activity of augmentationfactor by DFR. One milliliter of the immune serum was transferred intravenously (iv) via the tail vein into recipient mice pretreated ip with 100 mg/kg of CY 4 days before. Three to six hours later, mice were immunized with 1 X lo7 MMC-AH130 tumor cells or 2.5 X lo6 CRBC in 0.05 ml PBS into the right hind footpad (recipient immunization). Three to four days after the immunization, DFR was elicited by injection of 1 X lo6 MMC-AH 130 tumor cells or 2.5 X lo7 CRBC in 0.05 ml PBS into the left hind footpad. The degree of swelling was measured 24 hr later with a dial thickness gauge. Reactions were expressed as the difference in thickness between the left and right footpad. The results are shown as the mean f the standard error of the mean (SEM) of five to eight animals. Tissueculture medium. RPM1 1640 medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% NU-SERUM (Collaborative Research, Inc., Lexington, Mass.), 20 mM Hepes, 100 units/ml penicillin, and 100 pg/ml streptomycin was used for in vitro experiments. Efictor cellsfor cell-mediated cytostatic (CMS) assays.Peritoneal exudate cells (PEC) were used as effector cells in most experiments. The PEC were induced by injecting ip with 1 ml of 2% boiled and autoclaved starch (Wako Pure Chemical Industries, Ltd., Osaka, Japan) 2 days before the assay. In some experiments, the whole PEC were fractionated into adherent cells and nonadherent cells. The whole PEC were suspended in the RPM1 culture medium at a concentration of 1 X lo6 cells/ml and incubated in plastic dishes (Falcon 300 1, Becton-Dickinson, Oxnard, Calif.) for 60 min at 37°C. The nonadherent cells were put into other plastic dishes for the second incubation, and the adherent cells were washed three times with warm HBSS (37°C) to remove the residual nonadherent cells. The adherent cells were incubated in PBS containing 0.6 mM EDTA on ice for 15 min and harvested by gently pipetting. The nonadherent cells were harvested at the end of the second incubation at 37°C for 60 min. For reconstitution experiments, the adherent cells were added to the nonadherent cells at the ratio of 1:1. In some experiments, spleen cells and inguinal and popliteal lymph nodes (LN) cells were used as the effector cells for the CMS assay. Assaysfor the activity of augmentationfactor by cytostaticactivity. The CMS assay was carried out as described (3, 4, 11, 12) with slight modifications. AH 130 tumor cells were incubated at 37°C for 1 hr in plastic dishes (Falcon 300 1) at a concentration of 1 X 1O6cells/ml to remove macrophages of host origin and used as target cells. Cell suspensions containing 2 X 1O4unlabeled target cells and 5 or 10 X 1O4effector cells
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(effector:target ratio of 2.5: 1 or 5: 1) in 0.2-ml aliquots of the RPM1 culture medium in flat-bottom microculture plates (Cell Wells 25860, Corning Glass Works, Corning, N.Y.) were cultured in a humidified atmosphere of 5% CO2 at 37°C for 24 hr. DNA synthesis was assessedby adding 0.4 &i [‘251]5-iodo-2’deoxyuridine ([iZ51]UdR; Amersham International plc, Amersham, England) per well 9 hr before the end of the culture (23). The cultures were harvested using a Labo Mash Cell Harvester (Lab0 Science Co., Ltd., Tokyo, Japan) and the radioactivity was measured with a well-type gamma-counter (Shimadzu, Tokyo, Japan). Percentage of cytostasis (% CTS) was calculated, using the following formula: %CTS = 100 ’
[i2?]UdR uptake by target cells with immune effector cells ’ - [i2’I]UdR uptake by target cells with nonimmune effector cells *
Assay for the activity of augmentation factor by macrophage-activating factor(s) (MAF)-production by spleen and LN cells. Spleen and LN cells from serum recipients immunized according to the same protocol as that for DFR were cultured at 5 X lo6 cells/ml for 24 hr at 37°C under 5% C02, with or without 5 X lo6 MMC-AH130 tumor cells/ml. MAF activity in these supematants was evaluated as the ability to induce nonspecific cytolytic activity in peptone-induced BDFl macrophages toward P8 15 tumor cells, according to Pace and Russel(24) and Schreiber et al. (25), with some modifications. P8 15 tumor cells were suspended at a concentration of 1 X 10’ cells/ml and incubated at 37°C for 1 hr with 50 &i of Na25’CrG4(“Cr; Japan Atomic Energy Research Institute, Tokyo, Japan). Thereafter, these cells were washed three times with HBSS and used as “0-labeled target cells. The peptone-induced PEC were obtained 3 days after injecting ip with 1 ml of 10% proteose peptone (Difco), and washed three times with HBSS. The PEC were adjusted to a concentration of 2 X lo6 cells/ml in the RPM1 culture medium and the suspension was put into flatbottom microculture plates (Cell Wells 25860) at a volume of 0.1 ml per well. After culturing for 3 hr, the nonadherent cells were aspirated, and the adherent monolayers were washed twice with the RPM1 culture medium. Such monolayers were composed of more than 98% of monocyte-macrophage series, determined morphologically after staining with Giemsa. After the final wash and aspiration, serial dilutions of 0.2ml test media were added and the monolayers were cultured for 12 hr at 37°C. The monolayers were then washed and 2 X lo4 5’Cr-labeled P8 15 target cells suspended in 0.2-ml aliquots of the RPM1 culture medium supplemented with 10 rig/ml LPS (Escherichia coli strain 0 111:B4, Difco) were added to each well. The cell mixtures were cultured for 18 hr and “Cr releasewas determined in 0.1 ml of the supematant. The radioactivity was measured with a well-type gamma counter. Percentagespecific lysis was calculated, using the following formula: % specific lysis =
experimental release - control release x 100. maximum release - control release
Control releasewas the amount released from labeled target cells cultured with nonactivated macrophages treated only with the RPM1 culture medium. Maximum release was the amount released from labeled target cells when incubated with 0.1 ml of 10% Triton X-100 (Wako Pure Chemical Industries, Ltd.). The data shown were calculated from six replicate determinations.
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Treatment of spleenand LN cells with nylon wool column. Nylon wool nonadherent T-cell-enriched fraction was prepared by a modification of the method of Julius et al. (26). After double passageover a nylon wool column, from 10 to 15% of the cells were recovered in the eluate, of which more than 90% were Thy- 1.2 positive, as determined by fluorescence staining and flow cytometric analysis. Anti- Thy-l .2 antibody plus complementtreatment of spleenand LN cells.Separate aliquots of cells ( 1 X 1O7cells/ml) were incubated in a 1:2000 dilution of monoclonal anti-Thy- 1.2 antibody (clone F7D5, Olac 1976 Ltd., Oxan, U.K.) at 4°C for 40 min. After washing, these cells were incubated further for 60 min at 37°C with 1:10 diluted Low-Tox rabbit complement (Cedarlane Laboratories, Ltd., Homby, Ontario, Canada). Thereafter, the cells were washed three times with HBSS, resuspended in the RPM1 culture medium, and then used as T-cell-depleted spleen and LN cells. Absorption of immune serum by anti-mouseIg-conjugated Sepharose.Anti-mouse Ig-conjugated Sepharose was prepared as described (19). Absorption of immune serum by anti-mouse Ig-conjugated Sepharosewas performed at 4°C for 12 hr. Statistics.The statistical significance of the data was determined by Student’s t test. A P value of lessthan 0.05 was taken as significant. RESULTS
Antigen-specific augmentation of DFR in mice transferred with immune serum. When recipients are transferred with the serum from donors immunized and elicited with heterologous erythrocytes and immunized with the same antigen, DFR became positive 3 days later. At 3 days after the immunization, however, DFR was not positive in mice without such a transfer, as reported (19,20). Accordingly, a similar transfer experiment was carried out to determine whether the immune serum possessed the ability to augment DFR, when xenogeneic tumor cells were used as the antigen. Immune sera were obtained as described under Materials and Methods. As shown in Table 1, the transfer of immune serum from AH 130-immunized donors significantly augmented AH 130-specific DFR but did not augment CRBC-specific DFR in transferred recipients. Inversely, the transfer of immune serum from CRBC-immunized donors could augment only CRBC-specific DFR. Thus, the serum from donors immunized and elicited with AH 130 tumor cells contained the augmentation factor capable of augmenting DFR antigen specificity in transferred recipients. Experiments with the same protocol were performed three times and similar results were obtained. In further experiments, data were repeatedly confirmed at least twice. Augmentation of anti-tumor cytostatic activity in PEC from mice transferred with immune serum. The CMS assaywas carried out by using PEC from mice transferred with the immune serum and immunized with MMC-AH130 tumor cells 5 days before. The immune serum used in the assay for augmentation of DFR was also used in this experiment. The PEC were obtained by injecting ip with 1 ml of 2% starch 2 days before the assay.As shown in Table 2, the immune serum augmented cytostatic activity in PEC, as well as DFR, compared to that in PEC from mice immunized without such a transfer. Cytostatic activity was not detected in PEC from mice transferred with the immune serum, without the immunization of recipients. Cytostatic activity in PEC was always higher in mice transferred with the immune serum and immunized than that in mice immunized without such a transfer at 4 to 6 days after the antigenic challenge. These results suggestedthat the immune serum possessedthe
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TABLE 1 Antigen-Specific Augmentation of DFR in Mice Transferred with Immune Serum Transferred serum”
Antigen b
DFR’ (X 0.1 mm)
Nontransferred controls Nonimmunized donors AH I30-immunized CRBC-immunized
AH130 AH130 AH130 AH130
0.4 + 0.3 0.2 + 0.2d 3.9 -t 0.3’ 0.5 * 0.2d
Nontransferred controls Nonimmunized donors AH 130-immunized CRBC-immunized
CRBC CRBC CRBC CRBC
0.4+0.1 0.2 zk0.2d 0.4 r 0.2d 3.5 * 0.4e
’ CY-pretreated C57BL/6 mice were immunized SCwith 1 X 10’ MMC-AH130 tumor cells or 1 X 10s CRBC emulsified with CFA as serum donors. At 7 days after the immunization, these mice were injected with the same antigen without CFA, i.e., 1 X lo6 MMC-AH130 tumor cells or 2.5 X 10’ CRBC, into a hind footpad for elicitation of DFR. One day later, sera were obtained from these mice. ’ One milliliter of immune serum was transferred iv to CY-pretreated C57BL/6 mice. Three to six hours later, the recipients were immunized with 1 X 10’ MMC-AH130 tumor ceils or 2.5 X lo6 CRBC. Three days after the immunization, these mice were injected with the same antigen, i.e., 1 X lo6 MMC-AH130 tumor cells or 2.5 X 10’ CRBC into a hind footpad for elicitation of DFR. ’ DFR was expressed as footpad swelling 24 hr after the elicitation. Each value represents the mean f SEM of footpad swelling in five mice. d Not significant. eValues that differ significantly from nontransferred controls, P < 0.00 1.
ability to augment cytostatic activity, and that the expression of augmentation effect in this system required the antigenic challenge in transferred recipients. When effector cells were cultured with target cells at the effector:target ratio of 5: 1, the baseline of [‘*‘I]UdR uptake by target cells was decreased,as compared with that TABLE 2 Augmentation of Cytostatic Activity in PEC from Mice Transferred with Immune Serum Transfer of immune serum’
Immunization of recipient b
-
-
+ +
+ +
[i2SI]UdR uptake (% CTS) ET, 5:l
E:T, 2.5: 1
9842 + 392’ 9216 zk237d (6.3) 4008+211’ (59.3) 1680 -+ 1lO”‘(82.9)
14442 f 758 14992 + 621 d (-3.8) 13002 + 327d (10.0) 6722 + 625
a Seefootnote a of Table 1. The immune serum from AH 130-immunized donors was used in this experiment. b One milliliter of immune serum was transferred iv to CY-pretreated C57BL/6 mice. Three to six hours later, the recipients were immunized with 1 X 10’ MMC-AH 130 tumor cells. Starch-induced PEC were harvested at 5 days after the immunization and were used as effector cells. Each group included five mice. ’ Each value represents the mean of sii replicates + SEM. d Values that are not significant, compared with a nontransferred and nonimmunized control group. pValues that differ significantly from a nontransferred and nonimmunized control group, P < 0.00 1. ‘Values that differ significantly from a nontransferred, but immunized group, P < 0.001.
DELAYED-TYPE
HYPERSENSITIVITY
AUGMENTATION
FACTOR
317
TABLE 3 Absorption of Immune Serum with Rabbit Anti-Mouse Ig-Conjugated Sepharose4B
Transferred serum’
Immunization of recipientsb
-
-
Control serum Native immune serum Rabbit anti-mouse IgSepharoseabsorbed
+ + + +
[1251]UdRuptake (% CTS) E:T, 5: 1
7960 2 399’ 8016 + 671 (-0.6) 7234 -t 444d (9.2) 5263 f 325e(33.9) 4135 *218”(48.1)
’ Immune serum was obtained as described in footnote a of Table 2. This immune serum was absorbed with rabbit anti-mouse Ig-conjugated Sepharose 4B at 4’C for 12 hr. Control serum was obtained from normal mice. bSee footnote b of Table 2. cEach value represents the mean of six replicates k SEM. d Values that are not significant, compared with a nontransferred but immunized group. eValues that differ significantly from a nontransferred hut immunized group, P < 0.005.
at the effector:target ratio of 2.5: 1. This decrease may be caused by changing the culture conditions or by natural cytotoxicity of macrophages, since the total number of cells in each well increases approximately twice, under the assay condition at the effector:target ratio of 5: 1.
Absorption of immune serum with rabbit anti-mouseIg-conjugated Sepharose4B. The immune serum containing the augmentation factor for cytostatic activity was absorbed with rabbit anti-mouse IgG (K, X, y specific)-conjugated Sepharose 4B at 4°C for 12 hr, and the activity was assessed.As shown in Table 3, the augmentation activity was not absorbed with rabbit anti-mouse Ig-Sepharose. These results suggest that the augmentation factor differed from conventional immunoglobulins.
Augmentation activity of various kinds of immune serumfor cytostatic activity in PEC. Experiments were done to determine the augmentation activity of various kinds of immune serum (Table 4). The immune serum from donor mice immunized with MMC-AH 130 tumor cells in CFA and elicited 24 hr before the serum harvest possesseda high degree of augmentation activity, as described above. On the other hand, such an activity was not seenwhen such donors were not injected with eliciting antigens 1 day before the serum harvest. Accordingly, the elicitation with antigen is necessaryfor the production of augmentation factor, when donors were immunized with MMC-AH 130 tumor cells in CFA. However, such an activity was evident in the serum from donors without the elicitation with antigen, when such donors were inoculated with viable AH 130 tumor cells, although such an activity was somewhat weak compared with that in the serum from antigen-elicited donors. In mice inoculated SCwith 1 X 10’ viable AH1 30 tumor cells, tumors grew for 7 days after the inoculation, thereafter rapidly regressed,and were completely rejected at 15 days after the inoculation. The augmentation activity was evident in the serum from donors inoculated 5 or 8 days previously; however, it was not detected
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TABLE 4 Augmentation Effect of Various Rinds of Immune Serum on Cytostatic Activity in PEC Treatment of serum donors Expt. I
Immunization
Elicitation’
Nontransferred Nontransferred MMC-AH 130 in CFAd
Nontransferred Nontransferred MMC-AH 130 MMC-AH 130 Nontransferred Nontransferred MMC-AH 130 Nontransferred Nontransferred -
-
II
III
MMC-AH 130 in CFA Nontransferred Nontransferred Viable AH 1308 Viable AH 130 Nontransferred Nontransferred Viable AH 130 (5 days) Viable AH 130 (8 days) Viable AH 130 ( 15 days)
-
Immunization of recipient@
[‘*‘I]UdR uptake (% CTS) ET, 5: I
+ + + + + + + + + + + +
106.52r 897” 7348 r 698 (30.2) 7432 r 525’ (30.2) 6982 + 595’ (34.5) 3453 f 623’(67.6) 10022 + 466 81402454 (18.8) 8334 + 442’ (16.8) 5846 f 254’(41.7) 4147 k 281/(58.6) 12045 f 735 9574 z!z675 (20.5) 4185 +- 379’(65.3) 4356 +- 562’(63.8) 9648 5 957’ (19.9)
’ In Experiments I and II, AH 130-immunized serum donors were injected with 1 X 10’ MMC-AH130 tumor cells into a hind footpad at 7 days after the immunization for elicitation of DIR. One day later, sera were obtained from these mice. Sera from nonelicited mice were obtained at 8 days after the immunization. ’ Seefootnote b of Table 2. ’ Each value represents the mean of six replicates +- SEM. d C57BL/6 mice were immunized sc with 1 X 10’ MMC-AH130 tumor cells emulsified with CFA as serum donors. Two days before the immunization, these mice were pretreated with CY. ’ Values that are not significant, compared with a nontransferred but immunized group. ‘Values that differ significantly from a nontransferred but immunized group, P c 0.005. g C57BL/6 mice were inoculated scwith 1 X 10’ viable AH 130 tumor cells. Two days before the inoculation, these mice were pretreated with CY. In Experiment III, sera from nonelicited mice were obtained at 5,8, and 15 days after the inoculation.
in the serum from donors inoculated 15 days previously without the elicitation with antigen.
Cell fraction analysis of augmentedcytostatic activity in PEC induced by transfer of immune serum. When whole PEC were separated into plastic dish-adherent and -nonadherent cells, cytostatic activity was not detected in each fraction of adherent and nonadherent cells (Table 5). However, when adherent cells of PEC from normal mice were added at the ratio of 1:1 to nonadherent cells from mice transferred with the immune serum and immunized with MMC-AH 130 tumor cells, cytostatic activity was restored and the augmenting effect by the transfer of immune serum also became detectable. When adherent cells of PEC from mice transferred with the immune serum and immunized or mice immunized without such a transfer were added to these nonadherent cells, almost the same results were obtained, compared to that in adherent cells of PEC from normal mice. Taken together, the expression of cytostatic activity in our system required the collaboration of adherent and nonadherent cells, and the adherent cells of PEC, even from normal mice, were available as the source of adherent cells for the restoration of cytostatic activity in the presence of immune nonadherent cells.
DELAYED-TYPE
HYPERSENSITIVITY
AUGMENTATION
FACTOR
319
TABLE 5 Cell Fraction Analysis of Augmented Cytostatic Activity in PEC Induced by Transfer of Immune Serum Transfer of immune serum’
Immunization of recipientsb
Cell fraction of PEC’
-
-
Whole Adherent Nonadherent Nonadherent + normal adherent Whole Adherent Nonadherent Nonadherent + normal adherent Whole Adherent Nonadherent Nonadherent + normal adherent
-
+
[ ‘ZSI]UdR uptake (96CTS)d E:T, 5: 1 12921 -t 456’ 12102 + 478 16952 + 548 13301+- 819 10897 + 465 (15.7) 11749 + 754/(2.9) 17405 + 715’(-2.7) 10335 + 230 (22.3) 8041 +318x(37.8) 11355 2 657’(6.2) 16868 2 985/(0.5) 6410+ 34jg(51.8)
DSee footnote a of Table 2. b Seefootnote b of Table 2. Each group in this experiment included 20 mice. c Whole PEC of each group were fractionated and nonadherent cells were reconstituted with adherent cells of starched-induced PEC from normal mice, as described under Materials and Methods. d Each % CTS was calculated, compared with the same fraction of a nonimmunized control group. ’ Each value represents the mean of six replicates -t SEM. ‘Values that are not significant, compared with the same fraction of a nonimmunized control group. gValues that differ significantly from the same fraction of a nontransferred but immunized group, P
Augmentation of cytostatic activity in spleen and LN cellsfrom mice transferred with immune serum. Cytostatic activity could not be detected in spleen and LN cells from mice transferred with the immune serum and immunized with MMC-AH 130 tumor cells or mice immunized without such a transfer (Table 6). However, when the adherent cells of PEC from normal mice were added to the spleen and LN cells at the ratio of 1:1, cytostatic activity could be detected, and the activity of such cells from mice transferred with the immune serum and immunized was higher than that of cells from mice immunized without such a transfer. Thus, the spleen and LN cells from immune mice included primary effector cells for cytostatic activity, but a sufficient number of adherent cells was always required for functional expression. Furthermore, these results suggestthat the appearance of primary effector cells for cytostatic activity is also accelerated in spleen and LN cells by the transfer of immune serum. When the immune spleen and LN cells were treated with monoclonal anti-Thy1.2 and complement, such cells could not render the cytostatic activity to the adherent cells of PEC from normal mice (Table 7), suggesting that primary effector cells for cytostatic activity are Thy- 1-positive T cells or cytostatic activity is T-cell dependent. For further confirmation, nylon wool nonadherent T-cell-enriched fractions of spleen and LN cells were used as effector cells. When the adherent cells of PEC from normal mice were added to such cells, cytostatic activity was detected. These results
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TABLE 6 Augmentation ofcytostatic Activiy in Spleen and LN Cells from Mice Transferred with Immune Serum Transfer of immune serum0
-
-
+
Immunization of recipientsb
Effector cells’
-
PEC Spleen + LN Spleen + LN + normal adherent PEC Spleen + LN Spleen + LN + normal adherent PEC Spleen + LN Spleen + LN + normal adherent
[rZ51]UdRuptake (% CTS)d ET, 5:l 10139 + 571e 151422401 10965 + 581 6895 rfr508 (32.0) 16215 + 323’(-7.1) 8538 + 453 (22.1) 2399 + 1428(76.3) 15669 + 403/(-3.5) 4210 + 304#(40.9)
a Seefootnote a of Table 2. b Seefootnote b of Table 2. ‘Whole PEC and spleen and LN cells were used as effector cells. Spleen and LN cells were added to adherent cells of starch-induced PEC from normal mice, at the ratio of 1:1. d Each % CTS was calculated, compared with the same kind of effector cells of a nonimmunized control group. eEach value represents the mean of six replicates + SEM. lValues that are not significant, compared with the same kind of effector cells of a nonimmunized control group. 8 Values that differ from the same kind of effector cells of a nontransferred but immunized group, P
indicate that lymphokines such as MAF released from T cells contribute to the expression of cytostatic activity by activating macrophages, and that the transfer of immune serum acceleratesthe generation of such T cells. Augmentation of MAF production by spleen and LN cells from mice transferred with immune serum. To determine that MAF production was also augmented by the transfer of immune serum, the following experiments were carried out. MAF activity could be assessedby its ability to induce nonspecific anti-tumor cytotoxic (cytostatic and/or cytolytic) potentials in macrophages. In this study, MAF activity was assessed by both cytostatic and cytolytic assay and similar results were obtained. Only data obtained with the cytolytic assay, a more popular assay system (24, 29, are shown. Immune spleen and LN cells were cultured with MMC-AH 130 tumor cells and MAF activity in these cultured-supematants was assessed(Fig. 1). Such an activity was detected in l/2 and l/4 dilutions of the supematant from the immune spleen and LN cells, when these cells were harvested from mice transferred with the immune serum and immunized with MMCAH130 tumor cells at 4 days after the antigenic challenge (Fig. 1A). On the other hand, when the immune cells were harvested from mice immunized without such a transfer, a low degree of MAF activity was detected only in a l/2 dilution of the supematant. When the immune cells were harvested from mice transferred with the immune serum and immunized with MMC-AH 130 tumor cells at 5 days after their antigenic
DELAYED-TYPE
HYPERSENSITIVITY
AUGMENTATION
FACTOR
321
TABLE 1 Expression of Augmented Cytostatic Activity Requiring Sensitized T Lymphocytes Transfer of immune serum’
Immunization of recipientsb
-
-
-
+
+
+
-
-
-
+
+
+
Treatment of spleen and LN cells’ Untreated Untreated C alone Anti-Thy- 1 + C Untreated C alone Anti-Thy- 1 + C Unfractionated Nylon-nonadherent Unfractionated Nylon-nonadherent Unfractionated Nylon-nonadherent
[“‘I]UdR
uptake (% CTS)d E:T, 5: 1
10034 f 457e 703 1 + 378 (29.9) 6742 f 375 (32.8) 10127 f 468/(-0.9) 2642 + 172g(73.7) 2930 f 1638(70.8) 9821 k 389’-(2.1) 10632 f 597 10323 IL 699 9418+842 (11.4) 8487 f 7 12 (20.2) 3514 f 124g(66.7) 2889 + 1048(72.8)
DSeefootnote a of Table 2. b See footnote b of Table 2. ’ Spleen and LN cells of each group were treated with anti-Thy- 1 and complement or complement alone (the first experiment), or with nylon wool column (the second experiment), then added to adherent cells of starch-induced PEC from normal mice, at the ratio of l:l, and used as effector cells. Untreated or unfractionated spleen and LN cells were used as positive control. dEach % CTS was calculated, compared with untreated or unfractionated spleen and LN cells of a nonimmunized control group. ’ Each value represents the mean of six replicates + SEM. /Values that are not significant, compared with untreated spleen and LN cells from a nonimmunized control group. 8 Values that differ significantly from spleen and LN cells received the same treatment from a nontransferred but immunized group, P < 0.00 1.
challenge (Fig. 1B), a high degreeof MAF activity could be detected in a 1/ 16 dilution of supematant from these immune cells. In contrast, when the immune cells were harvested from mice immunized without such a transfer, MAF activity was detected in l/2 and l/4 dilutions of the supematant, although not in a l/8 dilution of the supematant. Therefore, the former supematant may have approximately two- to fourfold MAF activity, compared with the latter. MAF production was also dependent on Thy- 1-positive T cells (Figs. 2A, B), aswell asprimary effector cells for cytostatic activity. These results suggestthat the cytostatic activity in this study is compatible with MAF activity which can be assessedby “Cr-release assay and that the transfer of immune serum accelerates the generation of lymphokine-producing T cells responsible for cytostatic activity and MAF production. DISCUSSION We detected a factor(s) in the serum of mice sensitized with xenogeneic tumor cells to induce DFR, which was capable of augmenting DFR antigen specificity in transferred recipients (Table l), as similarly shown in the experiments with heterolo-
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ET AL.
0 2
4
I
13
REClPROCll
32
2
4
OF SUPERNATANT
5
13
32
DllUTlON
FIG. 1. Augmentation of MAF production by spleen and LN cells from mice transferred with immune serum. Transfer system and transferred immune serum were the same as in Table 2. Spleen and LN cells were harvested at 4 days (A) or 5 days (B) after the immunization of recipients, cultured with MMC-AH 130 tumor cells, and MAF activity of these supematants was evaluated as described under Materials and Methods. Spleen and LN cells from mice transferred with the immune serum and immunized with MMCAH 130 tumor cells (Cl), mice immunized without such a transfer (A), and nonimmunized mice (0) were used. Closed symbols represent MAF activity of the supematant from each group of spleen and LN cells cultured without antigens. The data are expressed as the means of six replicates + SD.
B
2
4
8
RECIPIIOCAL
13
32 OF SUPERNATANT
OlLUTlON
FIG. 2. Effect of anti-Thy- 1.2 plus complement on MAF production by spleen and LN cells. Transfer system and transferred immune serum were the same as in Table 3. Spleen and LN cells were harvested at 5 days after the immunization from the recipients transferred with the immune serum and immunized with MMC-AH130 tumor cells (A) or mice immunized without such a transfer (B). These cells treated or untreated (-) were cultured with anti-Thy- 1.2 and complement (. . . ), complement alone (-.-), with MMC-AH 130 tumor cells, and MAF activity of these supematants was evaluated as described under Materials and Methods. The data are expressed as the means of six replicates + SD.
DELAYED-TYPE
HYPERSENSITIVITY
AUGMENTATION
FACTOR
323
gous erythrocytes (19,20). This factor could also augment in vitro anti-tumor cytostatic activity. This augmented cytostatic activity appeared at 4 days after the antigenie challenge of serum recipients, in the whole PEC (Table 2, Fig. 1). The activity was attenuated or abolished when plastic dish-adherent or nonadherent PEC alone were used as effector cells, and was restored when adherent PEC from normal mice were added to the immune nonadherent PEC (Table 5). The appearance of primary effector cells responsible for cytostatic activity was also accelerated in spleen and LN cells from mice transferred with the immune serum before the antigenic challenge (Table 6). However, sufficient numbers of adherent PEC, in either caseof nonadherent PEC and spleen and LN cells, were always necessary as the direct effecters for functional expression and adherent PEC even from normal mice were available as the source of adherent cells. These primary effecters were T lymphocytes (Table 7). Lymphokine(s) such as MAF releasedfrom such sensitized T lymphocytes contribute to the expression of cytostatic activity by activating adherent cells (macrophages) (Figs. 2,3). Thus, the augmentation factor may exert its effect in the induction phase to raise sensitized T lymphocytes, and may accelerate the generation of lymphokineproducing T lymphocytes responsible for cytostatic activity. The contribution of lymphotoxin, cytotoxic T cells, natural killer (NK) cells, arming macrophages, or antibody-dependent cell-mediated cytotoxicity (ADCC) may be negligible, since this augmented cytostatic activity requires the collaboration of both antigen-specific sensitized T lymphocytes and macrophages. Furthermore, cytotoxic activity assessedby a 4-hr “Cr release from labeled AH 130 target cells was hardly detectable in PEC and spleen and LN cells from these immunized mice (data not shown), as reported earlier ( 11). NK activity in PEC and spleen cells assessedby a 4-hr 5‘Cr releasefrom labeled YAC-1 target cells was not augmented by the transfer of immune serum (data not shown). Cytotoxic antibodies could not be detected in assay systems for ADCC and for complement-dependent cytotoxic antibodies, in the immune serum we used in the present study (data not shown). The proposed participation of cytostatic effector cells in the functional expression of DTH may be supported by our previous studies (3,4, 11, 12), and in vitro cytostatic activity may be an useful in vitro assay system for in vivo DTH reaction. In that system which involves use of a murine allotransplantable tumor, sarcoma 180, the inhibition pattern of in vivo tumor growth correlated closely with the in vitro cytostatic activity and in vivo DFR in mice with an acquired anti-tumor resistance against such a tumor. The cytostatic activity was also seenin macrophages activated by sensitized lymphocytes, and cytotoxic lymphocytes were not detected (3). The same result was obtained even in syngeneic tumor systemswith Meth A tumor of BALB/c origin and others (4, 12). We observed the augmentation activity of our factor in a transfer system, as described above, but have not yet determined its role in the immunized hosts (donors of the augmentation factor). When serum donors were immunized with MMC-AH 130 tumor cells in CFA, the elicitation with antigen was essentially required for production of the augmentation factor in the serum donors (Table 4), as required in systems with heterologous erythrocytes (19,20). In contrast, when serum donors were inoculated with viable AH 130 tumor cells 5 or 8 days previously, a sufficient degree of augmentation activity was detected, without elicitation of serum donors. However, when serum donors were inoculated 15 days previously, such an activity was not detected without elicitation of serum donors. In serum donors inoculated with viable
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AH130 tumor cells 5 or 8 days previously, tumor masseswere palpable, but not in mice inoculated 15 days previously. At 5 or 8 days after the inoculation, sensitized T lymphocytes may be restimulated at the site of direct elimination of tumor cells and produce the augmentation factor. These findings suggestthat the augmentation factor may exert its effect on the induction phase, in the immunized hosts. Lawrence reported that a low-molecular-weight substance from leucocyte lysate was able to transfer DTH (termed transfer factor) (27). Such a transfer factor can introduce DTH antigen specificity in recipients without additional antigenic challenge. On the other hand, our augmentation factor required the antigenic challenge of recipients for the expression of activity. Our factor exerts its effect on the induction phase of DTH, while “transfer factor” is effective on the expression phase. Ptak and co-workers reported that an antigen-binding factor produced by T cells was able to transfer an antigen-specific immediate hypersensitivity-like skin reaction in mice (16). Their factor leads to mast cell activation and the release of vasoactive amines, however, has no effect on the induction phase and does not accelerate the induction of sensitized lymphocytes after immunization. Antigen-specific factors released in the serum of donors immunized and elicited in the same manner as described in the present paper may participate in the acceleration and augmentation of DTH and acquired resistance against various kinds of particulate antigens. In our studies, the presence of such a factor was suggestedin experimental systems with Listeriu monocytogenes to which acquired resistance can be attributed to the cooperation of lymphokine-producing sensitized T lymphocytes and macrophages (Yamada et al., manuscript in preparation). Furthermore, there is the possibility that the same type of antigen-specific factor contributes to the resistance to some kinds of syngeneic tumors, since lymphokine-induced cytostatic macrophageswere confirmed to be responsible for the elimination of syngeneic tumor cells such as Meth A (4, 12,28). The biological meaning of this factor in the host resistance will be defined by further studies. ACKNOWLEDGMENTS We thank Shigeko Ueda, Chikako Ishikawa, Izumi Yoshimatsu, and Atsuko Yamada for technical assistance and secretarial services and Mariko Ohara for comments on the manuscript.
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