394
Eur. J. Immunol. 1977. 7: 394-400
H.S. Koren and RJ. Hodes
H.S. Koren’ and R.J. Hodes’ Division of Immunology, Duke University Medi-lCenter. Dumam’and lmmunolow Branch, National Cancer Institute, National Institutes of Health, Bethesda’
Effect of tumor cells on the generation of cytotoxic T lymphocytes in vitro
1. Accessorv cell functions of mouse tumor cells in the generation of cytotoxic T lymphocytes in vitro: replacement of adherent phagocytic cells by tumor cells or 2-mercaptoethanol* In agreement with previous reports, the primary in vitro response t o alloantigens has been shown t o be dependent o n the presence of m a c r e phages (Mphs). Splenocytes extensively depleted of adherent phagocytic cells did n o t generate cytotoxic T lymphocytes, and this activity could be completely restored by small numbers of adherent peritoneal cells (accessory cells). Either P388D1 (Mph-like tumor), P388 (“null” tumor) or P8 15 (mastocytoma) tumor cells, or 2-mercaptoethanol, could completely replace the accessory function normally mediated by accessory cells. These tumor cells did not nonspecifically “enhance” the cytotoxic activity generated with normal nondepleted spleen cells. The restored cultures maintained killing specificity t o H-2 targets which was mediated by effector T cells as shown by sensitivity t o anti-@ and complement. Therefore, Mphs seem n o t t o be the sole cells capable of mediating an accessory function in a primary response t o alloantigens in vitro.
1. Introduction
The role of the accessory cells in cellular immunity has drawn the attention of many investigators. Mosier first described the requirement of an adherent cell t o collaborate with a nonadherent population in the initiation of an antibody response t o sheep red cells (SRC) in vitro [I]. It was later established that other in vitro responses such as the mixed lymphocyte reaction (MLR) [2-41, mitogen responses [5,6] and generation of cytotoxic T lymphocytes (CTL) [7- l l ] required the presence of adherent macrophage (Mphhlike cells or their culture supernates [ 12, 131. In addition, there is evidence that cells of the reticuloendothelial system are needed f o r a succes% ful immune response in vivo [ 141. Recently, Heber-Katz and Click have shown that reducing agents such as Zmercaptoetha no1 (2-ME) could substitute for serum in t h e medium for MLR o r enhance an immune response (IR) by a synergistic interaction with fetal calf serum (FCS) [ 151. Chen and Hirsch [ 161 and more recently Lemke and Opitz [ 171 were able t o show that 2-ME can substitute for Mph in a primary response to SRC in vitro. Bevan et al. have shown that 2-ME could restore the proliferative MLR as well as the generation of CTL in a [I 16611
* This work was supported by the grant USPHSCA-14049. Correspondence: Richard J. Hodes, Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014, USA Abbreviations: SRC: Sheep red cells MLR: Mixed lymphocyte reac tion CTL: Cytotoxic T lymphocytes Mph(s): Macrophage(s) I R Immune response FCS: Fetal calf serum BA8: Braittassociated theta antigen MEM: Eagle’s minimum essential medium PC: Perk toneal cells BSS Balanced salt solution RAMB: Rabbit anti-mouse brain GpC: Guinea pig complement CMC: Cebmediated cytotoxicity POL: Polymerized flagellin SIg: Surface lgbearing 2-ME:2-Mercapte ethanol C: Complement
murine spleen cell population depleted of adherent cells [ 181. Moller et al., also using the primary response t o SRC system, have shown that Mphs can be substituted for not only by 2-ME but also by fibroblasts and supernates of these cells [ 191. The latter results suggest that the accessory function in a primary in vifro response t o SRC is n o t an exclusive property o f Mphs, as previously thought. In this paper we describe the restora tion of cytotoxit activity to Mphs-depleted DBA/2 spleens by 2-ME and by three DBA/2 tumor cells (P388D1, P388 and P8 15). Suppressive effect mediated by P815 tumor cells resulting from mycoplasma infection will be described in a SB parate paper (manuscript in preparation).
2 Materials and methods 2.1. Previously described materials and methods
Reagents and methods used t o characterize cell populations with respect t o their phagocytic activity, surface Ig, brainassociated theta (BAO) and Fc-receptor have already been described [20]. 2.2. Animals
Mice of inbred strain DBA/2J (H-2d) were obtained from Jackson Laboratories, Bar Harbor, Maine and NIH Animal Production, Bethesda, Md. Males aged 8- 12 weeks were used throughout this study.
2.3. Tumor cells All the tumor cells used in this study (Table 1 ) were grown in v i a 0 rather than using t u m o r cells from ascites fluid. This was done in order t o avoid contamination of the tumor cells
Replacement of accessory function by 2-mercaptoethanol and tumor cells
Eur. J. Immunol. 1977. 7: 394-400
under study with accessory cells of host origin. They were passaged bi-weekly in tissue culture using Eagle's minimum essential medium (MEM, Flow Laboratories, Rockville, Md.) supplemented with 10 % fetal calf serum (MEM-10 % FCS), 2 mM L-glutamine, 100 unitslml penicillin, 100 pg/ml streptomycin (Microbiological Associates, Bethesda, Md.). P388D1, a Mph-like tumor cell line, was grown in spinner cultures as previously described (21 1. All other tumor lines were grown in 75 ml plastic flasks (Falcon Plastics, Oxnard, Cal., no. 3024) in suspension cult ire. All the tumor cell cultures were maintained at 37 " C . Table 1. Characteristics of cultured tumor cell line used Tumor
Strain Latex of origin phage cytosisa)
P388D1 P388
DBA/2 DBA/2
-ke) -
-
DBA/2
-
(parent)
P815 L1210 LSTRA EL 4 a)
b)
c) d)
e)
DBA/2 BALB/c C51BL/6
-
Slgb) BA@)
Fcd)
Cell classifit cation
-
-
Mph "nulr'
-
-
+ + +
+
-
-+
+
+
-
mste
cytoma B "null" T
Latex phagocytosis, ability to phagocytize latex beads during 4 5 min incubation at 37 "C. Cells ingesting more than three beads were considered positive. SIg, surface lg-bearing; stains with fluorescein-conjugatedpolyvalent rabbit antcmouse lg. BAQ, brain-associated @bearing; reacts with RAMB serum. Fc, bears a receptor for heat-aggregated human Ig and/or antibody-antigen complexes (7 SEA). (+), possess the cell marker characteristic under study; (-), does not possess the cell marker under study. For each cell marker a minimum of 200 cells was scored
395
which efficiently avoided leakage of Sephadex o u t of the syringe. The depletion procedure was carried out at room temperature. The column, consisting of 35 ml of Sephadex G 1 0 , was equilibrated with 100 ml of prewarmed BSS-5 % FCS. Up t o 600 x lo6 spleen cells were loaded on 1 column and the nonadherent cells were eluted with approximately 25 ml of BSS-5 % FCS. The column-passed cells were spun once, and u p to 400 x l o 6 cells were resuspended in 8 ml of BSS2O % FCS. Thirty-two ml of lymphocyte separator reagent (carbonyl iron, Technicon Instruments Corp., Tarrytown, NY) was added t o the 8 ml of cells in a 50 ml conical tube (Falcon, no. 2074) a n d rotated at 3 7 "C for 1 h. A strong magnet was then used to pellet excess Fe and Fe-ingested cells. Cells in the supernatant were carefully removed, spun once and adjusted to 20 x 1O6 cells/ml in BSS- 1 0 % FCS. Two ml of this suspension were layered on t o p of 3 ml of FicollHypaque (Bionetics Kensington, Md.) in 1 5 ml conical tubes (Falcon, no. 2095). Cells were then spun a t 2200 x g for 3 min. Bands at the interface were collected, washed twice with excess of BSS-10 % FCS and finally resuspended in the complete medium (described below) used for the in virro sensitization. The approximate cell recoveries following Sephadex G I O , carbonyl iron and FicolkHypaque were usually 50 %, 7 0 % and 80 %, respectively. Therefore, the final yield was about 20 % of the original number of cells. The cells obtained through the above procedures will be termed depleted cells.
NON-DEPLETED
G-10 S f P n l t y X 6 - 0 X IO'CELLS
mcu
1EYP
WBONYL IRON
miAn AT
+ CELLS x 0 c Fm i nn
2.4. Spleen a n d peritoneal cell preparations
Sterile single cell suspensions of DBA/2 mouse spleen cells were prepared, and red cells osmotically lysed, as previously described [22]. Peritoneal cells (PC) were obtained by i p jecting 5 ml of balanced salt solution (BSS) (NIH Media Unit) and 5 % FCS i.p. and then aspirating the cell-containing fluid. This procedure was repeated 3 times, usually resulting in a total yield of 2 x l o 6 cells per mouse. These cells were spun at 1000 x g for 7 min, then irradiated with 1000 rads followed by treatment with rabbit anti-mouse brain serum (RAMB) and complement (C) (see below). The resultant population of PC was added in various concentrations t o Linbro plates used for sensitization and incubated in a humidified COz incubator for 2 h. Nonadherent cells were then removed by washing with MEM-10 % FCS; those remaining adherent cells will be termed purified PC. At this stage responding and stimulator cells were added to the wells.
Scheme 1. Schematic representation of the procedure used to deplete
DBA/2 spleen cells of the accessory function
2.6. Rabbit anthmouse brain serum (RAMB) The production and T cell specificity of RAMB as well as its use with guinea pig C (GpC) to obliterate T cell responses have been presented in a n earlier publication [24]. We used our RAMB f o r treatment of spleen cells and PC at a final dilution of 1 :60 a n d reconstituted lyophilized GpC (Gibco, Grand Island, NY) at a final dilution of 1 :8.
2.5. Removal of adherent phagocytic cells from DBA/2 splenoc ytes
2.7. In vitro sensitization
A general scheme for the depletion procedures used in this study is shown in Scheme 1. Modified Sephadex G 1 0 columns as described by Mishell and Ly were used [23]. The modification involved replacing the glass beads with a nylon wool plug
Sensitization was carried o u t by c e c u l t u r e of responding DBA/2 (H-2d) spleen cells with 10 000 rad-irradiated ( 13'C0 source, 1500 R / m h exposure rate) sensitizing EL4 (H-2b) tumor cells, in a total volume of 2 ml in each well of multiwell dishes (FB16-24-TC, Linbro Chemicals, New Haven,
396
Eur. J. Immunol. 1977. 7: 394-400
H.S. Koren and RJ. Hodes
Conn.). The medium used for the in vitro sensitization was RPMI- 1640 o r MEM supplemented with 2 mM L-glu tamine, 100 units/ml penicillin, 100 pg/ml streptomycin, 1 mM sodium pyruvate, 1 x nonessential amino acids (Microbiological Associates) and 10 % FCS (Reheis Chemical Co., Phoenix, Arizona, lot no. 505). This lot was shown t o support the generation of cytotoxic cells in the absence of 2-ME. Responding cells were either normal or depleted spleen cells. 2-ME at a final concentration of 5 x M (Eastman Kodak Co., Rochester, NY), o r tumor cells or PC were added t o the sensitizing cultures in specific experiments as indicated in Sect. 3. Sensitization cultures were incubated at 37 "C in humidified air - 5 % COz for 5 days. At the end of the sensitization period, cells were harvested from culture by repeated pipetting, then centrifuged and resuspended in MEM-I 0 % FCS. Replicate wells were pooled at this stage. Viable cells were counted by trypan blue exclusion and used as effector cells in a cytotoxicity assay.
2.8. Cytotoxicity assay Cell-mediated cytotoxicity (CMC) was assayed by a procedure previously reported [24]. Target cells were labeled by incubation in MEM-10 % FCS a t a concentration of 30 x 106 to 7 0 x 106/ml with 0.1 mCi/ml 51Cr (as sodium dichromate, AmershamSearle, Arlington Heights, Ill., approximately 125 pCi/pg Cr) for 3 0 min at 3 7 "C. Labeled cells were then centrifuged and washed 4 times in MEM-10 % FCS, and viable cells were counted. The same procedure was used when EL4, P815 or LSTRA tumor cells were used as targets. Five x l o 4 SICr-labeled target cells were mixed with effector cells at various effector to target cell ratios in a total volume of 200 pl of MEM- 10 % FCS in each well of round-bottomed microtiter plates (IS-MRC-96; Linbro Chemicals). Target cells alone (without effector cells) were incubated t o determine "spontaneous release" of "Cr label. All groups were set up in triplicate. Plates were centrifuged at 25 x g f o r 2 min a t 15 "C and then incubated for 4 h at 3 7 "C in humidified air - 5 % Table .2 Attempts to abrogate the ability of mouse spleen cellsa) to generate cytotoxic activity by various separation techniques
Exp. Separation no. method 1
2
3
4:l
EL 4 2:l
%Specific Iysisb) P815 or 1:l LSTRA 4: I
Siwivalc) (%)
324 G10 2 ~ d ) 41.3
22.0 9.6 37.4 16.0
- 0.3 - 1.5
40 12
Fee) (;lo+ Fe
46.1 54.2 -0.1
29.6 13.2 38.1 19.8 0.5 -0.3
- 0.8 - 0.3 -0.1
20 31 23
70.6 1.2
59.4 33.2 1.2 0.7
- 0.6 - 0.2
71 32
G 1 0 + FC
COz. At the end of this incubation period plates were centrifuged at 7 0 0 x g for 10 min at 4 "C. One hundred microliters of supernatant were removed from each well and transferred t o a 1 6 x 125 m m plastic tube for counting in a well-type gamma counter (Amersham Searle). "Total label" was determined by direct counting of 5 x 1 O4 51Cr-labeled target cells. Cytotoxic activity was calculated as: (Counts released in experimental group) - (Counts in spontaneous release) % specific lysis =
x 100.
(Counts in total label)
*
The standard deviation of triplicate cultures was 2 % of the mean. In each experiment, the specificity of cytotoxicity induced was verified by testing each effector population against LSTRA or P815 target cells which are syngeneic to the effectors.
3. Results 3.1. Attempts t o deplete spleen cells of the ability t o generate cytotoxic activity
Preliminary experiments have shown that passing DBA/2 spleen cells once through a Sephadex G-10 column does not result in lack of activity upon subsequent sensitization. Interestingly, spleen cells from BALB/c mice were easily depleted of accessory cells by one passage through a Sephadex G 1 0 column (our unpublished observation). As can be seen in the first experiment in Table 2, cells passed twice through Sephadex G 1 0 columns and used as responder cells had still maintained their ability t o generate cytotoxic activity. Exp. 2 describes an attempt t o deplete accessory cells for CMC based on selective removal of phagocytic cells by carbonyl iron particles. This technique was followed by Ficoll-Hypaque gradient t o ensure total removal o f excess iron particles and cells containing iron particles after the magnet step. This procedure also failed t o eliminate generation of cytotoxic activity; however, a combined depletion technique whereby cells were first passed through a Sephadex G 1 0 column and then treated with carbonyl iron totally obliterated the generation of cytotoxic activity, as can be seen in the last line of Expts. 2 and 3. The number of viable cells recovered from depleted cultures was usually depressed too. Table 3 shows the results of a marker study performed with nondepleted and depleted cells The number of latex-positive cells dropped below levels of detection. The SIg' cells were unchanged and the brain-associated ' 0 (BAO') cells were slightly enriched in the depleted population. Table 3. Marker study of mouse spleen cells before and after fractionation by Sephadex G10 column and carbonyl iron
Celisa)
latex phagocytosis
Sk (5%)
BAO
65 65
23 36
(W
(%)
a) 4 x 106 DBA/2 spleen cells were sensitized against 0.5 x 106 irradiated EL4 tumor cells. b) Spontaneous release of EL4 in Expts. 1, 2 and 3 was: 7.0, 6.3 and 8.3, respectively. LSTRA target cell was used in Expts. 1 and 2; P815 was used in Exp. 3. The spontaneous release was 5.6, 7.8, and 17.4, respectively. c) Percent viable cell recovery after 5-day sensitization culture. d) Cells were passed twice through Sephadex G10 columns e) Cells separated by carbonyl iron.
DBA/2 nondepleted DBA/2 depleted
5.5
< 0.5
a) DBA/2 spleen cells were first filtered through a Sephadex G 1 0 column, then incubated with carbonyl iron followed by separation on a Ficoll-Hypaque gradient. For details see Sect. 2.5. and footnote of Table 1.
Replacement of accessory function by 2-mercaptoethanol and tumor cells
Eur. J. Immunol. 1977. 7: 394-400
Since we were interested in total depletion of accessory cell function, this combined technique was used throughout this study. Previous studies attempting to deplete accessory cells for CMC in vitro using adherence depletion alone, achieved partial depletion only (9, 251. Wagner e t al. have also shown that they achieved efficient depletion only when they combined glass bead column fractionation with anti-Mph serum; neither of these techniques was as efficient as the combination of the two [8]. Rosenstreich et al. have noticed that they had t o employ two adherence columns to achieve complete depletion of Mphs from guinea pig lymph node cells [ 6). 3.2. Restoration of cytolytic capacity of depleted spleen cells by purified PC and >ME
To demonstrate that the inability of depleted populations to generate cytolytic cells actually reflects a specific deficit in adherent accessory cells, experiments were performed attempting t o reconstitute activity with PC. Since PC are a heterogeneous population of cells [ 161 they were treated in various ways ensuring a highly enriched population of adherent Mph-like cells. They were first treated with RAMB and C to eliminate T cells, irradiated with 10 000 rads and left o n plastic for 3 h at 37 OC, after which nonadherent cells were removed. Therefore, the number of cells as shown in Fig. 1 reflects a relatively high estimate since PC were counted prior t o adherence in the wells. Results of one such representative experiment are presented in Table 4 and a diagrammatic representation in Fig. 1. The data demonstrate that various degrees of restoration were achieved by using different amounts of purified PC. The doseresponse curve shows that low and high doses of PC achieved levels of cytotoxicity up t o 29.6 % specific lysis at 4: 1 compared to 49.1 %specific lysis of the nondepleted cells at the same ratio. Almost complete restoration (45.4 %)was achieved at the optimal dose of 0.8 x l o 5 PC. The survival of cultured sensitized cells appears t o correlate with the levels of cytotoxic activity. The fact that high doses of PC were suboptimal suggests that crowding conditions may have suppressed the development of cytotoxicity. In addition, 2-ME at a final concentration of 5 x 1 0-5 M substituted for accessory cells too, and resulted in a level of cytotoxic activity similar to the one seen by using optimal doses of PC. 2-ME has previously been shown
DEILLTCD
0s
OQZ K).
2
4
OF PERITONEAL CELLS A D E D PER CULrUr(XIOs)
Figure 1. DBA/2 spleen cells were depleted according to the procedure described in Scheme 1, then stimulated with irradiated EL4 cells for 5 days in vifro. To some of the cultures graded numbers of adherent, irradiated and RAMB + C-treated F T were added Other cultures received 2-ME (5 x 10- 5 M), the remaining depleted cells were left with out reconstitution Cytotoxic activity was tested on day 5 of sensitiza tion against 51Crlabeled EL4 targets
397
Table 4. Restoration of the ability to generate cytotoxic activity of 4 x 106 DBN2 responder cells by PC and 2-ME
Depletion
Additives 4:l
-
+ + + + + +
-
~ c a ) 4~ 105 pc 2~ 105 ~c o . 8 ~ 105 Pc 0.2x 105
2-ME S X 10-5
Spontaneous r elea se
49.2 0.2 20.5 29.6 45.4 20.8 45.1
Specific lysis (7%) E L4 P815 Survival 2:l 1:l 4:l (7c) 25.6
12.6
-0.1 -0.6
-
1.5
-1.1
10.5 4.3 - 1.3 15.5 7.5 - 1.8 25.8 1 2 4 -2.2 11.2 5.7 -1.0 1.4 20.6 1 2 5 15.6
58 15 48 46 51 51 65
13.0
a) Purified PC were plated for 2 h, then nonadherent cells were removed. t o have a Mph-independent accessory function in restoring a primary antibody response t o SRC [ 171 as well as MLR [ 181 in vitro.
3.3. Effect of adding various murine tumor lines on depleted spleen cells Since we were able t o show that the combined depletion techniques resulted in a population of cells incapable of mounting a primary in v i a o response t o alloantigens, but was restorable t o normal values with purified PC as well as 2-ME, we tested the effect of 5 tumor lines with different functional and surface characteristics o n the accessory cell-depleted cultures. All the tumor cells were of responder haplotype (H-2d) and, therefore, should n o t be able t o sensitize the responding cells t o foreign alloantigens. The tumors had been propagated in culture for a sufficient period of time to eliminate the possibility of introducing host cells into this assay. The tumor cells were irradiated and added in graded numbers t o the cultures o n day 0 of sensitization. Table 5 shows a representative experiment performed i n this study. P388D1, a Mph tumor line previously described by us [21], its parent line, P388, and a mastocytoma, PSI 5, demonstrated restorative activities resulting in cytotoxic activities comparable t o the nondepleted cultures. L1210, SIg’ tumor, and LSTRA which is a “null” tumor demonstrated only very low levels of restorative activity in the range of concentrations used in these experiments compared t o the other tumor cells. Similar t o the data presented in Tables 2 and 4, survival correlated closely t o the cytotoxic activity. For example, P388D1 cells restored optimally at a concentration of 2 x lo5 with a survival of 68 % which even exceeded the survival of the nondepleted group, 57 %. Relatively low levels of survival were obtained in cultures where L1210 and LSTRA were added, 33 % a n d 29 %, respectively. Specificity of the cytotoxicity generated in the restored cultures was demonstrated by lack of activity against the syngeneic P815 cells. This pattern of activity was reproducible in several experiments although the number of added cells needed for optimal restoration of sensitization varied. Table 6 lists experiments demonstrating successful restorations of accessory function mediated by the different tumor cell lines and the purified PC. As can be seen, purified PC restored each time tested, L1210 and LSTRA never restored, and the lines P388D1, P388 and P815 restored in most of the experi-
398
Eur. J. Immunol. 1977. 7: 394-400
H.S Koren and RJ. Hodes
ments. It is of interest that the variability of restoring ability seen with P8 15 was later shown t o correlate with the presence o r absence o f mycoplasma. The effect of mycoplasma o n the generation of allogeneic cytotoxic cells will be the subject of a separate paper.
Table 5. Restoration of the ability to generate cytotoxic activitya) by various murine tumor cells
(46)
-
-
-
0.2 4.0 20
30.6 19.6 -0.7 0.0 6.0 2 4 31.0 l a 4 17.4 9.2 -0.1 1.0 327 2 8 7 10.5 6 5 - 1.6 -0.2 - 1.4 -0.1 3 8 5 21.9 44.5 25.2 121 5.8 16.8 9.9 4.2 2 6 0.7 0.1
0.8
-0.8
0.2 LSTRA 4.0 LSTRA 20 LSTRA 0.8 LSTRA 0.2 Spontaneous release
-0.5
- 1.0
6.1
3.1 4.1
+
P388D1 P388D1 P388Dl P388D1 P388 P388 P388 P388 P815 P815 P815 P815 L1210 L1210 L1210 L1210
4.0 20 0.8
0.2 4.0 20 0.8
0.2 4.0 20 0.8
7.9 3.3 -0.2
-0.6
0.2
0.2 63
10.0 -4.0 0.7 - 3.4 2 5 - 1.7 7.3 - 2 8 5.3 -21 0.5 -0.6 1 4 7 -4.5 2 4 -3.2 0.3 - 1.8 0.6 -1.5 10.6 -2.5 14.1 -1.9 3.0 -6.0 4.9 0.4 1.7 -1.6 -0.4 5.5 -0.1 -1.1 0.3 1.0 1.8 -1.1 2 2 -1.8 1.5 - 1.2 -0.1 - 1.1 31.3
57 9 33 68 38 19 44 31 15 12 52 44 36 24 33 27 14 16 29 27 19 15
a) 4 x 106 DBA/2 spleen cells were sensitized against 0.5 x 106 irradiated EL4 tumor cells b) + Denotes depletion by Sephadex G 1 0 columns and carbonyl iron. c) Irradiated tumor cells added to depleted spleen cells on day of sensitization d) Percent viable cell recovery after 5-day sensitization culture.
Table 6. Reproducibility of experiments restoring the ability to generate cytotoxic activity with tumor and FC
successul restoration Number % !
P388D1
P388
Restorilrg cdls P815 Ll210
lO(12)a) lO(12) 6(8) 83.3 83.3 75
O(4) 0
LSTRA
PC
O(4) 0
5 (5) 100
a) Numbers in brackets denote the total number of experiments performed
3.4. Effect of RAMB and C on the cytotoxic cells from cultures restored with P388D1 Allogeneic cytotoxic cells raised in vivo as well as in vitro have repeatedly been shown t o be T cells [26]. Since depleted spleen cells were able t o respond t o alloantigens upon reconstitution with tumor cells, in addition t o conventional restora-
tion methods with purified PC, it was important t o establish the nature of the effector cell in this system. Effector cells from depleted cultures sensitized for 5 days in the presence of P388D1 were treated with RAMB and C to see whether the effector cells were indeed T cells. Table 7 shows the results of such an experiment. P388D1 cells restored the depleted cultures optimally at 2 x lo5. Treatment of the effector cells by RAMB and C obliterated their activity against EL4 target cells. Controls with RAMB or C alone did not significantly alter the cytolytic activity of these sensitized cells. This experiment does not prove that cultures restored with other tumor cells such as P388 or P8 15 would lead t o the generation of effector T cells. However, it is unlikely that the mechanism of restoration by these cells would differ from the one with P388D1.
Table 7. Effect of RAMB and C treatment on the cytotoxic cellsa) generated with P388D1 tumor cells restored cultured P388DI Effector DepIetionb) cells cds added treatmentd) 4:l x 10-5c)
-
+
2.0
-
325 -0.5 30.1 RAMB+C 1.5 RAMB 2a1 C 29.5 Spontaneous releave
specifii lyds (%) EL4 P815 Sur2:l 1:l 4:l viVd (%)
15.7 a2 -0.1 -0.2 7.5 14.8 0.2 -0.9 14.0 6.5 14.6 7.0 65
-1.5 56 -3.4 15 - 2 5 49 -3.1 -20 - 1.5 82
a) 4 x 106 DBA/2 spleen cells were sensitized against 0.5 x 106 irradiated EL4 tumor cells b) + Denote spleen cells depleted by Sephadex G 1 0 column and carbonyl iron c) Irradiated P388D1 cells were added to depleted cultures on day 0. d) Effector cellswere treated on day 5 with RAMB at a final dilution of 1:60 and GpC at a final concentration of 1:8.
3.5. Do tumor cells that restore accessory function “enhance” nondepleted spleen cells? The term “restoration”, as used in this study, refers t o the ability of certain cells or reagents t o substitute for adherent phagocytic cells in the generation of CTL in vifro. The question raised is whether this activity is restorative o r a more generalized “enhancing” activity which would be expressed with nondepleted responder cells as well. To test this possibility, tumor cells were added t o nondepleted DBA/2 r e sponder cells o n day 0 of sensitization t o EL4 tumor. Table 8 shows the results of 3 such experiments. As seen, none of the cell lines had a significant “enhancing” o r an immunopotentiating effect o n cytotoxic activity as measured on day 5. In certain cases such as with P388D1, at 0 . 8 x l o 5 cells in Exp. 3, survival was higher compared t o control without causing enhanced cytotoxic activity. The two cell lines which were shown t o lack restorative activity - L1210 and LSTRA (see Table 5) did n o t express a markedly suppressive effect in the range of concentrations used in E x p t s 2 and 3. This suggests a lack of restorative ability rather than active suppression on the part of these two cell lines.
Replacement of accessory function by 2-mercaptoethanol and tumor cells
Eur. J. Immunol. 1977. 7: 394-400
Table 8. Effect of adding tumor cells to nondepleted culturesa) Specific lysis (%) I-xp. no. 1
2
3
Tumor cells addedx IO-sb) 4:l
EL4 2:1
1:l
4.0 2.0 0.8 0.2 4.0 2.0 0.8 0.2 4.0 0.8 0.2 4.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2
28.7 2.1 7.8 16.9 21.2 10.4 13.6 21.7 27.4 14.1 5.0 8.0 10.3 12.2 11.9 13.9 12.3 7.5 3.0 8.9 14.0 13.2 29.5 24.1 16.1 15.1 31.6 18.9 4.9 16.9 10.3 2.5 8.7 23.4 32.5 11.6 14.1 23.2
16.9 1.3 4.2 8.8 16.0 6.5 8.1 13.8 14.0 6.2 2.0 4.0 5.3 4.7 6.6 6.8 6.0 3.8 1.8 4.5 7.3 6.5 14.4 12.4 7.2 7.4 14.3 8.0 2.9 8.4 4.3 2.0 3.9 10.5 16.8 5.7 7.3 12.4
-
P388D1 P388D1 P388D1 P388D1 P388 P388 P388 P388 P388D1 P388D1 P388Dl P388 P388 P388 P8l5 P815 P815 LSTRA LSTRA LSTRA P388D1 P388Dl P388D1 P388 P388 P388 pni 5 P815 P815 LSIRA LSIRA LSTRA LIZ10 Ll2lO L1210
40.1 3.4 13.8 27.1 41.7 19.2 24.1 35.6 38.3 28.8 4.9 15.0 19.8 19.6 24.7 26.1 25.6 15.7 6.5 17.6 27.9 25.6 48.1 41.6 33.5 32.0 48.3 34.1 10.7 37.6 19.3 6.4 18.2 44.7 53.5 24.1 30.3 41.2
P815 Survival 4:l (%) - 3.7
- 2.9 - 1.7 - 3.3 - 2.6 - 3.4 - 2.6 -2.3 - 2.8 -1.7 - 1.7 - 3.3 - 1.4 - 0.7 - 0.9 0.4 - 1.2 - 2.0 - 2.1 - 3.2 - 1.7 - 1.7 0.2 - 1.0 0.9 0.3 0.8 0.0 0.6 0.7 0.0 0.2 2.1 4.9 9.5 -0.7 -1.2 - 1.2
41.2 28 34 37 36 33 40 36 36 57 39 49 48 37 42 61 59 55 40 35 56 58
70 68 101 49 58 42 51 40 36 30 56 71 69 40 48 61
4 x 106 DBA/2 spleen cells were sensitized against 0.5 x 106 irradiated EL4 tumor cells. b) Irradiated tumor cells were added to cultures on day 0.
a)
4. Discussion
The role of adherent phagocytic Mph-like cells has often been shown to be obligatory in various cellular events (for review see [ 271). These cells have also been termed accessory o r auxiliary cells. Their role in the IR in vitro was first demonstrated by Mosier working with an anti-SRC response in vitro [ 1). Feldmann et al. have shown that secondary responses t o intact SRBC in vitro also depend o n Mph [28]. Accessory cells have since been shown to play a n auxiliary role in the induction of T helper cells [29]. However, secondary antibody responses to solubilized SRC o r t o POL, both T-independent antigens, are Mph-independent [28]. In contrast, Lee e t al. reported that solubilized SRC, trinitrophenylated (TNP) Ficoll and POL antigens were dependent o n adherent accessory cells, but to a lesser degree than the presence t o SRC [ 251. Chused et al. demonstrated accessory cell dependence in the antibody response to TNP-Ficoll but accessory cell independ-
399
ence in the response t o T and B cell mitogens [30]. Rosenstreich et al. have shown an absolute Mph dependency of T lymphocyte activation by mitogens [ 61. These various observations may be a reflection of the different depletion techniques employed, and the nature of the antigens used. More uniform results in respect t o the role of accessory cells are obtained in systems concerned with the generation of CTL in vitro. Lonai and Feldman have shown the requirement of Mphs in an in vitro graft rejection system. They found that the ratio of added Mphs t o depleted lymphoid cells was critical [ 71. Wagner et al. have shown a total abolition of cytotoxic activity resulting from a thorough depletion of accessory cells in mouse spleen; activity was completely restored by addition of peritoneal Mphs [ 81. McDonald et al. were also able to demonstrate a n obligatory cooperation of “progenitor killer cells” with a nonspecific “Mph-like cell” in an allograft response in vitro [9]. All these studies suggest that the accessory role in cell-mediated immune response is Mph-specific. There are, however, several reports suggesting that certain reducing agents such as 2-ME and even fibroblasts o r their supernates can substitute for Mphs, shedding a different light o n the role of the Mphs as an exclusive or specific cell required for a primary response in v i a o [ 17, 301. W e have employed an efficient technique for the removal of adherent phagocytic cells, consisting of cell filtration through a Sephadex G 1 0 column followed by carbony1 iron treatment. The resultant population of cells was highly viable (>95 %) and lacked detectable phagocytic cells (Table 3). These responder cells were unable to generate any cytotoxic activity against allogeneic E L 4 antigenic cells. We have shown that this inability does not result from lack o f precursor T cells because the ability t o generate CTL was restored with highly purified, RAMB + C-treated and irradiated PC. The results in Table 3 and Fig. 2 also show that 2-ME restores the ability t o generate cytotoxic cells maintaining the specificity of killing and resulting in high levels of survival of cultured depleted cells. It should be noted that the impaired cell survival in depleted cultures is restored by 2-ME o r by those cell populations which restore functional activity in cell-mediated lympholysis (CML). This suggests that at least a portion of the CML restorative activity may be due t o an effect on responder cell survival in culture. The restorative activity of 2-ME for lymphoid cells depleted of Mphs has so far only been shown i n antibody-producing systems in vztro as described by Chen and Hirsch [ 161, Hoffman et al. [3 11 and by Lemke and Opitz [ 171. 2-ME has been shown t o have an “enhancing” activity in antibody response in vitro [32], MLR [ 15, 331, CMC [ 18, 341, in promoting growth of tumor cells [ 3 5 , 3 6 ] and B lymphocyte colony formation in vitro
[381. Mocarelli et al. have shown that a n SV4Gtransformed Mph line will restore a primary response t o SRC in vitro [38]. We found that the Mph-like tumor P388D1, the present line, P388, and a mastocytoma, P815, did very efficiently restore ability t o cytotoxic T cells (Tables 5 and 6) when added t o depleted cells; t w o other cell lines L1210 and LSTRA did not. The restoration was dose and tumor cell type-dependent. Usually high concentrations had a n inhibitory activity similar t o the inhibition seen with high concentrations of PC (Table 3, Fig. 2 and [7, 81) and fibroblasts [ 191. A possible explanation for the inhibition may be simply overcrowding conditions in the reconstituted cultures. The restoration by tumor cells is highly reproducible except for the P8 15 cells when they are infected with mycoplasma. This observation will be discussed
400
Eur. J . Immunol. 1977. 7: 394-400
H.S Koren and R J. Hodes
in detail elsewhere. From our results it seems that the acce4 sory function of Mphs in the generation of CTL is not exclusively mediated by normal or a transformed Mph since 2-ME and other tumor cells reconstituted the cytotoxic activity as well. These results are in contrast t o the findings of Rosenstreich et al. who have demonstrated that 2-ME cannot replace Mphs of guinea pig lymph node lymphocytes in their response t o T cell mitogens [6].This discrepancy between their and our findings may be due to the different species, antigens and subpopulations of T cells studied. Mphs have been postulated to function as accessory cells by: (a) presenting antigen t o T cells, and (b) providing a “second signal” t o T cells by secretion of a stimulatory factor [ 271. In the case of allogeneic stimulator cells the first function of Mphs may not be required since viable cells can present their antigens in an immunogenic way; the requirement for Mphs t o provide the “second signal” can be bypassed by 2-ME and certain tumor cells in our system. Our findings are in line with earlier observations by Moller et aL showing that both fibroblast and their supernates are capable of restoring a primary anti-SRC response in v i f r o [ 181. We were not able t o find a common denominator for cells that were able t o restore except that they were Fc’, as were L1210 and LSTRA which lacked accessory activity. It would therefore appear that several cells, benign and tumor, are capable of supporting the induction of cytotoxic cells in vitro. The cell which is affected by these tumors has not been characterized nor have we identified any tumor cell products o r their modes of action. However, under normal test conditions using a FCS lot which is 2-ME-independent, none of the cell lines seemed t o have an augmenting effect when added t o now depleted cells, suggesting that these cells d o not act as general nonspecific immune stimulators. However, under suboptimal test conditions which would result, for example, when 2-ME is omitted in cultures supplemented with 2-ME-dependent serum, the addition of P388D1 and P388 has an augmenting effect resulting in normal levels of cytotoxicity (unpublished observations, H.K.) similar t o the findings reported by Hams et al. [34]. In conclusion, the mechanism by which cells or reagents can replace accessory cells in the generation of CTL is still poorly understood. It is possible, for example, that tumor cells or 2-ME act by cooperating with precursor cells or with a small residual of mature Mphs remaining in the depleted populations. However, the fact that Mphs can be replaced by various and defined populations of cells o r by 2-ME suggests that the accessory function of Mphs is not an exclusive property of these-cells.
5 References 1 Mosier, D.E., Science 1967. 1 5 8 1573. 2 Alter, B.J. and Bach, F.H., Cell Immunol 1970. I : 207. 3 Jones, A.L, Clin Exp. ImmunoL 1971. 8: 927.
4 Ritter, J., LohmamMatthes, ML, Sonntag, HC. and Fisher, H, CelL ImmunoL 1975.16: 153. 5 Oppenheim, J.J., Leventhal, RC. and Hersh, EM, J. ImmunoL 1968. I01 : 262
6 Rosenstreich. D.L, Farrar, J.J. and Dougherty, S., J. ImmunoL 1976 116: 131. 7 Lonai P. and Feldman, M., Immunology 1971. 21: 861. 8 Wagner, H., Feldmann, M,Boyle, W. and Schrader, J.W., J. Erp. Med 1972 136: 331. 9 McDonald, H R . Phillips, R A and Miller, R G , J. ImmunoL 1973. 111: 575. 10 Miller, C L and Mishell, RI.. J. ImmunoL 1975. 114: 692 11 Miller, RC., Schilling, RM. and Phillips RA.,J. ImmunoL 1977. 118: 166 12 Hoffmaa M and Dutton, RW.. Science 1971. I72 1047. 13 Sjoberg, O., Anderson, J. and Moller, G , J. ImmunoL 1972 109: 1379. 14 Friedman, H. and Sabet, T.Y., Immunology 1970. 1 8 883. 15 Heber-Katz, E. and Click, R E , CelL ImmunoL 1972 5 : 410. 16 Chen, C and Hisc h , J.G., J. Ekp. Med 1972 136: 604. 17 Lemke, H and Opitz, H-G, 1 ImmunoL 1976 I 1 7 : 388 18 Bevan, M.J., Epstein, R and Cohen, M., J. Exp. Med 1974.139: 1025. 19 Moller, G, Lemke, H. and Opitz, H.-C.. &and J. ImmunoL 1976 5: 269. 20 Handwerger, R S and Koren, RS, CIin ImmunoL ImrnunopathoL 1976 5: 272 21 Koren, HS, Handwerger, BS and Wunderlich, J.R, J. Immund 1975.114: 894. 22 Hodes, RJ. and Terry, W.D., J ImmunoL 1974 113: 39. 23 Ly, LA and Mishell, RI., J. ImmunoL Methods 1974 5: 239. 24 Hodeg RJ., Handwrger, R S and Terry, W.D., 1 Exp. Med 1974. 1 4 0 1646 25 Lee, K-C, Shiozawa, C , Shaw, A and Diener, E., Eur. J. Imrnunol 1976 6: 63. 26 Cerottini, J.-C and Banner, KT., in Dixon, F.J. and Kunkel, HG. (Eds), Advances in Immunology, Academic Press, New York 1974, VoL 18, p. 67. 27 Rosenstreich, D.L and Oppenheim, J.J., In Nelson, IXS (Ed), Immunobiology of Phe Macrophage, Academic Press New York 1976, p. 162 28 Feldmann, M and Palmer, J., Immunology 1971.21: 685. 29 Erb, P. and Feldmann, M., Nature 1975.254: 352 30 Chused, M.T., Kassan, SS and Mosier, D.E., J. Imrnund 1976. 116: 1579. 31 Hoffmann, MK., Hiimmerling, U., Simon, M and Oettgen, HF., J. ImmunoL 1976 116: 1447. 32 Click, R E , Benck, L and Alter, B.J., CelL ImmunoL 1972 3: 155. 33 Soulillou, J.-P., Carpenter, C R , Lundin, AP. and Strom, T.R, J. ImmunoL 1975.115: 1566. 34 Harris, J.W., McDonald, HR,Enger$ HD., Fitcb F.W. and Cerottini, J.-C, J. Immunol 1976 116: 1071.
We thank Drs Teny and Amos for invaluable support in providing the environment in which to cony out this work Appreciation is expressed to Ms Karen S Hathcock and Ms R Sconyers for excellent technical assistance and Ms J. Kerber and M s M. Schoenfelder for excellent secretarial assistance.
36 Nathan, CF. and Terry, W.D.. J. Exp. Med 1975. 142: 887. 37 Metcalf, D., J. IrnmunoL 1976.116: 635.
Received February 24, 1977.
38 Mocarelli, P., Palmer, J. and Defendi V.,ImmunoL Commun 1973. 2: 441.
35 Broome, J.D. and Jeng. MW., J. Ekp. Med 1973.138: 574.
Eur. J. Immunol. 1977. 7: 394-400
H.S. Koren and RJ. Hodes
H.S. Koren’ and R.J. Hodes’ Division of Immunology, Duke University Medi-lCenter. Dumam’and lmmunolow Branch, National Cancer Institute, National Institutes of Health, Bethesda’
Effect of tumor cells on the generation of cytotoxic T lymphocytes in vitro
1. Accessorv cell functions of mouse tumor cells in the generation of cytotoxic T lymphocytes in vitro: replacement of adherent phagocytic cells by tumor cells or 2-mercaptoethanol* In agreement with previous reports, the primary in vitro response t o alloantigens has been shown t o be dependent o n the presence of m a c r e phages (Mphs). Splenocytes extensively depleted of adherent phagocytic cells did n o t generate cytotoxic T lymphocytes, and this activity could be completely restored by small numbers of adherent peritoneal cells (accessory cells). Either P388D1 (Mph-like tumor), P388 (“null” tumor) or P8 15 (mastocytoma) tumor cells, or 2-mercaptoethanol, could completely replace the accessory function normally mediated by accessory cells. These tumor cells did not nonspecifically “enhance” the cytotoxic activity generated with normal nondepleted spleen cells. The restored cultures maintained killing specificity t o H-2 targets which was mediated by effector T cells as shown by sensitivity t o anti-@ and complement. Therefore, Mphs seem n o t t o be the sole cells capable of mediating an accessory function in a primary response t o alloantigens in vitro.
1. Introduction
The role of the accessory cells in cellular immunity has drawn the attention of many investigators. Mosier first described the requirement of an adherent cell t o collaborate with a nonadherent population in the initiation of an antibody response t o sheep red cells (SRC) in vitro [I]. It was later established that other in vitro responses such as the mixed lymphocyte reaction (MLR) [2-41, mitogen responses [5,6] and generation of cytotoxic T lymphocytes (CTL) [7- l l ] required the presence of adherent macrophage (Mphhlike cells or their culture supernates [ 12, 131. In addition, there is evidence that cells of the reticuloendothelial system are needed f o r a succes% ful immune response in vivo [ 141. Recently, Heber-Katz and Click have shown that reducing agents such as Zmercaptoetha no1 (2-ME) could substitute for serum in t h e medium for MLR o r enhance an immune response (IR) by a synergistic interaction with fetal calf serum (FCS) [ 151. Chen and Hirsch [ 161 and more recently Lemke and Opitz [ 171 were able t o show that 2-ME can substitute for Mph in a primary response to SRC in vitro. Bevan et al. have shown that 2-ME could restore the proliferative MLR as well as the generation of CTL in a [I 16611
* This work was supported by the grant USPHSCA-14049. Correspondence: Richard J. Hodes, Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20014, USA Abbreviations: SRC: Sheep red cells MLR: Mixed lymphocyte reac tion CTL: Cytotoxic T lymphocytes Mph(s): Macrophage(s) I R Immune response FCS: Fetal calf serum BA8: Braittassociated theta antigen MEM: Eagle’s minimum essential medium PC: Perk toneal cells BSS Balanced salt solution RAMB: Rabbit anti-mouse brain GpC: Guinea pig complement CMC: Cebmediated cytotoxicity POL: Polymerized flagellin SIg: Surface lgbearing 2-ME:2-Mercapte ethanol C: Complement
murine spleen cell population depleted of adherent cells [ 181. Moller et al., also using the primary response t o SRC system, have shown that Mphs can be substituted for not only by 2-ME but also by fibroblasts and supernates of these cells [ 191. The latter results suggest that the accessory function in a primary in vifro response t o SRC is n o t an exclusive property o f Mphs, as previously thought. In this paper we describe the restora tion of cytotoxit activity to Mphs-depleted DBA/2 spleens by 2-ME and by three DBA/2 tumor cells (P388D1, P388 and P8 15). Suppressive effect mediated by P815 tumor cells resulting from mycoplasma infection will be described in a SB parate paper (manuscript in preparation).
2 Materials and methods 2.1. Previously described materials and methods
Reagents and methods used t o characterize cell populations with respect t o their phagocytic activity, surface Ig, brainassociated theta (BAO) and Fc-receptor have already been described [20]. 2.2. Animals
Mice of inbred strain DBA/2J (H-2d) were obtained from Jackson Laboratories, Bar Harbor, Maine and NIH Animal Production, Bethesda, Md. Males aged 8- 12 weeks were used throughout this study.
2.3. Tumor cells All the tumor cells used in this study (Table 1 ) were grown in v i a 0 rather than using t u m o r cells from ascites fluid. This was done in order t o avoid contamination of the tumor cells
Replacement of accessory function by 2-mercaptoethanol and tumor cells
Eur. J. Immunol. 1977. 7: 394-400
under study with accessory cells of host origin. They were passaged bi-weekly in tissue culture using Eagle's minimum essential medium (MEM, Flow Laboratories, Rockville, Md.) supplemented with 10 % fetal calf serum (MEM-10 % FCS), 2 mM L-glutamine, 100 unitslml penicillin, 100 pg/ml streptomycin (Microbiological Associates, Bethesda, Md.). P388D1, a Mph-like tumor cell line, was grown in spinner cultures as previously described (21 1. All other tumor lines were grown in 75 ml plastic flasks (Falcon Plastics, Oxnard, Cal., no. 3024) in suspension cult ire. All the tumor cell cultures were maintained at 37 " C . Table 1. Characteristics of cultured tumor cell line used Tumor
Strain Latex of origin phage cytosisa)
P388D1 P388
DBA/2 DBA/2
-ke) -
-
DBA/2
-
(parent)
P815 L1210 LSTRA EL 4 a)
b)
c) d)
e)
DBA/2 BALB/c C51BL/6
-
Slgb) BA@)
Fcd)
Cell classifit cation
-
-
Mph "nulr'
-
-
+ + +
+
-
-+
+
+
-
mste
cytoma B "null" T
Latex phagocytosis, ability to phagocytize latex beads during 4 5 min incubation at 37 "C. Cells ingesting more than three beads were considered positive. SIg, surface lg-bearing; stains with fluorescein-conjugatedpolyvalent rabbit antcmouse lg. BAQ, brain-associated @bearing; reacts with RAMB serum. Fc, bears a receptor for heat-aggregated human Ig and/or antibody-antigen complexes (7 SEA). (+), possess the cell marker characteristic under study; (-), does not possess the cell marker under study. For each cell marker a minimum of 200 cells was scored
395
which efficiently avoided leakage of Sephadex o u t of the syringe. The depletion procedure was carried out at room temperature. The column, consisting of 35 ml of Sephadex G 1 0 , was equilibrated with 100 ml of prewarmed BSS-5 % FCS. Up t o 600 x lo6 spleen cells were loaded on 1 column and the nonadherent cells were eluted with approximately 25 ml of BSS-5 % FCS. The column-passed cells were spun once, and u p to 400 x l o 6 cells were resuspended in 8 ml of BSS2O % FCS. Thirty-two ml of lymphocyte separator reagent (carbonyl iron, Technicon Instruments Corp., Tarrytown, NY) was added t o the 8 ml of cells in a 50 ml conical tube (Falcon, no. 2074) a n d rotated at 3 7 "C for 1 h. A strong magnet was then used to pellet excess Fe and Fe-ingested cells. Cells in the supernatant were carefully removed, spun once and adjusted to 20 x 1O6 cells/ml in BSS- 1 0 % FCS. Two ml of this suspension were layered on t o p of 3 ml of FicollHypaque (Bionetics Kensington, Md.) in 1 5 ml conical tubes (Falcon, no. 2095). Cells were then spun a t 2200 x g for 3 min. Bands at the interface were collected, washed twice with excess of BSS-10 % FCS and finally resuspended in the complete medium (described below) used for the in virro sensitization. The approximate cell recoveries following Sephadex G I O , carbonyl iron and FicolkHypaque were usually 50 %, 7 0 % and 80 %, respectively. Therefore, the final yield was about 20 % of the original number of cells. The cells obtained through the above procedures will be termed depleted cells.
NON-DEPLETED
G-10 S f P n l t y X 6 - 0 X IO'CELLS
mcu
1EYP
WBONYL IRON
miAn AT
+ CELLS x 0 c Fm i nn
2.4. Spleen a n d peritoneal cell preparations
Sterile single cell suspensions of DBA/2 mouse spleen cells were prepared, and red cells osmotically lysed, as previously described [22]. Peritoneal cells (PC) were obtained by i p jecting 5 ml of balanced salt solution (BSS) (NIH Media Unit) and 5 % FCS i.p. and then aspirating the cell-containing fluid. This procedure was repeated 3 times, usually resulting in a total yield of 2 x l o 6 cells per mouse. These cells were spun at 1000 x g for 7 min, then irradiated with 1000 rads followed by treatment with rabbit anti-mouse brain serum (RAMB) and complement (C) (see below). The resultant population of PC was added in various concentrations t o Linbro plates used for sensitization and incubated in a humidified COz incubator for 2 h. Nonadherent cells were then removed by washing with MEM-10 % FCS; those remaining adherent cells will be termed purified PC. At this stage responding and stimulator cells were added to the wells.
Scheme 1. Schematic representation of the procedure used to deplete
DBA/2 spleen cells of the accessory function
2.6. Rabbit anthmouse brain serum (RAMB) The production and T cell specificity of RAMB as well as its use with guinea pig C (GpC) to obliterate T cell responses have been presented in a n earlier publication [24]. We used our RAMB f o r treatment of spleen cells and PC at a final dilution of 1 :60 a n d reconstituted lyophilized GpC (Gibco, Grand Island, NY) at a final dilution of 1 :8.
2.5. Removal of adherent phagocytic cells from DBA/2 splenoc ytes
2.7. In vitro sensitization
A general scheme for the depletion procedures used in this study is shown in Scheme 1. Modified Sephadex G 1 0 columns as described by Mishell and Ly were used [23]. The modification involved replacing the glass beads with a nylon wool plug
Sensitization was carried o u t by c e c u l t u r e of responding DBA/2 (H-2d) spleen cells with 10 000 rad-irradiated ( 13'C0 source, 1500 R / m h exposure rate) sensitizing EL4 (H-2b) tumor cells, in a total volume of 2 ml in each well of multiwell dishes (FB16-24-TC, Linbro Chemicals, New Haven,
396
Eur. J. Immunol. 1977. 7: 394-400
H.S. Koren and RJ. Hodes
Conn.). The medium used for the in vitro sensitization was RPMI- 1640 o r MEM supplemented with 2 mM L-glu tamine, 100 units/ml penicillin, 100 pg/ml streptomycin, 1 mM sodium pyruvate, 1 x nonessential amino acids (Microbiological Associates) and 10 % FCS (Reheis Chemical Co., Phoenix, Arizona, lot no. 505). This lot was shown t o support the generation of cytotoxic cells in the absence of 2-ME. Responding cells were either normal or depleted spleen cells. 2-ME at a final concentration of 5 x M (Eastman Kodak Co., Rochester, NY), o r tumor cells or PC were added t o the sensitizing cultures in specific experiments as indicated in Sect. 3. Sensitization cultures were incubated at 37 "C in humidified air - 5 % COz for 5 days. At the end of the sensitization period, cells were harvested from culture by repeated pipetting, then centrifuged and resuspended in MEM-I 0 % FCS. Replicate wells were pooled at this stage. Viable cells were counted by trypan blue exclusion and used as effector cells in a cytotoxicity assay.
2.8. Cytotoxicity assay Cell-mediated cytotoxicity (CMC) was assayed by a procedure previously reported [24]. Target cells were labeled by incubation in MEM-10 % FCS a t a concentration of 30 x 106 to 7 0 x 106/ml with 0.1 mCi/ml 51Cr (as sodium dichromate, AmershamSearle, Arlington Heights, Ill., approximately 125 pCi/pg Cr) for 3 0 min at 3 7 "C. Labeled cells were then centrifuged and washed 4 times in MEM-10 % FCS, and viable cells were counted. The same procedure was used when EL4, P815 or LSTRA tumor cells were used as targets. Five x l o 4 SICr-labeled target cells were mixed with effector cells at various effector to target cell ratios in a total volume of 200 pl of MEM- 10 % FCS in each well of round-bottomed microtiter plates (IS-MRC-96; Linbro Chemicals). Target cells alone (without effector cells) were incubated t o determine "spontaneous release" of "Cr label. All groups were set up in triplicate. Plates were centrifuged at 25 x g f o r 2 min a t 15 "C and then incubated for 4 h at 3 7 "C in humidified air - 5 % Table .2 Attempts to abrogate the ability of mouse spleen cellsa) to generate cytotoxic activity by various separation techniques
Exp. Separation no. method 1
2
3
4:l
EL 4 2:l
%Specific Iysisb) P815 or 1:l LSTRA 4: I
Siwivalc) (%)
324 G10 2 ~ d ) 41.3
22.0 9.6 37.4 16.0
- 0.3 - 1.5
40 12
Fee) (;lo+ Fe
46.1 54.2 -0.1
29.6 13.2 38.1 19.8 0.5 -0.3
- 0.8 - 0.3 -0.1
20 31 23
70.6 1.2
59.4 33.2 1.2 0.7
- 0.6 - 0.2
71 32
G 1 0 + FC
COz. At the end of this incubation period plates were centrifuged at 7 0 0 x g for 10 min at 4 "C. One hundred microliters of supernatant were removed from each well and transferred t o a 1 6 x 125 m m plastic tube for counting in a well-type gamma counter (Amersham Searle). "Total label" was determined by direct counting of 5 x 1 O4 51Cr-labeled target cells. Cytotoxic activity was calculated as: (Counts released in experimental group) - (Counts in spontaneous release) % specific lysis =
x 100.
(Counts in total label)
*
The standard deviation of triplicate cultures was 2 % of the mean. In each experiment, the specificity of cytotoxicity induced was verified by testing each effector population against LSTRA or P815 target cells which are syngeneic to the effectors.
3. Results 3.1. Attempts t o deplete spleen cells of the ability t o generate cytotoxic activity
Preliminary experiments have shown that passing DBA/2 spleen cells once through a Sephadex G-10 column does not result in lack of activity upon subsequent sensitization. Interestingly, spleen cells from BALB/c mice were easily depleted of accessory cells by one passage through a Sephadex G 1 0 column (our unpublished observation). As can be seen in the first experiment in Table 2, cells passed twice through Sephadex G 1 0 columns and used as responder cells had still maintained their ability t o generate cytotoxic activity. Exp. 2 describes an attempt t o deplete accessory cells for CMC based on selective removal of phagocytic cells by carbonyl iron particles. This technique was followed by Ficoll-Hypaque gradient t o ensure total removal o f excess iron particles and cells containing iron particles after the magnet step. This procedure also failed t o eliminate generation of cytotoxic activity; however, a combined depletion technique whereby cells were first passed through a Sephadex G 1 0 column and then treated with carbonyl iron totally obliterated the generation of cytotoxic activity, as can be seen in the last line of Expts. 2 and 3. The number of viable cells recovered from depleted cultures was usually depressed too. Table 3 shows the results of a marker study performed with nondepleted and depleted cells The number of latex-positive cells dropped below levels of detection. The SIg' cells were unchanged and the brain-associated ' 0 (BAO') cells were slightly enriched in the depleted population. Table 3. Marker study of mouse spleen cells before and after fractionation by Sephadex G10 column and carbonyl iron
Celisa)
latex phagocytosis
Sk (5%)
BAO
65 65
23 36
(W
(%)
a) 4 x 106 DBA/2 spleen cells were sensitized against 0.5 x 106 irradiated EL4 tumor cells. b) Spontaneous release of EL4 in Expts. 1, 2 and 3 was: 7.0, 6.3 and 8.3, respectively. LSTRA target cell was used in Expts. 1 and 2; P815 was used in Exp. 3. The spontaneous release was 5.6, 7.8, and 17.4, respectively. c) Percent viable cell recovery after 5-day sensitization culture. d) Cells were passed twice through Sephadex G10 columns e) Cells separated by carbonyl iron.
DBA/2 nondepleted DBA/2 depleted
5.5
< 0.5
a) DBA/2 spleen cells were first filtered through a Sephadex G 1 0 column, then incubated with carbonyl iron followed by separation on a Ficoll-Hypaque gradient. For details see Sect. 2.5. and footnote of Table 1.
Replacement of accessory function by 2-mercaptoethanol and tumor cells
Eur. J. Immunol. 1977. 7: 394-400
Since we were interested in total depletion of accessory cell function, this combined technique was used throughout this study. Previous studies attempting to deplete accessory cells for CMC in vitro using adherence depletion alone, achieved partial depletion only (9, 251. Wagner e t al. have also shown that they achieved efficient depletion only when they combined glass bead column fractionation with anti-Mph serum; neither of these techniques was as efficient as the combination of the two [8]. Rosenstreich et al. have noticed that they had t o employ two adherence columns to achieve complete depletion of Mphs from guinea pig lymph node cells [ 6). 3.2. Restoration of cytolytic capacity of depleted spleen cells by purified PC and >ME
To demonstrate that the inability of depleted populations to generate cytolytic cells actually reflects a specific deficit in adherent accessory cells, experiments were performed attempting t o reconstitute activity with PC. Since PC are a heterogeneous population of cells [ 161 they were treated in various ways ensuring a highly enriched population of adherent Mph-like cells. They were first treated with RAMB and C to eliminate T cells, irradiated with 10 000 rads and left o n plastic for 3 h at 37 OC, after which nonadherent cells were removed. Therefore, the number of cells as shown in Fig. 1 reflects a relatively high estimate since PC were counted prior t o adherence in the wells. Results of one such representative experiment are presented in Table 4 and a diagrammatic representation in Fig. 1. The data demonstrate that various degrees of restoration were achieved by using different amounts of purified PC. The doseresponse curve shows that low and high doses of PC achieved levels of cytotoxicity up t o 29.6 % specific lysis at 4: 1 compared to 49.1 %specific lysis of the nondepleted cells at the same ratio. Almost complete restoration (45.4 %)was achieved at the optimal dose of 0.8 x l o 5 PC. The survival of cultured sensitized cells appears t o correlate with the levels of cytotoxic activity. The fact that high doses of PC were suboptimal suggests that crowding conditions may have suppressed the development of cytotoxicity. In addition, 2-ME at a final concentration of 5 x 1 0-5 M substituted for accessory cells too, and resulted in a level of cytotoxic activity similar to the one seen by using optimal doses of PC. 2-ME has previously been shown
DEILLTCD
0s
OQZ K).
2
4
OF PERITONEAL CELLS A D E D PER CULrUr(XIOs)
Figure 1. DBA/2 spleen cells were depleted according to the procedure described in Scheme 1, then stimulated with irradiated EL4 cells for 5 days in vifro. To some of the cultures graded numbers of adherent, irradiated and RAMB + C-treated F T were added Other cultures received 2-ME (5 x 10- 5 M), the remaining depleted cells were left with out reconstitution Cytotoxic activity was tested on day 5 of sensitiza tion against 51Crlabeled EL4 targets
397
Table 4. Restoration of the ability to generate cytotoxic activity of 4 x 106 DBN2 responder cells by PC and 2-ME
Depletion
Additives 4:l
-
+ + + + + +
-
~ c a ) 4~ 105 pc 2~ 105 ~c o . 8 ~ 105 Pc 0.2x 105
2-ME S X 10-5
Spontaneous r elea se
49.2 0.2 20.5 29.6 45.4 20.8 45.1
Specific lysis (7%) E L4 P815 Survival 2:l 1:l 4:l (7c) 25.6
12.6
-0.1 -0.6
-
1.5
-1.1
10.5 4.3 - 1.3 15.5 7.5 - 1.8 25.8 1 2 4 -2.2 11.2 5.7 -1.0 1.4 20.6 1 2 5 15.6
58 15 48 46 51 51 65
13.0
a) Purified PC were plated for 2 h, then nonadherent cells were removed. t o have a Mph-independent accessory function in restoring a primary antibody response t o SRC [ 171 as well as MLR [ 181 in vitro.
3.3. Effect of adding various murine tumor lines on depleted spleen cells Since we were able t o show that the combined depletion techniques resulted in a population of cells incapable of mounting a primary in v i a o response t o alloantigens, but was restorable t o normal values with purified PC as well as 2-ME, we tested the effect of 5 tumor lines with different functional and surface characteristics o n the accessory cell-depleted cultures. All the tumor cells were of responder haplotype (H-2d) and, therefore, should n o t be able t o sensitize the responding cells t o foreign alloantigens. The tumors had been propagated in culture for a sufficient period of time to eliminate the possibility of introducing host cells into this assay. The tumor cells were irradiated and added in graded numbers t o the cultures o n day 0 of sensitization. Table 5 shows a representative experiment performed i n this study. P388D1, a Mph tumor line previously described by us [21], its parent line, P388, and a mastocytoma, PSI 5, demonstrated restorative activities resulting in cytotoxic activities comparable t o the nondepleted cultures. L1210, SIg’ tumor, and LSTRA which is a “null” tumor demonstrated only very low levels of restorative activity in the range of concentrations used in these experiments compared t o the other tumor cells. Similar t o the data presented in Tables 2 and 4, survival correlated closely t o the cytotoxic activity. For example, P388D1 cells restored optimally at a concentration of 2 x lo5 with a survival of 68 % which even exceeded the survival of the nondepleted group, 57 %. Relatively low levels of survival were obtained in cultures where L1210 and LSTRA were added, 33 % a n d 29 %, respectively. Specificity of the cytotoxicity generated in the restored cultures was demonstrated by lack of activity against the syngeneic P815 cells. This pattern of activity was reproducible in several experiments although the number of added cells needed for optimal restoration of sensitization varied. Table 6 lists experiments demonstrating successful restorations of accessory function mediated by the different tumor cell lines and the purified PC. As can be seen, purified PC restored each time tested, L1210 and LSTRA never restored, and the lines P388D1, P388 and P815 restored in most of the experi-
398
Eur. J. Immunol. 1977. 7: 394-400
H.S Koren and RJ. Hodes
ments. It is of interest that the variability of restoring ability seen with P8 15 was later shown t o correlate with the presence o r absence o f mycoplasma. The effect of mycoplasma o n the generation of allogeneic cytotoxic cells will be the subject of a separate paper.
Table 5. Restoration of the ability to generate cytotoxic activitya) by various murine tumor cells
(46)
-
-
-
0.2 4.0 20
30.6 19.6 -0.7 0.0 6.0 2 4 31.0 l a 4 17.4 9.2 -0.1 1.0 327 2 8 7 10.5 6 5 - 1.6 -0.2 - 1.4 -0.1 3 8 5 21.9 44.5 25.2 121 5.8 16.8 9.9 4.2 2 6 0.7 0.1
0.8
-0.8
0.2 LSTRA 4.0 LSTRA 20 LSTRA 0.8 LSTRA 0.2 Spontaneous release
-0.5
- 1.0
6.1
3.1 4.1
+
P388D1 P388D1 P388Dl P388D1 P388 P388 P388 P388 P815 P815 P815 P815 L1210 L1210 L1210 L1210
4.0 20 0.8
0.2 4.0 20 0.8
0.2 4.0 20 0.8
7.9 3.3 -0.2
-0.6
0.2
0.2 63
10.0 -4.0 0.7 - 3.4 2 5 - 1.7 7.3 - 2 8 5.3 -21 0.5 -0.6 1 4 7 -4.5 2 4 -3.2 0.3 - 1.8 0.6 -1.5 10.6 -2.5 14.1 -1.9 3.0 -6.0 4.9 0.4 1.7 -1.6 -0.4 5.5 -0.1 -1.1 0.3 1.0 1.8 -1.1 2 2 -1.8 1.5 - 1.2 -0.1 - 1.1 31.3
57 9 33 68 38 19 44 31 15 12 52 44 36 24 33 27 14 16 29 27 19 15
a) 4 x 106 DBA/2 spleen cells were sensitized against 0.5 x 106 irradiated EL4 tumor cells b) + Denotes depletion by Sephadex G 1 0 columns and carbonyl iron. c) Irradiated tumor cells added to depleted spleen cells on day of sensitization d) Percent viable cell recovery after 5-day sensitization culture.
Table 6. Reproducibility of experiments restoring the ability to generate cytotoxic activity with tumor and FC
successul restoration Number % !
P388D1
P388
Restorilrg cdls P815 Ll210
lO(12)a) lO(12) 6(8) 83.3 83.3 75
O(4) 0
LSTRA
PC
O(4) 0
5 (5) 100
a) Numbers in brackets denote the total number of experiments performed
3.4. Effect of RAMB and C on the cytotoxic cells from cultures restored with P388D1 Allogeneic cytotoxic cells raised in vivo as well as in vitro have repeatedly been shown t o be T cells [26]. Since depleted spleen cells were able t o respond t o alloantigens upon reconstitution with tumor cells, in addition t o conventional restora-
tion methods with purified PC, it was important t o establish the nature of the effector cell in this system. Effector cells from depleted cultures sensitized for 5 days in the presence of P388D1 were treated with RAMB and C to see whether the effector cells were indeed T cells. Table 7 shows the results of such an experiment. P388D1 cells restored the depleted cultures optimally at 2 x lo5. Treatment of the effector cells by RAMB and C obliterated their activity against EL4 target cells. Controls with RAMB or C alone did not significantly alter the cytolytic activity of these sensitized cells. This experiment does not prove that cultures restored with other tumor cells such as P388 or P8 15 would lead t o the generation of effector T cells. However, it is unlikely that the mechanism of restoration by these cells would differ from the one with P388D1.
Table 7. Effect of RAMB and C treatment on the cytotoxic cellsa) generated with P388D1 tumor cells restored cultured P388DI Effector DepIetionb) cells cds added treatmentd) 4:l x 10-5c)
-
+
2.0
-
325 -0.5 30.1 RAMB+C 1.5 RAMB 2a1 C 29.5 Spontaneous releave
specifii lyds (%) EL4 P815 Sur2:l 1:l 4:l viVd (%)
15.7 a2 -0.1 -0.2 7.5 14.8 0.2 -0.9 14.0 6.5 14.6 7.0 65
-1.5 56 -3.4 15 - 2 5 49 -3.1 -20 - 1.5 82
a) 4 x 106 DBA/2 spleen cells were sensitized against 0.5 x 106 irradiated EL4 tumor cells b) + Denote spleen cells depleted by Sephadex G 1 0 column and carbonyl iron c) Irradiated P388D1 cells were added to depleted cultures on day 0. d) Effector cellswere treated on day 5 with RAMB at a final dilution of 1:60 and GpC at a final concentration of 1:8.
3.5. Do tumor cells that restore accessory function “enhance” nondepleted spleen cells? The term “restoration”, as used in this study, refers t o the ability of certain cells or reagents t o substitute for adherent phagocytic cells in the generation of CTL in vifro. The question raised is whether this activity is restorative o r a more generalized “enhancing” activity which would be expressed with nondepleted responder cells as well. To test this possibility, tumor cells were added t o nondepleted DBA/2 r e sponder cells o n day 0 of sensitization t o EL4 tumor. Table 8 shows the results of 3 such experiments. As seen, none of the cell lines had a significant “enhancing” o r an immunopotentiating effect o n cytotoxic activity as measured on day 5. In certain cases such as with P388D1, at 0 . 8 x l o 5 cells in Exp. 3, survival was higher compared t o control without causing enhanced cytotoxic activity. The two cell lines which were shown t o lack restorative activity - L1210 and LSTRA (see Table 5) did n o t express a markedly suppressive effect in the range of concentrations used in E x p t s 2 and 3. This suggests a lack of restorative ability rather than active suppression on the part of these two cell lines.
Replacement of accessory function by 2-mercaptoethanol and tumor cells
Eur. J. Immunol. 1977. 7: 394-400
Table 8. Effect of adding tumor cells to nondepleted culturesa) Specific lysis (%) I-xp. no. 1
2
3
Tumor cells addedx IO-sb) 4:l
EL4 2:1
1:l
4.0 2.0 0.8 0.2 4.0 2.0 0.8 0.2 4.0 0.8 0.2 4.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2 2.0 0.8 0.2
28.7 2.1 7.8 16.9 21.2 10.4 13.6 21.7 27.4 14.1 5.0 8.0 10.3 12.2 11.9 13.9 12.3 7.5 3.0 8.9 14.0 13.2 29.5 24.1 16.1 15.1 31.6 18.9 4.9 16.9 10.3 2.5 8.7 23.4 32.5 11.6 14.1 23.2
16.9 1.3 4.2 8.8 16.0 6.5 8.1 13.8 14.0 6.2 2.0 4.0 5.3 4.7 6.6 6.8 6.0 3.8 1.8 4.5 7.3 6.5 14.4 12.4 7.2 7.4 14.3 8.0 2.9 8.4 4.3 2.0 3.9 10.5 16.8 5.7 7.3 12.4
-
P388D1 P388D1 P388D1 P388D1 P388 P388 P388 P388 P388D1 P388D1 P388Dl P388 P388 P388 P8l5 P815 P815 LSTRA LSTRA LSTRA P388D1 P388Dl P388D1 P388 P388 P388 pni 5 P815 P815 LSIRA LSIRA LSTRA LIZ10 Ll2lO L1210
40.1 3.4 13.8 27.1 41.7 19.2 24.1 35.6 38.3 28.8 4.9 15.0 19.8 19.6 24.7 26.1 25.6 15.7 6.5 17.6 27.9 25.6 48.1 41.6 33.5 32.0 48.3 34.1 10.7 37.6 19.3 6.4 18.2 44.7 53.5 24.1 30.3 41.2
P815 Survival 4:l (%) - 3.7
- 2.9 - 1.7 - 3.3 - 2.6 - 3.4 - 2.6 -2.3 - 2.8 -1.7 - 1.7 - 3.3 - 1.4 - 0.7 - 0.9 0.4 - 1.2 - 2.0 - 2.1 - 3.2 - 1.7 - 1.7 0.2 - 1.0 0.9 0.3 0.8 0.0 0.6 0.7 0.0 0.2 2.1 4.9 9.5 -0.7 -1.2 - 1.2
41.2 28 34 37 36 33 40 36 36 57 39 49 48 37 42 61 59 55 40 35 56 58
70 68 101 49 58 42 51 40 36 30 56 71 69 40 48 61
4 x 106 DBA/2 spleen cells were sensitized against 0.5 x 106 irradiated EL4 tumor cells. b) Irradiated tumor cells were added to cultures on day 0.
a)
4. Discussion
The role of adherent phagocytic Mph-like cells has often been shown to be obligatory in various cellular events (for review see [ 271). These cells have also been termed accessory o r auxiliary cells. Their role in the IR in vitro was first demonstrated by Mosier working with an anti-SRC response in vitro [ 1). Feldmann et al. have shown that secondary responses t o intact SRBC in vitro also depend o n Mph [28]. Accessory cells have since been shown to play a n auxiliary role in the induction of T helper cells [29]. However, secondary antibody responses to solubilized SRC o r t o POL, both T-independent antigens, are Mph-independent [28]. In contrast, Lee e t al. reported that solubilized SRC, trinitrophenylated (TNP) Ficoll and POL antigens were dependent o n adherent accessory cells, but to a lesser degree than the presence t o SRC [ 251. Chused et al. demonstrated accessory cell dependence in the antibody response to TNP-Ficoll but accessory cell independ-
399
ence in the response t o T and B cell mitogens [30]. Rosenstreich et al. have shown an absolute Mph dependency of T lymphocyte activation by mitogens [ 61. These various observations may be a reflection of the different depletion techniques employed, and the nature of the antigens used. More uniform results in respect t o the role of accessory cells are obtained in systems concerned with the generation of CTL in vitro. Lonai and Feldman have shown the requirement of Mphs in an in vitro graft rejection system. They found that the ratio of added Mphs t o depleted lymphoid cells was critical [ 71. Wagner et al. have shown a total abolition of cytotoxic activity resulting from a thorough depletion of accessory cells in mouse spleen; activity was completely restored by addition of peritoneal Mphs [ 81. McDonald et al. were also able to demonstrate a n obligatory cooperation of “progenitor killer cells” with a nonspecific “Mph-like cell” in an allograft response in vitro [9]. All these studies suggest that the accessory role in cell-mediated immune response is Mph-specific. There are, however, several reports suggesting that certain reducing agents such as 2-ME and even fibroblasts o r their supernates can substitute for Mphs, shedding a different light o n the role of the Mphs as an exclusive or specific cell required for a primary response in v i a o [ 17, 301. W e have employed an efficient technique for the removal of adherent phagocytic cells, consisting of cell filtration through a Sephadex G 1 0 column followed by carbony1 iron treatment. The resultant population of cells was highly viable (>95 %) and lacked detectable phagocytic cells (Table 3). These responder cells were unable to generate any cytotoxic activity against allogeneic E L 4 antigenic cells. We have shown that this inability does not result from lack o f precursor T cells because the ability t o generate CTL was restored with highly purified, RAMB + C-treated and irradiated PC. The results in Table 3 and Fig. 2 also show that 2-ME restores the ability t o generate cytotoxic cells maintaining the specificity of killing and resulting in high levels of survival of cultured depleted cells. It should be noted that the impaired cell survival in depleted cultures is restored by 2-ME o r by those cell populations which restore functional activity in cell-mediated lympholysis (CML). This suggests that at least a portion of the CML restorative activity may be due t o an effect on responder cell survival in culture. The restorative activity of 2-ME for lymphoid cells depleted of Mphs has so far only been shown i n antibody-producing systems in vztro as described by Chen and Hirsch [ 161, Hoffman et al. [3 11 and by Lemke and Opitz [ 171. 2-ME has been shown t o have an “enhancing” activity in antibody response in vitro [32], MLR [ 15, 331, CMC [ 18, 341, in promoting growth of tumor cells [ 3 5 , 3 6 ] and B lymphocyte colony formation in vitro
[381. Mocarelli et al. have shown that a n SV4Gtransformed Mph line will restore a primary response t o SRC in vitro [38]. We found that the Mph-like tumor P388D1, the present line, P388, and a mastocytoma, P815, did very efficiently restore ability t o cytotoxic T cells (Tables 5 and 6) when added t o depleted cells; t w o other cell lines L1210 and LSTRA did not. The restoration was dose and tumor cell type-dependent. Usually high concentrations had a n inhibitory activity similar t o the inhibition seen with high concentrations of PC (Table 3, Fig. 2 and [7, 81) and fibroblasts [ 191. A possible explanation for the inhibition may be simply overcrowding conditions in the reconstituted cultures. The restoration by tumor cells is highly reproducible except for the P8 15 cells when they are infected with mycoplasma. This observation will be discussed
400
Eur. J . Immunol. 1977. 7: 394-400
H.S Koren and R J. Hodes
in detail elsewhere. From our results it seems that the acce4 sory function of Mphs in the generation of CTL is not exclusively mediated by normal or a transformed Mph since 2-ME and other tumor cells reconstituted the cytotoxic activity as well. These results are in contrast t o the findings of Rosenstreich et al. who have demonstrated that 2-ME cannot replace Mphs of guinea pig lymph node lymphocytes in their response t o T cell mitogens [6].This discrepancy between their and our findings may be due to the different species, antigens and subpopulations of T cells studied. Mphs have been postulated to function as accessory cells by: (a) presenting antigen t o T cells, and (b) providing a “second signal” t o T cells by secretion of a stimulatory factor [ 271. In the case of allogeneic stimulator cells the first function of Mphs may not be required since viable cells can present their antigens in an immunogenic way; the requirement for Mphs t o provide the “second signal” can be bypassed by 2-ME and certain tumor cells in our system. Our findings are in line with earlier observations by Moller et aL showing that both fibroblast and their supernates are capable of restoring a primary anti-SRC response in v i f r o [ 181. We were not able t o find a common denominator for cells that were able t o restore except that they were Fc’, as were L1210 and LSTRA which lacked accessory activity. It would therefore appear that several cells, benign and tumor, are capable of supporting the induction of cytotoxic cells in vitro. The cell which is affected by these tumors has not been characterized nor have we identified any tumor cell products o r their modes of action. However, under normal test conditions using a FCS lot which is 2-ME-independent, none of the cell lines seemed t o have an augmenting effect when added t o now depleted cells, suggesting that these cells d o not act as general nonspecific immune stimulators. However, under suboptimal test conditions which would result, for example, when 2-ME is omitted in cultures supplemented with 2-ME-dependent serum, the addition of P388D1 and P388 has an augmenting effect resulting in normal levels of cytotoxicity (unpublished observations, H.K.) similar t o the findings reported by Hams et al. [34]. In conclusion, the mechanism by which cells or reagents can replace accessory cells in the generation of CTL is still poorly understood. It is possible, for example, that tumor cells or 2-ME act by cooperating with precursor cells or with a small residual of mature Mphs remaining in the depleted populations. However, the fact that Mphs can be replaced by various and defined populations of cells o r by 2-ME suggests that the accessory function of Mphs is not an exclusive property of these-cells.
5 References 1 Mosier, D.E., Science 1967. 1 5 8 1573. 2 Alter, B.J. and Bach, F.H., Cell Immunol 1970. I : 207. 3 Jones, A.L, Clin Exp. ImmunoL 1971. 8: 927.
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6 Rosenstreich. D.L, Farrar, J.J. and Dougherty, S., J. ImmunoL 1976 116: 131. 7 Lonai P. and Feldman, M., Immunology 1971. 21: 861. 8 Wagner, H., Feldmann, M,Boyle, W. and Schrader, J.W., J. Erp. Med 1972 136: 331. 9 McDonald, H R . Phillips, R A and Miller, R G , J. ImmunoL 1973. 111: 575. 10 Miller, C L and Mishell, RI.. J. ImmunoL 1975. 114: 692 11 Miller, RC., Schilling, RM. and Phillips RA.,J. ImmunoL 1977. 118: 166 12 Hoffmaa M and Dutton, RW.. Science 1971. I72 1047. 13 Sjoberg, O., Anderson, J. and Moller, G , J. ImmunoL 1972 109: 1379. 14 Friedman, H. and Sabet, T.Y., Immunology 1970. 1 8 883. 15 Heber-Katz, E. and Click, R E , CelL ImmunoL 1972 5 : 410. 16 Chen, C and Hisc h , J.G., J. Ekp. Med 1972 136: 604. 17 Lemke, H and Opitz, H-G, 1 ImmunoL 1976 I 1 7 : 388 18 Bevan, M.J., Epstein, R and Cohen, M., J. Exp. Med 1974.139: 1025. 19 Moller, G, Lemke, H. and Opitz, H.-C.. &and J. ImmunoL 1976 5: 269. 20 Handwerger, R S and Koren, RS, CIin ImmunoL ImrnunopathoL 1976 5: 272 21 Koren, HS, Handwerger, BS and Wunderlich, J.R, J. Immund 1975.114: 894. 22 Hodes, RJ. and Terry, W.D., J ImmunoL 1974 113: 39. 23 Ly, LA and Mishell, RI., J. ImmunoL Methods 1974 5: 239. 24 Hodeg RJ., Handwrger, R S and Terry, W.D., 1 Exp. Med 1974. 1 4 0 1646 25 Lee, K-C, Shiozawa, C , Shaw, A and Diener, E., Eur. J. Imrnunol 1976 6: 63. 26 Cerottini, J.-C and Banner, KT., in Dixon, F.J. and Kunkel, HG. (Eds), Advances in Immunology, Academic Press, New York 1974, VoL 18, p. 67. 27 Rosenstreich, D.L and Oppenheim, J.J., In Nelson, IXS (Ed), Immunobiology of Phe Macrophage, Academic Press New York 1976, p. 162 28 Feldmann, M and Palmer, J., Immunology 1971.21: 685. 29 Erb, P. and Feldmann, M., Nature 1975.254: 352 30 Chused, M.T., Kassan, SS and Mosier, D.E., J. Imrnund 1976. 116: 1579. 31 Hoffmann, MK., Hiimmerling, U., Simon, M and Oettgen, HF., J. ImmunoL 1976 116: 1447. 32 Click, R E , Benck, L and Alter, B.J., CelL ImmunoL 1972 3: 155. 33 Soulillou, J.-P., Carpenter, C R , Lundin, AP. and Strom, T.R, J. ImmunoL 1975.115: 1566. 34 Harris, J.W., McDonald, HR,Enger$ HD., Fitcb F.W. and Cerottini, J.-C, J. Immunol 1976 116: 1071.
We thank Drs Teny and Amos for invaluable support in providing the environment in which to cony out this work Appreciation is expressed to Ms Karen S Hathcock and Ms R Sconyers for excellent technical assistance and Ms J. Kerber and M s M. Schoenfelder for excellent secretarial assistance.
36 Nathan, CF. and Terry, W.D.. J. Exp. Med 1975. 142: 887. 37 Metcalf, D., J. IrnmunoL 1976.116: 635.
Received February 24, 1977.
38 Mocarelli, P., Palmer, J. and Defendi V.,ImmunoL Commun 1973. 2: 441.
35 Broome, J.D. and Jeng. MW., J. Ekp. Med 1973.138: 574.
