- Email: [email protected]
Metabolic and physiologic studies of nonimmune lymphoid cells cytotoxic for fibroblastic cells in vitro
CELLULAR
IMMUNOLOGY
Metabolic
13, 41-51 (1974)
and Physiologic Studies of Nonimmune tymphoid Cytotoxic for Fibroblastic Cells in Vitro1 E. MAYHEW
Cells
AND M. BENNETT
Department of Experimental Pathology, Roswell Park Memorial Institute, BujWo, New York 14203, and Department of Pathology, Boston University School of Medicine, Boston, Massachusetts 02118 Received November 20, 1973 .4n i vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in viva rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4°C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement. Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and “Sr both are able to prevent rejection of marrow allografts in tivo. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with ‘*Sr were effectively cytotoxic but lost practically all of their cytotoxicity afer incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in viva during marrow allograft rejections.
INTIiODUCTtON Allogeneic target cells may be killed or their proliferation may be inhibited in vitro by lymphocytes from sensitized donors (l-4) and lymphocytes from unsensitized donors in the presence of certain mitogens (5-7) and even in the absence of mitogens (7-9). The cytotoxic reaction previously described by us (10) involved lymphocytes from nonimmunized mice which were cytotoxic for allogeneic target fibroblastic cells in the absence of mitogens. Effector cells were found in bone marrow, spleen and lymph nodes but not in thymus. They were relatively radioresistant in function, did not depend upon thymic influences for their differentiation, and their function was fairly insensitive to suppression by rabbit anti-mouse thymocyte serum but sensitive to suppression by cyclophosphamide. Moreover, lymph 1 Supported in part by Grants CA 14405-01 from the National Institutes of Health, GB 35852 from the National Science Foundation, 1428-C-1 from the American Cancer Society, Massachusetts Division, and 1154 from the Northeastern Chapter of the Massachusetts Heart Association. 41 1974by AcademicPress, 0 reprcductionin any form
P
42
MAYHEW
AND
BENNETT
node cells from (C3H/He x C57BL/lO)Fl hybrid mice were cytotoxic for C57BL/IO but not for C3H/He parental strain target fibroblastic cells derived from freshly explanted embryonic tissue (10). This finding of “hybrid resistance” (11) in vitro and other data suggested that such lymphoid cells were the same as those effector cells responsible for the rejection of hemopoietic allografts in viva (12, 13). The finding of cytotoxic lymphoid cells of marrow origin led to the hypothesis that marrow was the central lymphoid organ for effector cells responsible for marrow allograft rejections. Treatment of mice with the bone-seeking isotope, 8gSr, did indeed prevent such mice from rejecting bone marrow allografts without suppressing the function of thymus-dependent (T) or bursal equivalent-dependent (B) lymphocytes (14). W e now call these effector cells “M cells,” since they depend upon the integrity of marrow lymphoid tissue for their differentiation. Recently, macrophages have been implicated as effector cells involved in marrow allograft reactions ( 15). However, YSr does not suppress macrophage or “accessory cell” functions (16) necessary for humoral antibody formation (unpublished observations). Therefore, M cells and macrophages may need to cooperate in the rejection of hemopoietic allografts. We have tested for the in vitro cytotoxic effects of lymphoid cells from mice treated with 8gSr to investigate this problem further. To determine the mechanism of cytotoxicity of these lymphoid cells, metabolic inhibitors and enzymes affecting cell surface parameters have been tested for their effects. A lymphotoxin is apparently secreted by nonimmune aggressor lymphocytes activated by phytohemagglutinin (17). Steps leading to secretion of the lymphotoxin probably include contact between aggressor and target cells, subsequent recognition of the target cells as self- or non-self by the aggressor cells, and activation of aggressor lymphocytes, which depends upon energy metabolism and protein synthesis, In our system free of mitogens, no toxic humoral substance has been isolated so far. Data presented here indicate that protein synthesis by aggressor lymphocytes is required for only a few hours. MATERIALS
AND
METHODS
Target Cells For most of these studies aneuploid L929 mouse fibroblasts origiinally derived from C3H mice grown in RPM1 1640 (18) supplemented with 20% fetal calf serum (FCS) were used as target cells. Washed, trypsinized L929 cells were diluted to l-2 X lo4 cells/ml and 10 ~1 aliquots were added to Falcon microtest 11 plate wells. The plates were incubated at 37°C in 5% COB/air for 24 hr before adding the aggressor cells (Y. infra) . Aggressor Cells Suspensions of axillary, brachial and mesenteric lymph node cells (LNC), bone marrow cells (BMC) or thymus cells (TC) from young adults C3H/He (C3H), C57BL/l0~~Cz(B10), or (C3H/He X C57BL/lO)Fl (C3BFl) mice were prepared as described previously (10).
CYTOTOXICITY
Cytotoxic
OF
NONIMMUNE
LYMPHOID
43
CELLS
Experiments
Aggressor cells were added to target cells in the microtest plates such that the 1ymphoid:target cell ratio was varied from 1000 to 8: 1. The plates were incubated for 24 hr, washed in fresh media, reincubated for 6 hr, rewashed and stained with Giemsa. Counts of the adhering fibroblasts were made in each well and the cytotoxic index determined (10). Cl = loo _ Mean number of fibroblasts/well (experimental) Mean number of fibroblasts/well (control)
x loo
(1)
Appropriate controls were made in all experiments. In each experiment six to twelve wells per experimental and control group were tested and counted. The number of experiments made are indicated in the legends to figures and tables. The standard errors of means were calculated for all points and were lo-‘20% of the mean in most instances. Significance of differences were calculated using Student’s t test. Actinomycin D was added to cultures to give a final concentration of 2 pg/ml, cycloheximide 10 pg/ml, neuraminidase (Vibro cholerae strain 24, General Biochemicals) 10 units/ml, and ribonuclease A (Worthington Biochemicals), 0.1 mg/ ml in separate experiments. The times of addition of inhibitor with respect to addition of the aggressor cells are mentioned in the Results section. Control wells in these inhibitor studies had inhibitor added under the same conditions as the test wells. The pH of the culture medium was varied over the range of pH 5-8 by use of 0.1 M phosphate buffer diluted in RPM1 1640 plus 20% FCS. It was not possible to measure the pH of the solutions directly in the microtest plate wells, but 5 ml tubes were set up with the same cell concentration/ml as in the microtest plates. The PH of these suspensions was measured before and after microtest plate experiments. The pH values shown in the figure are the initial pH; they changed during the course of the experiments by different amounts, 0.3 pH units or less. Aggressor and target cells were incubated together at 4°C in one study. Cell lines originally derived from embryonic skeletal muscle of C3H or BlO mice (10) were used as target cells in some experiments. Treatment of Mice with sQSr C57BL/6 J (B6) or (C57BL/6J X DBA/ZJ)Fl (BDFl) mice were injected iv with 100 PCi of 8gSr twice, at 28-day intervals. Cells were taken from such mice (and controls) one to three months after the second injection (14). Preparation
of Antiserum
DBA/ZJ mice were injected ip with lo* B6 spleen cells at two-week intervals four times. In addition, 0.2 ml of complete Freund’s adjuvant was injected ip to induce ascites (19). The serum and ascitic fluid were harvested 9 days after the last injection. Neither the serum nor the ascitic fluid was able to prevent marrow allograft rejection in rrivo. The serum was used in the in vitro cytotoxicity experiments without complement to test for its ability to prevent or “block” cytotoxic effects of nonimmune lymph node cells (LNC) .
MAYHEW
AND
1000 500
250
Lymphoid
BENNETT
125 62.5
cell / target
31.3
16
8
cell ratio
FIG. 1. Effect of metabolic inhibitors on LNC:L929 cytotoxicity. O-O Controls; A-A Treated with actinomycin D 2 pg/ml; O-O Treated with cycloheximide 10 pg/ml; m---m Incubated at 4°C with LNC. Each point represents the mean cytotoxic index from a total of 12 wells in 2 experiments.
RESULTS Metabolic Studies When metabolic activity was inhibited by cooling at 4”C, there was no measurable cytotoxicity (Fig. 1) at all LNC-target cell ratios (p > 0.5). In other experiments (data not shown) target and aggressor cells were incubated at 4°C for 24 hr, then at 37°C for another 24 hr. Cytotoxicity was observed, at LNC-target cell ratios of 250 : 1 and greater (p < O.Ol), indicating that no irreversible damage was done to the aggressor cells. Partial inhibition of DNA-directed RNA synthesis by actinomycin D did not affect cytotoxicity, whereas inhibition of protein synthesis by cycloheximide did prevent the cytotoxicity of aggressor cells (Fig. 1) at LNC-target cell ratios of 125 : 1 and greater (p < 0.01). In subsequent experiments cycloheximide was added O-5 hr after plating aggressor cells into wells containing target cells. Cycloheximide was able to suppress cytotoxicity (fi < 0.01) if added during the first two hr, but not afterwards (Fig. 2) (p > 0.05). Th ese data suggest that protein synthesis is required for a brief time in this system, Thymus cells, which are not toxic in this
I
I
ICXXI 500
Lymphoid
I
250
cell
I
I
125 62.5
I target
I
31.3
I
16
II
8
cell ratio
FIG. 2. Effect of time of addition of cycloheximide on subsequent LNC:L929 cytotoxicity. 0-O Controls (no cycloheximide added) ; O-O Cycloheximide added at 0 hr; A---A Cycloheximide addBedat 1 hr ; [7--n Cycloheximide added at 2 hr ; n - ----W Cycloheximide added at 3 hr ; A-----A Cycloheximide added at 5 hr. Each point represents the mean cytotoxic index from a total of 18 wells in 3 experiments.
CYTOTOXICITY
OF NONIMMUNE
I
I
I
LYl’dPHOID
I
I
I
CELLS
45
I
1000 500 250 125 62.5 31.3 16 8 Lymphoid cell I target cell ratio FIG. 3. Effect of Neuraminidase and Ribonuclease of LNC and TC:L929 cytotoxicity. LNC treated with a-0 LNC control; O- 0 LNC treated with neuraminidase; A-A ribonuclease; m-----B TC Control; m-----n TC treated with neuraminidase; A-----A TC treated with ribonuclease.Each point represents the mean cytotoxic index from a total of 18 wells in 3 experiments.
system (lo), remained so at 4°C or in the presence of actinomycin D or cycloheximide (p > 0.05) (data not shown). Alteration of the electrical charge of cells at their peripheries with neuraminidase or with ribonuclease did not alter the cytotoxicity of aggressor LNC (j > 0.05) (Fig. 3). These enzymes also did not alter the lack of cytotoxicity of TC (Fig. 3) (p > 0.05). Other agents tested which did not alter the cytotoxic reaction between LNC and L929 fibroblastic cells included the polycations poly-lysine (mol. wt. 2800) at 4 rg/ml (p > 0.05) and cytochrome C at 10 pg/ml (p > 0.05) (data not shown). The LNC cytotoxic reaction were sensitive to change in pH (Fig. 4). Peak activity was at pH 7 with a more rapid fall-off of cytotoxicity on the acidic than on the basic side of neutrality. Physiologic
Studies
Thymus cells are not cytotoxic in this system, and TC inhibit the restriction of proliferation of parental-strain marrow cells in irradiated Fl hybrid mice (19). Therefore, we wondered if TC would be able to inhibit the cytotoxicity of LNC. LNC and TC were mixed in ratios of 9: 1, 1: 1 and 1:9 LNC: TC and plated onto target L929 cells. The cytotoxic indices decreased as the percentage of TC increased (Fig. 5). For a given ratio of LNC: L929 cells the presence of TC decreased the observed cytotoxicity. This can be clearly seen if comparisons such
PH.
FIG. 4. Cytotoxicity of LNC against L929 cells at different pH’s. A-A LNC:L929 ratio 5OO:l; 0 -0 LNC:L929 ratio 125:l; O-O LNC:L929 ratio 31:l. Each point represents the mean cytotoxic
index
from
a total
of 12 wells
in 2 experiments.
46
MAYHEW
Lymphoid
AND
BENNETT
cell /target
cell ratio
FIG. 5. Cytotoxic effects of mixed LNC and TC on L929 cells. O-O LNC only; d----n LNC:TC, 9:1, m-----H LNC:TC, 1:l; O-----O LNC:TC, 1:9; A-----A TC only. Each point represents the mean cytotoxic index from a total of 12 wells in two experiments. The cell number used to calculate lymphoid to target cell ratio was the sum of LNC plus TC for each mixture. At a 1OOO:l lymphoid cell:target cell ratio there was (A) 1000 LNC/target cell for LNC only; (B) 900 LNC plus 100 TC/target cell for a LNC:TC 9:l (C) 500 LNC plus 500 TC/target cell for LNC:TC 1: 1; and, (D) 100 LNC plus 900 TC/target cell for LNC:TC 1:9.
as the following are made. (A) At a 500: 1 LNC only: target cell ratio the same number of LNC were present as for a 1000: 1 LNC:TC 1: 1 mixture to target cell ratio. Here the cytotoxicity of LNC only (100%) was significantly higher than for the mixture (42%) (p < 0.01). (B) At a 125: 1 LNC only: target cell ratio approximatley the same number of LNC were present as for a 1000: 1 LNC:TC 1:9 mixture to target cell ratio, In this case the cytotoxicity of LNC (58%) was significantly higher than for the mixture (14%) (p < 0.01). However, the inhibition by thymus cells of LNC cytotoxicity was certainly not complete at high aggressor cell to target cell ratios. Whereas effector cells responsible for marrow allograft rejection are radioresistant, their precursors are radiosensitive (12, 13). Sublethal irradiation (500700 R) 7-21 days before lethal irradiation (800-900 R) and marrow transplantation prevented the rejection process. Cytotoxic LNC were also found to be radioresistant in function (10). To determine if cytotoxic LNC have radiosensitive precursors, C3BFl mice were exposed to 0 R or 600 R seven days prior to plating their LNC into wells containing Lg.29 target cells. LNC from irradiated mice were much less cytotoxic than LNC from unirradiated mice (Table 1). This finding is in keeping with the concept that cytotoxic LNC in this system reflect effector cell function operative against marrow cells have radiosensitive precursors.
allografts.
It only
shows that the effector
The DBA/Z anti-C57BL/6 spleen cell serum was raised to provide an agent to inactivate cells responsible for marrow allograft rejection (20). Such an agent could be tested in vitro for its ability to prevent the cytotoxic reaction. This particular antiserum was ineffective in tivo but did have anti-C57BL activity as it lysed C57BL lymphocytes in the presence of guinea pig complement. Therefore, we used the serum without complement to test its ability to protect BlO target cells from the cytotoxic action of lymphoid cells. The antiserum was mixed with the target and aggressor cells at a final dilution of 1: 16 and was present throughout the culture period.
CYTOTOXICITY
OF
NONIMMUNE
TABLE LNC
CYTOTOXICITYOF
Cytotoxic
LNC:L929 cell ratio
0 Ra 1000: 1 500: 1 2.50:1 125:l
74.2 58.3 36.4 17.4
f 8.6 f 7.8 f 4.4 f 1.9
LYMPHOID
47
CELLS
1
FROM IRRADIATED
C3BFl
MICE Significance of difference@
index 600 R= 26.5 20.2 10.6 11.2
f 5.2 f 4.3 f 1.6 f 1.6
p < 0.01 p < 0.01 p < 0.01 0.05 > p > 0.02
a Dose of total-body x-rays 7 days before harvesting LNC (lymph node cells) ; istandard error of mean. * Significance of difference calculated using Student’s t test. Each value represents the mean cytotoxic index from a total of 12 wells in two experiments.
The antiserum protected BlO (p < 0.01) but not C3H (p > 0.05) target cells from the LNC taken from C3H, BlO, or C3BFl young adult mice (Table 2, Expt 1). In this and other experiments “syngeneic cytotoxicity” was observed, i.e., LNC were cytotoxic for fibroblasts originally taken from syngeneic embryos. The cells had been in culture for some time and apparently became susceptible to “syngeneic” LNC. It was apparent that the C3H cells were more susceptible to this cytotoxic reaction than El0 cells. However, the BlO LNC were more reactive against C3H than against BlO target cells, and C3H LNC were more cytotoxic to BlO than to C3H target cells. The C3BFl LNC were cytotoxic to both lines of target cells. Lymph node cells (p < 0.01)) but not bone marrow cells (p > 0.05) of the hypoplastic marrow, of C57BL/6 mice treated with 89Sr were cytotoxic for target C3H and BlO fibroblastic cells (Table 1, Expt. 2). Cytologically, nearly all nucleated marrow cells from mice treated with 89Sr are mature granulocytes. The DBA/Z anti-C57BL/6 spleen cell serum protected B10 cells from LNC of both normal and 8gSr-treated C57BL/6 mice. In a similar experiment, the cytotoxic effects of spleen cells (SC) and LNC from BDFl mice treated with ‘YSr were determined. Control and experimental LNC were almost equally cytotoxic (Table 2, Expt 3). The spleen cells from mice treated with 8gSr were significantly less cytotoxic than control spleen cells (0.05 > p > 0.02). This difference could be explained by a “dilution effect” due to extensive extra-myeloid myelopoiesis that occurs in spleens of such mice. It should be emphasized that the spleens of mice treated with 8gSr take over all stem cell functions (12). These results with 89Sr-treatment were surprising, since all of the preceding experiments indicated that this in vitro assay detected effector cells invoived in the resistance to marrow allografts. A second antiserum was kindly given to us by Dr. R. J. Eckner. This antiserum was produced in BALB/c mice by injecting lo8 C57BL/6 spleen cells with complete Freund’s adjuvant ip, initially. For the next 15 weeks, 108 C57BL/6 spleen cells were injected ip at weekly intervals. The serum was harvested seven days after the last injection and decomplemented (56”C, 30 min). This antiserum prevented the rejection of DBA/2 Ha marrow cells by irradiated C3BFl mice in vi710
48
MAYHEW
AND
BENNETT
(data not shown). LNC (10’) from both control and Y5--treated BDFl mice were incubated with the BALB/c anti-C57BL spleen cell serum (or normal BALB/c serum) for 30 min at 37°C in 2 ml of 1: 10 dilution of antiserum. In TABLE
2
EFFECT OF DBA/2 ANTI-CS~BL/~ SPLEEN CELL SERUM ON THE CYTOTOXIC EFFECT OF LYMPHOID CELLS FROM NORMAL MICE AND MICE TREATED WITH @SR Expt
Lymphoid Strain
1
BlO
C3H
2
3
cell donor
Pretreatmenr
-
-
Tissueb
LNC
LNC
C3BFl
-
LNC
C57BL/6
-
LNC
C57BL/6
sgSr
LNC
C57BL/6
-
BMC
C57BL/6
?Sr
BMC
BDFl BDFl BDFl BDFl
-
LNC SC LNC SC
*?Sr *?Sr
Target cell strain C3H C3H B10 BlO C3H C3H B10 B10 C3H C3H B10 B10 C3H C3H BlO B10 C3H C3H BlO BlO C3H BlO C3H BlO BlO BlO BlO B10
AntiserumC
+ + + + + + + i+ -t -
-
Cytotoxicd index
83.3 64.6 26.8 6.2 39.6 51.6 52.4 +16.1 84.3 78.1 34.6 15.8 79.0 92.0 36.3 f11.6 95.7 91.6 32.6 + 3.2 57.2 22.8 14.3 + 5.7 63.9 79.6 67.3 48.0
Q Mice were injected with 100 pCi sgSr iv at 2%day intervals and the cells were harvested 1 month later in Expt 2. In Expt 3, a third injection of 100 pCi *%r was performed 4 months after the second injection and the cells were harvested 1 month later. b LNC, lymph node cells; BMC, bone marrow cells; SC, spleen cells. c The serum at a 1: 16 dilution was added to each well at the same time as the LNC and was present throughout the culture period. No complement was present. The method of raising the antiserum is given in Materials and Methods. This antiserum did not prevent marrow allograft rejection in viva. d The standard errors of the mean cytotoxic index were lCrZO% of the mean value. The aggressor: target cell ratio was 1000: 1. Each value represents the mean cytotoxicity index from a total of 12 wells. + indicates enhancement of growth.
CYTOTOXICITY
OF NONIMMUNE
LYMPHOID
CELLS
49
-20[, , , , , , , ,] 1000
250 Lymphoid
62.5 ceil / target
16 cell ratio
FIG. 6. Effect of BhLB/c anti-CS7BL/6 spleen cell serum on cytotoxicity of BD’FI LNC against L929 cells. O-O Control LNC treated with normal BALB/c serum and compleLNC from “Sr-treated mice treated with normal BALBjc serum and comment ; H-M plement; O-O Control LNC treated with BALBjc anti-C57BL/6 spleen cell serum and complement. i-J---n LNC from Tr-treated mice treated with BALB/c anti-C57BL/6 spleen cell serum and complement. Each point represents the mean cytotoxic index from a total of 12 wells in 1 experiment.
addition, guinea pig serum (l/10 v/v) was added as a source of complement. The LNC were then washed and added to target L929 cells. LNC treated with the anti-spleen cell serum plus complement lost much of their cytotoxicity (Fig. 6) at all LNC:target cell ratios (p <0 01). However, cytotoxic indices of 50 to 60% were measured at the 500: 1 and 100: 1 ratios of control BDFl LNC to L929 cells. This result alerted us to the possibility that this in vitro system may detect more than one type of cytotoxic lymphoid cell. The BALB/c anti-C57BL/6 spleen cell serum plus complement suppressed the cytotoxic activity of LNC from 3r-treated mice to low levels (fl < 0.01) even at the 1000: 1 and 500: 1 LNC; target cell ratios (Fig. 6). In this experiment, the LNC from mice treated with 89Sr were less cytotoxic than control LNC (0.02 > p > O.Ol), but were nonetheless effective. DISCUSSION As the cytotoxic reaction between LNC and L cells is insensitive to actinomycin but sensitive to cycloheximide and cold, it can be suggested that metabolic activity, and probably protein synthesis (21) is involved in the cytotoxic action of nonimmune LNC. The results also suggest that the lack of toxicity of TC is not due to metabolically linked factors. The results from experiments where the time of addition of cycloheximide was varied can be interpreted to indicate that protein synthesis is involved in a process,
50
MAYHEW
AND
BENNETT
necessary for subsequent cytotoxicity, which occurs shortly after the aggressor cells reach the target cells at the microtest plate well surface. At present we have not investigated whether a Iymphotoxin is produced in this system but one could be produced by the cells at this time similar to that shown in the system investigated by Granger et al. (17). The data presented suggests that the pH optimum for the cytotoxic reaction is approximately 7. If there were significant deviations from neutrality, the cytoioxicity was reduced. This may be an important factor in in &YO cell-mediated cytotoxicity. There have been reports that the local environment of tumors may lymphocytes could be more acidic than that of normal tissue (22). Thus, cytotoxic be less effective against tumor cells for this reason alone. As neuraminidase and ribonuclease were without effect on the cytotoxic reaction this could indicate that the cell surface groups, sialic acids (23, 24) and RNase susceptible groups (25) respectively are not involved in the lymphocyte-target cell cytotoxicity in this system. Similarly, the positively charged polymers poly-lysine and cytochrome c also were without effect indicating that these agents which bind to cell surfaces (26) do not inhibit LNC cytotoxicity. These negative results would tend to indicate that cell surface charge is not a major factor in determining the outcome of cytotoxic reactions. The simplest interpretation of the results where the presence of TC inhibited LNC cytotoxicity is that TC can come in contact with the target cells and physically block the sites that LNC must contact to produce the cytotoxic reaction. However, the results presented in this report do not rule out direct interactions between the LNC and TC. These observations do suggest that one might be able to analyze how one type of immunocompetent cell population may regulate the function of another type. The observation of “syngeneic cytotoxicity” by LNC could be blocked by specific anti-target cell antisera (Table 2) suggests that the LNC are not “self-tolerant” when removed from the body and placed in an in vitro environment. Rat thymoantigens in vitro cytes apparently can be sensitized to “self” histocompatibility within 6 hr (27). We have recently observed that lethally irradiated rats restrict the proliferation of even syngeneic marrow stem cells for several days after transplantation (unpublished observations of P. Rodday, M. Bennett, and J. Vitale), which may or may not be relevant to these in vitro observations. It should be pointed out here that Cudkowicz’s pioneering observation that led to most studies of the immunobiology of marrow allograft rejection (12, 13) was the failure of parental-strain C57BL marrow cells to grow in lethally irradiated Fl hybrid mice (28). Two cell types from nonimmunized mice appear capable of in vitro cytotoxic reactions agianst target fibroblastic cells for the following reasons: (i) BALB/c anti-C57BL/6 spleen cell serum and complement destroyed much but not all of the cytotoxic effect of LNC ; (ii) sgSr-treatment alone had little effect on the cytotoxic potential of LNC; and (iii) BALB/c anti-C57BL/6 spleen cell serum and complement destroyed almost all of the cytotoxic effect of LNC from mice treated with 89Sr (Table 2 and Fig. 6). Since either 8gSr or BALB/c anti-C57BL/6 spleen cell serum prevents marrow allograft rejections, two different cell types may need to cooperate in v&o. This is a hypothesis that we are now testing.
CYTOTOXICITY
OF
NONIMMUNE
LYMPHOID
CELLS
51
ACKNOWLEDGMENTS We thank Dr. L. Weiss for useful discussions. We also thank J. Ciszkowski and K. Caruana for their technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Rosenau, W., and Moon, H. D., J. Nat. Cancer Inst. 27, 471, 1961. Wilson, D. B., J. Exp, Med. 122, 143, 1965. Hellstrom, I., Znt. J. Cancer 2, 65, 1967. Berke, G., Ginsberg, H., Yagil, G., and Feldman, M., Israel J. Med. Sci. 5, 135, 1969. Moller, E., Science 147, 873, 1965. Holm, G., and Perlman, P., Immunology 12, 525, 1967. Bach, F. H., In “Histocompatability Testing,” pp. 136, 320. Wilkins and Wilkins, Baltimore, 1965. 8. Bach, ‘F. H., and Hirschhorn, K., .Sem&ars Hemufol. 2, 68, 1965. 9. Jamieson, C. W., and Wallace, J. H., J. Zmmunol. 105, 7, 1970. 10. Mayhew, E., and Bennett, M., Immunology 21, 123, 1971. 11. Cudkowicz, G., and Stimpfling, J. H., Immunology 7, 291, 1964. 12. Cudkowicz, G., and Bennett, M., J. Exp. Med. 134, 83, 1971. 13. Cudkowicz, G., and Bennett, M. J. Exp. Med. 134, 1513, 1971. 14. Bennett, M., J. Zmmunol. 110, 510, 1973. 15. Lotzovo, E., and Cudkowicz, G., Fed. Proc. 32, 226, 1973. 16. Gorczynski, R. M., Miller, R. G., and Phillips, R. A., J. Exj. Med. 134, 1201, 1971. 17. Granger, G. A., and Kolb, W. P., J. Zmmzclzol. 101, 111, 1968. 18. Moore, G. E., Sandberg, A. A., and Ulrich, K., J. Nat. Cancer Znsf. 86, 405, 1966. 19. Goodman, J. W., Burch, K. T., and Bastorel, N. L., Blood 39, 850, 1972. 20. Gregory, C. J., McCulloch, E. A., and Till, J. E., Traranspluntatiolz 13, 138, 1972. 21. Tata, J. R., and Widnell, C. C., Biochem. I. 98, 604, 1966. 22. Eden, M., Hains, B., and Kahler, H., J. Natl. Cancer Inst. 16, 541, 1955. 23. Gottschalk, A., Physiol. Reu. 37, 66, 1957. 24. Weiss, L., and Cudney, T. L., Znt. J. Cancer 4, 776, 1969. 25. Mayhew, E., and Weiss, L., In “Surface Chemistry of Biological Systems” (M. Blank, Ed.), pp. 191-208. Plenum Press, New York, 1970. 26. Juliano, R., and Mayhew, E., Exp. Cell Res. 73, 3, 1972. 27. Cohen, I. R., Nature New Biol. 242, 60, 1973. 28. Cudkowicz, G., Proc. Sot. Exp. Biol. Med. 107, 968, 1961.
IMMUNOLOGY
Metabolic
13, 41-51 (1974)
and Physiologic Studies of Nonimmune tymphoid Cytotoxic for Fibroblastic Cells in Vitro1 E. MAYHEW
Cells
AND M. BENNETT
Department of Experimental Pathology, Roswell Park Memorial Institute, BujWo, New York 14203, and Department of Pathology, Boston University School of Medicine, Boston, Massachusetts 02118 Received November 20, 1973 .4n i vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in viva rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4°C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement. Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and “Sr both are able to prevent rejection of marrow allografts in tivo. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with ‘*Sr were effectively cytotoxic but lost practically all of their cytotoxicity afer incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in viva during marrow allograft rejections.
INTIiODUCTtON Allogeneic target cells may be killed or their proliferation may be inhibited in vitro by lymphocytes from sensitized donors (l-4) and lymphocytes from unsensitized donors in the presence of certain mitogens (5-7) and even in the absence of mitogens (7-9). The cytotoxic reaction previously described by us (10) involved lymphocytes from nonimmunized mice which were cytotoxic for allogeneic target fibroblastic cells in the absence of mitogens. Effector cells were found in bone marrow, spleen and lymph nodes but not in thymus. They were relatively radioresistant in function, did not depend upon thymic influences for their differentiation, and their function was fairly insensitive to suppression by rabbit anti-mouse thymocyte serum but sensitive to suppression by cyclophosphamide. Moreover, lymph 1 Supported in part by Grants CA 14405-01 from the National Institutes of Health, GB 35852 from the National Science Foundation, 1428-C-1 from the American Cancer Society, Massachusetts Division, and 1154 from the Northeastern Chapter of the Massachusetts Heart Association. 41 1974by AcademicPress, 0 reprcductionin any form
P
42
MAYHEW
AND
BENNETT
node cells from (C3H/He x C57BL/lO)Fl hybrid mice were cytotoxic for C57BL/IO but not for C3H/He parental strain target fibroblastic cells derived from freshly explanted embryonic tissue (10). This finding of “hybrid resistance” (11) in vitro and other data suggested that such lymphoid cells were the same as those effector cells responsible for the rejection of hemopoietic allografts in viva (12, 13). The finding of cytotoxic lymphoid cells of marrow origin led to the hypothesis that marrow was the central lymphoid organ for effector cells responsible for marrow allograft rejections. Treatment of mice with the bone-seeking isotope, 8gSr, did indeed prevent such mice from rejecting bone marrow allografts without suppressing the function of thymus-dependent (T) or bursal equivalent-dependent (B) lymphocytes (14). W e now call these effector cells “M cells,” since they depend upon the integrity of marrow lymphoid tissue for their differentiation. Recently, macrophages have been implicated as effector cells involved in marrow allograft reactions ( 15). However, YSr does not suppress macrophage or “accessory cell” functions (16) necessary for humoral antibody formation (unpublished observations). Therefore, M cells and macrophages may need to cooperate in the rejection of hemopoietic allografts. We have tested for the in vitro cytotoxic effects of lymphoid cells from mice treated with 8gSr to investigate this problem further. To determine the mechanism of cytotoxicity of these lymphoid cells, metabolic inhibitors and enzymes affecting cell surface parameters have been tested for their effects. A lymphotoxin is apparently secreted by nonimmune aggressor lymphocytes activated by phytohemagglutinin (17). Steps leading to secretion of the lymphotoxin probably include contact between aggressor and target cells, subsequent recognition of the target cells as self- or non-self by the aggressor cells, and activation of aggressor lymphocytes, which depends upon energy metabolism and protein synthesis, In our system free of mitogens, no toxic humoral substance has been isolated so far. Data presented here indicate that protein synthesis by aggressor lymphocytes is required for only a few hours. MATERIALS
AND
METHODS
Target Cells For most of these studies aneuploid L929 mouse fibroblasts origiinally derived from C3H mice grown in RPM1 1640 (18) supplemented with 20% fetal calf serum (FCS) were used as target cells. Washed, trypsinized L929 cells were diluted to l-2 X lo4 cells/ml and 10 ~1 aliquots were added to Falcon microtest 11 plate wells. The plates were incubated at 37°C in 5% COB/air for 24 hr before adding the aggressor cells (Y. infra) . Aggressor Cells Suspensions of axillary, brachial and mesenteric lymph node cells (LNC), bone marrow cells (BMC) or thymus cells (TC) from young adults C3H/He (C3H), C57BL/l0~~Cz(B10), or (C3H/He X C57BL/lO)Fl (C3BFl) mice were prepared as described previously (10).
CYTOTOXICITY
Cytotoxic
OF
NONIMMUNE
LYMPHOID
43
CELLS
Experiments
Aggressor cells were added to target cells in the microtest plates such that the 1ymphoid:target cell ratio was varied from 1000 to 8: 1. The plates were incubated for 24 hr, washed in fresh media, reincubated for 6 hr, rewashed and stained with Giemsa. Counts of the adhering fibroblasts were made in each well and the cytotoxic index determined (10). Cl = loo _ Mean number of fibroblasts/well (experimental) Mean number of fibroblasts/well (control)
x loo
(1)
Appropriate controls were made in all experiments. In each experiment six to twelve wells per experimental and control group were tested and counted. The number of experiments made are indicated in the legends to figures and tables. The standard errors of means were calculated for all points and were lo-‘20% of the mean in most instances. Significance of differences were calculated using Student’s t test. Actinomycin D was added to cultures to give a final concentration of 2 pg/ml, cycloheximide 10 pg/ml, neuraminidase (Vibro cholerae strain 24, General Biochemicals) 10 units/ml, and ribonuclease A (Worthington Biochemicals), 0.1 mg/ ml in separate experiments. The times of addition of inhibitor with respect to addition of the aggressor cells are mentioned in the Results section. Control wells in these inhibitor studies had inhibitor added under the same conditions as the test wells. The pH of the culture medium was varied over the range of pH 5-8 by use of 0.1 M phosphate buffer diluted in RPM1 1640 plus 20% FCS. It was not possible to measure the pH of the solutions directly in the microtest plate wells, but 5 ml tubes were set up with the same cell concentration/ml as in the microtest plates. The PH of these suspensions was measured before and after microtest plate experiments. The pH values shown in the figure are the initial pH; they changed during the course of the experiments by different amounts, 0.3 pH units or less. Aggressor and target cells were incubated together at 4°C in one study. Cell lines originally derived from embryonic skeletal muscle of C3H or BlO mice (10) were used as target cells in some experiments. Treatment of Mice with sQSr C57BL/6 J (B6) or (C57BL/6J X DBA/ZJ)Fl (BDFl) mice were injected iv with 100 PCi of 8gSr twice, at 28-day intervals. Cells were taken from such mice (and controls) one to three months after the second injection (14). Preparation
of Antiserum
DBA/ZJ mice were injected ip with lo* B6 spleen cells at two-week intervals four times. In addition, 0.2 ml of complete Freund’s adjuvant was injected ip to induce ascites (19). The serum and ascitic fluid were harvested 9 days after the last injection. Neither the serum nor the ascitic fluid was able to prevent marrow allograft rejection in rrivo. The serum was used in the in vitro cytotoxicity experiments without complement to test for its ability to prevent or “block” cytotoxic effects of nonimmune lymph node cells (LNC) .
MAYHEW
AND
1000 500
250
Lymphoid
BENNETT
125 62.5
cell / target
31.3
16
8
cell ratio
FIG. 1. Effect of metabolic inhibitors on LNC:L929 cytotoxicity. O-O Controls; A-A Treated with actinomycin D 2 pg/ml; O-O Treated with cycloheximide 10 pg/ml; m---m Incubated at 4°C with LNC. Each point represents the mean cytotoxic index from a total of 12 wells in 2 experiments.
RESULTS Metabolic Studies When metabolic activity was inhibited by cooling at 4”C, there was no measurable cytotoxicity (Fig. 1) at all LNC-target cell ratios (p > 0.5). In other experiments (data not shown) target and aggressor cells were incubated at 4°C for 24 hr, then at 37°C for another 24 hr. Cytotoxicity was observed, at LNC-target cell ratios of 250 : 1 and greater (p < O.Ol), indicating that no irreversible damage was done to the aggressor cells. Partial inhibition of DNA-directed RNA synthesis by actinomycin D did not affect cytotoxicity, whereas inhibition of protein synthesis by cycloheximide did prevent the cytotoxicity of aggressor cells (Fig. 1) at LNC-target cell ratios of 125 : 1 and greater (p < 0.01). In subsequent experiments cycloheximide was added O-5 hr after plating aggressor cells into wells containing target cells. Cycloheximide was able to suppress cytotoxicity (fi < 0.01) if added during the first two hr, but not afterwards (Fig. 2) (p > 0.05). Th ese data suggest that protein synthesis is required for a brief time in this system, Thymus cells, which are not toxic in this
I
I
ICXXI 500
Lymphoid
I
250
cell
I
I
125 62.5
I target
I
31.3
I
16
II
8
cell ratio
FIG. 2. Effect of time of addition of cycloheximide on subsequent LNC:L929 cytotoxicity. 0-O Controls (no cycloheximide added) ; O-O Cycloheximide added at 0 hr; A---A Cycloheximide addBedat 1 hr ; [7--n Cycloheximide added at 2 hr ; n - ----W Cycloheximide added at 3 hr ; A-----A Cycloheximide added at 5 hr. Each point represents the mean cytotoxic index from a total of 18 wells in 3 experiments.
CYTOTOXICITY
OF NONIMMUNE
I
I
I
LYl’dPHOID
I
I
I
CELLS
45
I
1000 500 250 125 62.5 31.3 16 8 Lymphoid cell I target cell ratio FIG. 3. Effect of Neuraminidase and Ribonuclease of LNC and TC:L929 cytotoxicity. LNC treated with a-0 LNC control; O- 0 LNC treated with neuraminidase; A-A ribonuclease; m-----B TC Control; m-----n TC treated with neuraminidase; A-----A TC treated with ribonuclease.Each point represents the mean cytotoxic index from a total of 18 wells in 3 experiments.
system (lo), remained so at 4°C or in the presence of actinomycin D or cycloheximide (p > 0.05) (data not shown). Alteration of the electrical charge of cells at their peripheries with neuraminidase or with ribonuclease did not alter the cytotoxicity of aggressor LNC (j > 0.05) (Fig. 3). These enzymes also did not alter the lack of cytotoxicity of TC (Fig. 3) (p > 0.05). Other agents tested which did not alter the cytotoxic reaction between LNC and L929 fibroblastic cells included the polycations poly-lysine (mol. wt. 2800) at 4 rg/ml (p > 0.05) and cytochrome C at 10 pg/ml (p > 0.05) (data not shown). The LNC cytotoxic reaction were sensitive to change in pH (Fig. 4). Peak activity was at pH 7 with a more rapid fall-off of cytotoxicity on the acidic than on the basic side of neutrality. Physiologic
Studies
Thymus cells are not cytotoxic in this system, and TC inhibit the restriction of proliferation of parental-strain marrow cells in irradiated Fl hybrid mice (19). Therefore, we wondered if TC would be able to inhibit the cytotoxicity of LNC. LNC and TC were mixed in ratios of 9: 1, 1: 1 and 1:9 LNC: TC and plated onto target L929 cells. The cytotoxic indices decreased as the percentage of TC increased (Fig. 5). For a given ratio of LNC: L929 cells the presence of TC decreased the observed cytotoxicity. This can be clearly seen if comparisons such
PH.
FIG. 4. Cytotoxicity of LNC against L929 cells at different pH’s. A-A LNC:L929 ratio 5OO:l; 0 -0 LNC:L929 ratio 125:l; O-O LNC:L929 ratio 31:l. Each point represents the mean cytotoxic
index
from
a total
of 12 wells
in 2 experiments.
46
MAYHEW
Lymphoid
AND
BENNETT
cell /target
cell ratio
FIG. 5. Cytotoxic effects of mixed LNC and TC on L929 cells. O-O LNC only; d----n LNC:TC, 9:1, m-----H LNC:TC, 1:l; O-----O LNC:TC, 1:9; A-----A TC only. Each point represents the mean cytotoxic index from a total of 12 wells in two experiments. The cell number used to calculate lymphoid to target cell ratio was the sum of LNC plus TC for each mixture. At a 1OOO:l lymphoid cell:target cell ratio there was (A) 1000 LNC/target cell for LNC only; (B) 900 LNC plus 100 TC/target cell for a LNC:TC 9:l (C) 500 LNC plus 500 TC/target cell for LNC:TC 1: 1; and, (D) 100 LNC plus 900 TC/target cell for LNC:TC 1:9.
as the following are made. (A) At a 500: 1 LNC only: target cell ratio the same number of LNC were present as for a 1000: 1 LNC:TC 1: 1 mixture to target cell ratio. Here the cytotoxicity of LNC only (100%) was significantly higher than for the mixture (42%) (p < 0.01). (B) At a 125: 1 LNC only: target cell ratio approximatley the same number of LNC were present as for a 1000: 1 LNC:TC 1:9 mixture to target cell ratio, In this case the cytotoxicity of LNC (58%) was significantly higher than for the mixture (14%) (p < 0.01). However, the inhibition by thymus cells of LNC cytotoxicity was certainly not complete at high aggressor cell to target cell ratios. Whereas effector cells responsible for marrow allograft rejection are radioresistant, their precursors are radiosensitive (12, 13). Sublethal irradiation (500700 R) 7-21 days before lethal irradiation (800-900 R) and marrow transplantation prevented the rejection process. Cytotoxic LNC were also found to be radioresistant in function (10). To determine if cytotoxic LNC have radiosensitive precursors, C3BFl mice were exposed to 0 R or 600 R seven days prior to plating their LNC into wells containing Lg.29 target cells. LNC from irradiated mice were much less cytotoxic than LNC from unirradiated mice (Table 1). This finding is in keeping with the concept that cytotoxic LNC in this system reflect effector cell function operative against marrow cells have radiosensitive precursors.
allografts.
It only
shows that the effector
The DBA/Z anti-C57BL/6 spleen cell serum was raised to provide an agent to inactivate cells responsible for marrow allograft rejection (20). Such an agent could be tested in vitro for its ability to prevent the cytotoxic reaction. This particular antiserum was ineffective in tivo but did have anti-C57BL activity as it lysed C57BL lymphocytes in the presence of guinea pig complement. Therefore, we used the serum without complement to test its ability to protect BlO target cells from the cytotoxic action of lymphoid cells. The antiserum was mixed with the target and aggressor cells at a final dilution of 1: 16 and was present throughout the culture period.
CYTOTOXICITY
OF
NONIMMUNE
TABLE LNC
CYTOTOXICITYOF
Cytotoxic
LNC:L929 cell ratio
0 Ra 1000: 1 500: 1 2.50:1 125:l
74.2 58.3 36.4 17.4
f 8.6 f 7.8 f 4.4 f 1.9
LYMPHOID
47
CELLS
1
FROM IRRADIATED
C3BFl
MICE Significance of difference@
index 600 R= 26.5 20.2 10.6 11.2
f 5.2 f 4.3 f 1.6 f 1.6
p < 0.01 p < 0.01 p < 0.01 0.05 > p > 0.02
a Dose of total-body x-rays 7 days before harvesting LNC (lymph node cells) ; istandard error of mean. * Significance of difference calculated using Student’s t test. Each value represents the mean cytotoxic index from a total of 12 wells in two experiments.
The antiserum protected BlO (p < 0.01) but not C3H (p > 0.05) target cells from the LNC taken from C3H, BlO, or C3BFl young adult mice (Table 2, Expt 1). In this and other experiments “syngeneic cytotoxicity” was observed, i.e., LNC were cytotoxic for fibroblasts originally taken from syngeneic embryos. The cells had been in culture for some time and apparently became susceptible to “syngeneic” LNC. It was apparent that the C3H cells were more susceptible to this cytotoxic reaction than El0 cells. However, the BlO LNC were more reactive against C3H than against BlO target cells, and C3H LNC were more cytotoxic to BlO than to C3H target cells. The C3BFl LNC were cytotoxic to both lines of target cells. Lymph node cells (p < 0.01)) but not bone marrow cells (p > 0.05) of the hypoplastic marrow, of C57BL/6 mice treated with 89Sr were cytotoxic for target C3H and BlO fibroblastic cells (Table 1, Expt. 2). Cytologically, nearly all nucleated marrow cells from mice treated with 89Sr are mature granulocytes. The DBA/Z anti-C57BL/6 spleen cell serum protected B10 cells from LNC of both normal and 8gSr-treated C57BL/6 mice. In a similar experiment, the cytotoxic effects of spleen cells (SC) and LNC from BDFl mice treated with ‘YSr were determined. Control and experimental LNC were almost equally cytotoxic (Table 2, Expt 3). The spleen cells from mice treated with 8gSr were significantly less cytotoxic than control spleen cells (0.05 > p > 0.02). This difference could be explained by a “dilution effect” due to extensive extra-myeloid myelopoiesis that occurs in spleens of such mice. It should be emphasized that the spleens of mice treated with 8gSr take over all stem cell functions (12). These results with 89Sr-treatment were surprising, since all of the preceding experiments indicated that this in vitro assay detected effector cells invoived in the resistance to marrow allografts. A second antiserum was kindly given to us by Dr. R. J. Eckner. This antiserum was produced in BALB/c mice by injecting lo8 C57BL/6 spleen cells with complete Freund’s adjuvant ip, initially. For the next 15 weeks, 108 C57BL/6 spleen cells were injected ip at weekly intervals. The serum was harvested seven days after the last injection and decomplemented (56”C, 30 min). This antiserum prevented the rejection of DBA/2 Ha marrow cells by irradiated C3BFl mice in vi710
48
MAYHEW
AND
BENNETT
(data not shown). LNC (10’) from both control and Y5--treated BDFl mice were incubated with the BALB/c anti-C57BL spleen cell serum (or normal BALB/c serum) for 30 min at 37°C in 2 ml of 1: 10 dilution of antiserum. In TABLE
2
EFFECT OF DBA/2 ANTI-CS~BL/~ SPLEEN CELL SERUM ON THE CYTOTOXIC EFFECT OF LYMPHOID CELLS FROM NORMAL MICE AND MICE TREATED WITH @SR Expt
Lymphoid Strain
1
BlO
C3H
2
3
cell donor
Pretreatmenr
-
-
Tissueb
LNC
LNC
C3BFl
-
LNC
C57BL/6
-
LNC
C57BL/6
sgSr
LNC
C57BL/6
-
BMC
C57BL/6
?Sr
BMC
BDFl BDFl BDFl BDFl
-
LNC SC LNC SC
*?Sr *?Sr
Target cell strain C3H C3H B10 BlO C3H C3H B10 B10 C3H C3H B10 B10 C3H C3H BlO B10 C3H C3H BlO BlO C3H BlO C3H BlO BlO BlO BlO B10
AntiserumC
+ + + + + + + i+ -t -
-
Cytotoxicd index
83.3 64.6 26.8 6.2 39.6 51.6 52.4 +16.1 84.3 78.1 34.6 15.8 79.0 92.0 36.3 f11.6 95.7 91.6 32.6 + 3.2 57.2 22.8 14.3 + 5.7 63.9 79.6 67.3 48.0
Q Mice were injected with 100 pCi sgSr iv at 2%day intervals and the cells were harvested 1 month later in Expt 2. In Expt 3, a third injection of 100 pCi *%r was performed 4 months after the second injection and the cells were harvested 1 month later. b LNC, lymph node cells; BMC, bone marrow cells; SC, spleen cells. c The serum at a 1: 16 dilution was added to each well at the same time as the LNC and was present throughout the culture period. No complement was present. The method of raising the antiserum is given in Materials and Methods. This antiserum did not prevent marrow allograft rejection in viva. d The standard errors of the mean cytotoxic index were lCrZO% of the mean value. The aggressor: target cell ratio was 1000: 1. Each value represents the mean cytotoxicity index from a total of 12 wells. + indicates enhancement of growth.
CYTOTOXICITY
OF NONIMMUNE
LYMPHOID
CELLS
49
-20[, , , , , , , ,] 1000
250 Lymphoid
62.5 ceil / target
16 cell ratio
FIG. 6. Effect of BhLB/c anti-CS7BL/6 spleen cell serum on cytotoxicity of BD’FI LNC against L929 cells. O-O Control LNC treated with normal BALB/c serum and compleLNC from “Sr-treated mice treated with normal BALBjc serum and comment ; H-M plement; O-O Control LNC treated with BALBjc anti-C57BL/6 spleen cell serum and complement. i-J---n LNC from Tr-treated mice treated with BALB/c anti-C57BL/6 spleen cell serum and complement. Each point represents the mean cytotoxic index from a total of 12 wells in 1 experiment.
addition, guinea pig serum (l/10 v/v) was added as a source of complement. The LNC were then washed and added to target L929 cells. LNC treated with the anti-spleen cell serum plus complement lost much of their cytotoxicity (Fig. 6) at all LNC:target cell ratios (p <0 01). However, cytotoxic indices of 50 to 60% were measured at the 500: 1 and 100: 1 ratios of control BDFl LNC to L929 cells. This result alerted us to the possibility that this in vitro system may detect more than one type of cytotoxic lymphoid cell. The BALB/c anti-C57BL/6 spleen cell serum plus complement suppressed the cytotoxic activity of LNC from 3r-treated mice to low levels (fl < 0.01) even at the 1000: 1 and 500: 1 LNC; target cell ratios (Fig. 6). In this experiment, the LNC from mice treated with 89Sr were less cytotoxic than control LNC (0.02 > p > O.Ol), but were nonetheless effective. DISCUSSION As the cytotoxic reaction between LNC and L cells is insensitive to actinomycin but sensitive to cycloheximide and cold, it can be suggested that metabolic activity, and probably protein synthesis (21) is involved in the cytotoxic action of nonimmune LNC. The results also suggest that the lack of toxicity of TC is not due to metabolically linked factors. The results from experiments where the time of addition of cycloheximide was varied can be interpreted to indicate that protein synthesis is involved in a process,
50
MAYHEW
AND
BENNETT
necessary for subsequent cytotoxicity, which occurs shortly after the aggressor cells reach the target cells at the microtest plate well surface. At present we have not investigated whether a Iymphotoxin is produced in this system but one could be produced by the cells at this time similar to that shown in the system investigated by Granger et al. (17). The data presented suggests that the pH optimum for the cytotoxic reaction is approximately 7. If there were significant deviations from neutrality, the cytoioxicity was reduced. This may be an important factor in in &YO cell-mediated cytotoxicity. There have been reports that the local environment of tumors may lymphocytes could be more acidic than that of normal tissue (22). Thus, cytotoxic be less effective against tumor cells for this reason alone. As neuraminidase and ribonuclease were without effect on the cytotoxic reaction this could indicate that the cell surface groups, sialic acids (23, 24) and RNase susceptible groups (25) respectively are not involved in the lymphocyte-target cell cytotoxicity in this system. Similarly, the positively charged polymers poly-lysine and cytochrome c also were without effect indicating that these agents which bind to cell surfaces (26) do not inhibit LNC cytotoxicity. These negative results would tend to indicate that cell surface charge is not a major factor in determining the outcome of cytotoxic reactions. The simplest interpretation of the results where the presence of TC inhibited LNC cytotoxicity is that TC can come in contact with the target cells and physically block the sites that LNC must contact to produce the cytotoxic reaction. However, the results presented in this report do not rule out direct interactions between the LNC and TC. These observations do suggest that one might be able to analyze how one type of immunocompetent cell population may regulate the function of another type. The observation of “syngeneic cytotoxicity” by LNC could be blocked by specific anti-target cell antisera (Table 2) suggests that the LNC are not “self-tolerant” when removed from the body and placed in an in vitro environment. Rat thymoantigens in vitro cytes apparently can be sensitized to “self” histocompatibility within 6 hr (27). We have recently observed that lethally irradiated rats restrict the proliferation of even syngeneic marrow stem cells for several days after transplantation (unpublished observations of P. Rodday, M. Bennett, and J. Vitale), which may or may not be relevant to these in vitro observations. It should be pointed out here that Cudkowicz’s pioneering observation that led to most studies of the immunobiology of marrow allograft rejection (12, 13) was the failure of parental-strain C57BL marrow cells to grow in lethally irradiated Fl hybrid mice (28). Two cell types from nonimmunized mice appear capable of in vitro cytotoxic reactions agianst target fibroblastic cells for the following reasons: (i) BALB/c anti-C57BL/6 spleen cell serum and complement destroyed much but not all of the cytotoxic effect of LNC ; (ii) sgSr-treatment alone had little effect on the cytotoxic potential of LNC; and (iii) BALB/c anti-C57BL/6 spleen cell serum and complement destroyed almost all of the cytotoxic effect of LNC from mice treated with 89Sr (Table 2 and Fig. 6). Since either 8gSr or BALB/c anti-C57BL/6 spleen cell serum prevents marrow allograft rejections, two different cell types may need to cooperate in v&o. This is a hypothesis that we are now testing.
CYTOTOXICITY
OF
NONIMMUNE
LYMPHOID
CELLS
51
ACKNOWLEDGMENTS We thank Dr. L. Weiss for useful discussions. We also thank J. Ciszkowski and K. Caruana for their technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Rosenau, W., and Moon, H. D., J. Nat. Cancer Inst. 27, 471, 1961. Wilson, D. B., J. Exp, Med. 122, 143, 1965. Hellstrom, I., Znt. J. Cancer 2, 65, 1967. Berke, G., Ginsberg, H., Yagil, G., and Feldman, M., Israel J. Med. Sci. 5, 135, 1969. Moller, E., Science 147, 873, 1965. Holm, G., and Perlman, P., Immunology 12, 525, 1967. Bach, F. H., In “Histocompatability Testing,” pp. 136, 320. Wilkins and Wilkins, Baltimore, 1965. 8. Bach, ‘F. H., and Hirschhorn, K., .Sem&ars Hemufol. 2, 68, 1965. 9. Jamieson, C. W., and Wallace, J. H., J. Zmmunol. 105, 7, 1970. 10. Mayhew, E., and Bennett, M., Immunology 21, 123, 1971. 11. Cudkowicz, G., and Stimpfling, J. H., Immunology 7, 291, 1964. 12. Cudkowicz, G., and Bennett, M., J. Exp. Med. 134, 83, 1971. 13. Cudkowicz, G., and Bennett, M. J. Exp. Med. 134, 1513, 1971. 14. Bennett, M., J. Zmmunol. 110, 510, 1973. 15. Lotzovo, E., and Cudkowicz, G., Fed. Proc. 32, 226, 1973. 16. Gorczynski, R. M., Miller, R. G., and Phillips, R. A., J. Exj. Med. 134, 1201, 1971. 17. Granger, G. A., and Kolb, W. P., J. Zmmzclzol. 101, 111, 1968. 18. Moore, G. E., Sandberg, A. A., and Ulrich, K., J. Nat. Cancer Znsf. 86, 405, 1966. 19. Goodman, J. W., Burch, K. T., and Bastorel, N. L., Blood 39, 850, 1972. 20. Gregory, C. J., McCulloch, E. A., and Till, J. E., Traranspluntatiolz 13, 138, 1972. 21. Tata, J. R., and Widnell, C. C., Biochem. I. 98, 604, 1966. 22. Eden, M., Hains, B., and Kahler, H., J. Natl. Cancer Inst. 16, 541, 1955. 23. Gottschalk, A., Physiol. Reu. 37, 66, 1957. 24. Weiss, L., and Cudney, T. L., Znt. J. Cancer 4, 776, 1969. 25. Mayhew, E., and Weiss, L., In “Surface Chemistry of Biological Systems” (M. Blank, Ed.), pp. 191-208. Plenum Press, New York, 1970. 26. Juliano, R., and Mayhew, E., Exp. Cell Res. 73, 3, 1972. 27. Cohen, I. R., Nature New Biol. 242, 60, 1973. 28. Cudkowicz, G., Proc. Sot. Exp. Biol. Med. 107, 968, 1961.