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Natural cell-mediated cytotoxicity in mice treated with total lymphoid irradiation (TLI)
CELLULAR
IMMUNOLOGY
70, 188-195 (1982)
Natural Cell-Mediated Cytotoxicity in Mice Treated Lymphoid Irradiation (TLI) LOLA WEISS, ELI KEDAR,
MARILYN
WEIGENSBERG,
with Total
AND SHIMON
SLAVIN’
The Immunobiology Research Laboratory, Department of Medicine A, and The Lautenberg Center for General and Tumor Immunology, Hebrew University of Jerusalem, Jerusalem, Israel Received December 9, 1981; accepted March 25. 1982 Fractionated total lymphoid irradiation (TLI) of adult (BALB/c X C57BL/6)FI mice resulted in transiently augmented natural killer (NK) and natural cytotoxic (NC) ceil activities. Thus, 1 day after completion of TLI, NK and NC activities in the spleens of treated mice were lower than controls but values increased and reached a maximum level of 23- to 190fold above control at 6 days after irradiation, returning to normal levels 9 days later. Cytotoxicity was enhanced after removal of the plastic adherent population. No cytotoxicity was observed against P 815 target cells, which are sensitive to activated macrophages but not to NK. The significance of this modulation of natural cell-mediated cytotoxicity following TLI is discussed.
INTRODUCTION Total lymphoid irradiation (TLI) is an immunomanipulative radiotherapy regimen used to induce tolerance to soluble protein antigens (1) and histoincompatible organ grafts (2-8) without subsequent development of lethal graft-versus-host disease (GVHD) (3, 5-8). The mechanism by which tolerance is achieved and the role of various cellular subsets in the generation of tolerance has not yet been elucidated. The immediate post TLI period is characterized by an almost absolute lymphocytopenia (2, 3) with profoundly depressed T and B lymphocyte functions both in vivo (3, 9) and in vitro (3). Additionally, spleen cells from TLI-treated mice are capable of potent nonspecific suppression of the mixed lymphocyte reaction (MLR) until approximately 30 days after irradiation ( 10, 11). Kinetic studies of the regenerating cellular subpopulations revealed an immediate slow linear increase in the number of circulating B cells as well as non-B, non-T (null) cells, while T cells began reappearing at only 3 weeks following radiotherapy (3). Morphologically, spleens from TLI-treated mice consist of a striking population of large mononuclear cells as can be seen by light microscopy and by scatter profiles in the fluorescence-activated cell sorter (unpublished observations). It seemed, therefore, that the altered immune status of the TLI-treated animal may be due to a combination of elimination of immunocompetent T and B lymphocytes on the one hand, and suppression of their functions by newly generated cellular subpopulations on ’ Address correspondence to: Dr. S. Slavin, Department of Medicine A, Hadassah University Hospital, Jerusalem, Israel. 188 OOOS-8749/82/090188-08SO2.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form rcscrvcd.
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the other. It has been previously suggested that natural killer (NK) cells may play an important role in the genetic resistance to allogeneic marrow grafts (12, 13). Murine NK cells, initially described by Herberman et al. (14) and Kiessling et al. (15) were found to be nonadherent, non B lymphocytes with a modest expression of Thy 1 antigen. These cells are cytotoxic for certain cultured lymphoma cells. More recently, another cell population designated natural cytotoxic (NC) cells were described by Stutman et al. (16). NC cells are cytotoxic for adherent tumor cell lines. In view of the possible role of natural killer mechanisms in allogeneic BM transplantation, we have investigated the kinetics of NK and NC activities in mice conditioned with TLI. MATERIALS
AND METHODS
Animals. Three- to six-month-old (BALB/c X C57BL/6)F1 (H-2”‘“) male mice (F,), obtained from the Weizmann Institute of Science (Rehovot, Israel), were housed in conventional animal facilities during all experiments. Total lymphoid irradiation. TLI consisted of eight daily fractions of 200 rads/ day directed at the major lymph nodes, thymus, and spleen, with shielding of the bone marrow, as previously described in detail (2, 3). Radiation was delivered by a Phillips X-ray unit (250 kV, 20 mA) at a rate of 52 rads/min using 0.2 mm Cu filter. The source to skin distance was 70 cm. Target cells for cytotoxicity assays. The RLd 1 cell line, originated from a radiation-induced BALB/c T-cell lymphoma, was kindly provided by Dr. R. Herberman, The National Cancer Institute, National Institutes of Health, Bethesda, Maryland. A Moloney virus-induced YAC lymphoma of A/J mice, kindly provided by Dr. D. Naor, The Lautenberg Center for General and Tumor Immunology, Jerusalem, Israel, was maintained by weekly intraperitoneal passagesin A/J mice. YAC targets were collected from peritoneal exudate on the day of experimentation. Adherent BALB/c methylcholanthrene-induced fibrosarcoma cells (FS) were obtained from a tumor-bearing animal and passagedin vitro. The P 8 15 target which originated from a DBA/2 mastocytoma was obtained from the Weizmann Institute of Science. RL$ 1, P 8 15, and FS cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and antibiotics and were used at log growth at the time of testing in the chromium release assays. RL6 1 and YAC lymphomas were employed to assessNK activity while FS cells were used to measure NC activity, although cytotoxic activity by NK on FS target cells was not excluded. P 815 was used as a target for activated macrophages. Preparation of efictor cells. Spleens were removed asceptically from untreated and from TLI mice and teased gently through a nylon mesh. The cells were washed twice and resuspended in RPMI-1640 containing 10% heat-inactivated FCS. Removal of plastic adherent spleen cells. Ten milliliters of 2 X lo6 spleen cells/ ml RPMI- 1640 containing 10% FCS were incubated for 1 hr on 90 X 15-mm plastic tissue culture petri dishes (Miniplast, Ein Shemer, Israel) at 37°C in 5% CO*. The nonadherent cells were harvested by gently rinsing the plates once with medium. Target cell labeling. RLb 1, P 8 15, and YAC cells were labeled in vitro with 200 &i 51Crin 0.2 ml medium containing 10% FCS and incubated with occasional shaking for at least 1 hr at 37°C in 5% Co* in air. FS cells were labeled overnight and collected by a brief trypsinization. After labeling, cells were washed three times in RPMI-1640 (5% FCS) and resuspended in RPMI-1640 ( 10% FCS).
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Cytotoxicity assay. Effector cells (0.1 ml) were added in triplicate to lo4 (0.1 ml) target cells at ratios of 20: 1 to 100:1 in Linbro 96-well microtest plates. Control wells for spontaneous release contained lo4 target cells in 0.2 ml medium. Total “Cr release was determined by adding 0.1 ml of 1% Triton X- 100 to 0.1 ml of target cells. The plates were centrifuged for 3 min at 1200 rpm and incubated at 37°C in 5% CO* in air for 4 hr (NK assay on YAC and RL$ 1 cells) or overnight (NC assay on FS cells). Cytotoxicity against P 8 15 was examined after incubations of4and18hr. Samples of 0.1 ml of the supernatants were transferred to small vials and radioactivity of the “Cr released into the supernatant was counted in a Beckman gamma counter. Percentage cytotoxicity was calculated as follows: Test release - control release 9%specific lysis = x 100. Total counts - control release Results are also expressed in lytic units. One lytic unit (LU) is defined as the number of effector cells needed to lyse 7.5-16% of lo4 target cells as calculated from the dose-response curve (17). These values were selected because they were in the linear part of the curves. LU were calculated per spleen and per lo6 spleen cells. In each experiment the ratio of LU in the TLI-treated spleens to LU in the control spleens was calculated in order to facilitate comparison between individual experiments. RESULTS NK and NC Activity in the Spleen of Mice Treated with TLl Spleen cells from 3- to 6-month-old TLI-treated (BALB/c X C57BL/6)F, mice were collected on Days l-l 5 after irradiation and their ability to kill RLB 1, YAC or FS cells was examined (Table 1). Splenocytes from unirradiated age-matched TABLE 1 Natural Killer (NK) and Natural Cytotoxic (NK) Activity of Splenocytes of TLI-Treated Mice as Compared to Untreated Controls Percentage specific lysis” Target cells
Untreated controls
RLb 1
lb
3
6
15
9.3 k 3.1’ (5.0-13.5)
0
18.1 + 3.7 ( 16.0-22.4)
19.1 f 2.0 (17.0-21.0)
10.7 f 5.3 (6.9-14.4) (2 exp.)
YAC
2.2 + 2.6 (O-5.7)
0
6.9 (1 ew.)
11.4 * 4.5 (8.4-16.6)
4.8 (1 exp.)
FS
2.0 A 0.6 (1.6-2.7)
0
8.7 (I em.)
9.1 k 5.2 (5.3-15.0)
2.9 (1 ew.)
’ Effecter/target cell ratio was 50/ 1 for all groups. Assay time was 4 hr with RL6 1 and YAC targets, and 18 hr with FS. b Days after TLI. c Except where otherwise noted, values represent the mean +_standard deviation of at least three separate experiments. Ranges are shown in parentheses.
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5
10 DAYS AFTER
1s TLI
FIG. 1. Kinetics of appearance of NK/NC activity in spleens of TLI-treated mice, expressed as lytic units (LU)/ 1O6spleen cells of TLI-treated mice as compared to untreated age-matched controls.
controls showed low to nearly undetectable cytotoxic reactivity at this age. At Day 1 after TLI, the total number of viable leukocytes in the spleen was very low (about 5% of untreated) and had no cytotoxic reactivity whatsoever against any of the target cells tested. Thereafter, the number of spleen cells as well as the cytotoxic activity gradually increased. At 3 days after irradiation, the NK assay against RL8 1 cells showed an average of 18.1% specific lysis at an effector to target ratio of 50: 1, compared with 9.3% in the unirradiated control mice. This activity was maintained at 6 days after TLI but fell to approximately the same level as control at 15 days after TLI. The same results were obtained when YAC cells were used as the NK target, i.e., chromium release was elevated at 3 and 6 days post-TLI but returned to normal levels by 15 days after TLI. Similar results were also obtained in the overnight NC assay directed against FS adherent cells. Because of the sharp decrease in spleen cell number in TLI-treated mice, these cytotoxic activities were also calculated to express lytic units (LU) per 1O6spleen cells and as LU per spleen. The maximum LU/ lo6 TLI spleen cells against YAC, RL8 1, and FS cells relative to normal F, was seen 6-7 days after irradiation (Fig. 1) with values reaching 193, 22, and 29 times greater than control, respectively. Similarly, the ratio of LU/ spleen (TLI):LU/spleen (control) (Table 2) showed values of 185 and 21 against YAC and FS, respectively, on Day 6, and 30 against RLB 1 on Day 7. By Day 15, however, no significant differences were noted between TLI and control mice. Natural Killing Activity of Nonadherent Spleen Cells When plastic adherent cells were removed from spleens of mice at 6 days after TLI (at the same time of the peak of NK activity), NK activity tested against RL$
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Natural Killer (NK) and Natural Cytotoxic (NC) Activity in TLI-Treated Mice as Compared to Untreated Normal Controls Expressed as Lytic Units (LU) per Spleen LU per spleen“ (ratio of TLI/normal) Target cells
lb
3
4
6
I
RLd 1 YAC FS
0 0 0
0.5 ND’ ND
1.7 39.2 1.8
ND 185.0 21.6
30.4 27.3 ND
‘Mean number of viable spleen cells recovered at 1, 3, 4, 6, and 7 days after TLI were 2.5, 10.0, 13.5, 18.0, and 42.0 X 106,respectively. Mean number of spleen cells recovered from untreated control was 50.0 X 106. b Days after TLI. ’ ND, not done.
1 target cells was significantly enhanced as compared to the unseparated spleen cells (Table 3). NK activity of nonadherent as compared to unseparated TLItreated spleen cells was also tested against RLB 1 and YAC target cells at 6,11, and 13 days after TLI and the results indicate that the cytotoxic effect was not due to adherent cells (data not shown). Natural Cytotoxic Activity against P 815 Target Cells Cytotoxicity against the NK-insensitive but activated macrophage-sensitive target cell P 8 15 (18) was tested at 11 and 13 days after irradiation in a 4-hr assay, and at 13 and 14 days after irradiation in an 18-hr assay. Spleen cells obtained from both TLI-treated and nontreated mice showed negligible cytotoxicity in both assays(0- 1.4% lysis at effector to target cell ratios of up to 100: 1) with no difference between them. Taken together, these results suggest that the cytotoxicity observed after TLI is effected by a nonadherent cell, and is to be distinguished from cytotoxicity by activated macrophages. TABLE 3 Natural Killer Activity of Plastic Nonadherent Splenocytes Obtained from TLI-Treated Mice as Compared to Untreated Controls Percentage specific lysis” Effector cells TLI’ TLI, plastic nonadherent’ Untreated controls Untreated controls, plastic nonadherent
50:lb 21.3 + 36.6 + 0.15 + 7.4 +
1.35” 3.4 0.1 1.1
0 RM 1 cells used as targets. ’ Effector to target cell ratio. ’ Spleen cells obtained on Day 6 post-TLI, at peak of NK activity. d Mean of two sets of different experiments k standard deviation.
25:1
12.5:l
23.5 f 3.5 30.1 + 2.8 0 2.9 -+ 0.3
16.5 k 2.1 19.1 + 2.3 0 0.5 + 0.2
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DISCUSSION There is increasing evidence that NK cells may play an important role in resistance to tumor growth and also in rejection of BM transplants (reviewed by Herberman et al., 19, 20). According to Stutman and Lattime (21), natural cellmediated cytotoxicity can be divided into at least two categories. The classical murine NK activity directed against T lymphoma-derived targets appears at approximately 3 weeks of age, peaks at 4-8 weeks, with a decline in activity beginning at lo- 12 weeks of age. NC activity directed against adherent targets derived from solid tumors is already apparent at birth and does not seem to decay with age. In fact, there may be various subpopulations of natural cell-mediated cytotoxic cells each directed at different target determinants (22, 23). The present study was undertaken in order to examine NK and NC activities after TLI and their possible roles in TLI-induced tolerance to BM allografts. Natural cell-mediated cytotoxicity can be affected by ionizing radiation, depending on the mode and dose of irradiation. Studies by Oehler and Herberman (24) have shown that NK activity in rats subjected to 500 rads whole body irradiation is only moderately if at all affected 18 hr later while 1000 rads of whole body irradiation causes a drop in NK activity. However, in both cases postirradiation induction of NK was easily accomplished by treatment with polyinosinic-polycytidilic acid suggesting a population of preNK cells resistant to X-irradiation. Split dose of total body irradiation in mice (175 rads weekly for 4 weeks) resulted in severe depression of NK cell activity (25). Spontaneous NK activity could be restored after syngeneic BM transfusion (25). In vitro irradiation of human peripheral blood leukocytes revealed a dosedependent effect on NK cell activity: loss of all NK activity after 3000 rads as opposed to enhanced activity at doses ranging from 500 to 2000 rads (26). In the present investigation, we have studied the cytotoxic ability of spleen cells from TLI-treated animals directed against YAC and RLB I lymphoid targets (NK) and against a BALB/c fibrosarcoma-derived adherent target (NC). Our results indicate that following disappearance of detectable cytotoxicity on Day 1 post-TLI, both types of activities were significantly elevated at 3 to 7 days, but returned to normal levels by 15 days postirradiation. Spleen cells from TLI-treated mice depleted of plastic adherent cells showed significantly increased NK activity at 6 days after irradiation. Additionally, no cytotoxicity was observed against P 8 15, a target cell which has been shown to be sensitive to activated macrophages but not to NK cells ( 18), thus diminishing the likelihood that macrophage-mediated cytotoxicity is involved. The transiently augmented NK seen after TLI includes activity against the in vivo carried YAC cell which is not a classically NK-sensitive target. The observed elevated activities might either be due to selective survival in the spleen of NK/NC precursor cells which then differentiate into fully mature effector cells or else due to de novo production from bone marrow-derived stem cells with rapid mobilization into the regenerating spleen tissues. Alternatively, enhanced cytotoxic activity seen up to Day 7 might also be explained by a selectively rapid generation of NK/NC cells prior to regeneration of radiation sensitive cells which may suppress NK activities (26). It is worth noting that the augmented activity after TLI occurs during a period when all other cellular immune functions tested are profoundly depressed,including cell-mediated proliferative responses to mitogens and allogeneic lymphocytes (3), killer T cell function (Slavin et al., unpublished observations), T-cell-dependent
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antibody responses(9), as well as the ability to reject a skin allograft (3). It is also interesting that during this time period an overwhelming population of non-B, nonT large mononuclear cells is apparent in the spleen of mice recovering from TLI (Slavin et al., in preparation). Such cells may be analogous to the large granular lymphocytes (LGL) recently described in human peripheral blood as the main source of NK cells (27, 28). A characteristic property of TLI treatment is the resulting ability to implant histoincompatible BM cells without subsequent GVHD or graft rejection (3-8). Two main hypotheses have been put forward to explain this phenomenon. One is that an active suppressor cell mechanism induced by TLI inhibits the alloreactivity against and by the foreign BM graft (6-8). Indeed, spleen cells isolated from TLItreated mice and TLI-conditioned BM transplanted mice are capable of profound suppression of the in vitro mixed lymphocyte reaction (10, 11). In this respect, it is interesting to note that NK cells may also act as nonspecific suppressor cells in some immune responses (20). It is thus possible that the large mononuclear cells appearing in the spleen may express both NK and suppressor activity. A second hypothesis addresses itself to the question of the specificity of tolerance observed in TLI-conditioned mice (1, 3). Elevated NK and/or NC cell activity as well as the suppressor cells generated by TLI may be responsible for elimination of antigenspecific alloreactive clones during the immediate postirradiation period. Thus, tolerance could be induced to histoincompatible grafts during the first few days following TLI but not to challenges of third party grafts at a later date. Since BM cells have been reported to be relatively NK sensitive (29), the complete absence of natural cytotoxicity 1 day following TLI, may offer an explanation for the fact that establishment of BM chimerism is most efficient 1 day post-TLI (30). We are presently involved in experiments designed to characterize the cellular subpopulations generated after TLI and their possible biological role in the induction and maintenance of the state of tolerance. ACKNOWLEDGMENTS This work was supported by NIH Grants AI- 15387, CA-303 13, the United States-Israel Science Foundation 1786/79, and by the Kroc Foundation.
Binational
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12.
Zan-Bar, I., Slavin, S., and Strober, S., J. Immunol. 121, 1400, 1978. Slavin, S., Strober, S., Fuks, Z., and Kaplan, H. S., Science 193, 1252, 1976. Slavin, S., Strober, S., Fuks, Z., and Kaplan, H. S., J. Exp. Med. 146, 34, 1978. Slavin, S., Reitz, B., Bieber, C. P., Kaplan, H. S., and Strober, S., J. Exp. Med. 147, 700, 1978. Slavin, S., Fuks, Z., Kaplan, H. S., and Strober, S., J. Exp. Med. 147, 963, 1978. Slavin, S., Gottlieb, M., Bieber, C., Hoppe, R., Kaplan, H. S., Strober, S., and Grumet, C., Transplantation 27, 139, 1979. Strober, S., Slavin, S., Gottlieb, M., Zan-Bar, I., King, D., Hoppe, R. T., Fuks, Z., Grumet, F. C., and Kaplan, H. S., Immunol. Rev. 46, 87, 1979. Slavin, S., Yatziv, S., Zan-Bar, I., Fuks, Z., Kaplan, H. S., and Strober, S., In “Immunology 1980Progress in Immunology IV” (M. Fougereau and J. Dausset, Eds.), pp. 1160-1170. Academic Press, London, 1980. Zan-Bar, I., Slavin, S., and Strober, S., Ceil. Immunol. 45, 167, 1979. Slavin, S., and Strober, S., In “T and B Lymphocytes: Recognition and Function” (F. Bach, B. Bonavida, E. S. Vitetta, and C. F. Fox, eds.), pp. 241-247. Academic Press, New York, 1979. Slavin, S., and Strober, S., J. Immunol. 123, 942, 1979. Kiessling, R., Hochman, P. S., Haller, O., Shearer, G. M., Wigzell, H., and Cudkowicz, G., Eur.
J. Immunol. 7, 655, 1977.
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Trentin, J. J., and Bennett, M., Transplant. Proc. 9, 1303, 1977. Herberman, R. B., Nunn, M. E., and Lavrin, D. H., Znt. .Z. Cancer. 15, 216, 1975. Kiessling, R., Klein, E., Pross, H., and Wigzell, H., Eur. J. Zmmunol. 5, 117, 1975. Stutman, O., Paige, C. J., and Figarella, E. F., J. Zmmunol. 121, 1819, 1978. Cerottini, J. C., and Brunner, K. T., Adv. Zmmunol. 18, 67, 1974. Roder, J. C., Lohmann-Matthes, M. L., Domzig, W., Kiessling, R., and Haller, O., Eur. J Zmmunol. 9, 283, 1979. 19. Herberman, R. B., Djeu, J. Y., Kay, H. D., Ortaldo, J. R., Riccardi, C., Bonnard, G. D., Holden, H. T., Fignani, R., Santoni, A., and Puccetti, P., Zmmunol. Rev. 44, 43, 1979. 20. Herberman, R. B., Brunda M. J., Djeu, J. Y., Domzig, W., Goldfarb, R. H., Holden, H. T., Ortaldo, J. R., Reynolds, C. W., Riccardi, C., Santoni, A., Stadler, B. M., and Timonen, T., In “NK Cells: Fundamental Aspects and Role in Cancer” (B. Serrou, C. Rosenfeld, R. B. Herberman, Eds.). Human Cancer Immunology, Vol. 6. Elsevier/North Holland, Amsterdam, 1982 (in press). 21. Stutman, O., and Lattime, E. C., Fed. Proc. 40, 2699, 1981. 22. Lopez, C., Kirkpatrick, D., Livnat, S., and Storb, R., Lancer 2, 1025, 1981. 23. Ortaldo, J. R., and Herberman, R. B., In “NK Cells: Fundamental Aspects and Role in Cancer” (B. Serrou, C. Rosenfeld, R. B. Herberman, Eds.). Human Cancer Immunology, Vol. 6. Elsevier/ North Holland, Amsterdam, 1982 (in press). 24. Oehler, J. R., and Herberman, R. B., Znt. J. Cancer 21, 221, 1978. 25. Parkinson, D. R., Brightman, R. P., and Waksal, S. D., J. Zmmunol. 126, 1460, 1981. 26. Brovall, C., and Schacter, B., J. Zmmunol. 126, 2236, 1981. 27. Timonen, T., Saksela, E., Ranki, A., and Hayry, P., Cell. Zmmunol. 48, 133, 1979. 28. Timonen, T., Ortaldo, J. R., and Herberman, R. B., J. Exp. Med. 153, 569, 1981. 29. Riccardi, C., Santoni, A., Barlozzari, T., and Herberman, R. B., Cell. Zmmunol. 60, 136, 1981. 30. Gottlieb, M., Strober, S., and Kaplan, H. S., J. Zmmunol. 123, 379, 1979.
IMMUNOLOGY
70, 188-195 (1982)
Natural Cell-Mediated Cytotoxicity in Mice Treated Lymphoid Irradiation (TLI) LOLA WEISS, ELI KEDAR,
MARILYN
WEIGENSBERG,
with Total
AND SHIMON
SLAVIN’
The Immunobiology Research Laboratory, Department of Medicine A, and The Lautenberg Center for General and Tumor Immunology, Hebrew University of Jerusalem, Jerusalem, Israel Received December 9, 1981; accepted March 25. 1982 Fractionated total lymphoid irradiation (TLI) of adult (BALB/c X C57BL/6)FI mice resulted in transiently augmented natural killer (NK) and natural cytotoxic (NC) ceil activities. Thus, 1 day after completion of TLI, NK and NC activities in the spleens of treated mice were lower than controls but values increased and reached a maximum level of 23- to 190fold above control at 6 days after irradiation, returning to normal levels 9 days later. Cytotoxicity was enhanced after removal of the plastic adherent population. No cytotoxicity was observed against P 815 target cells, which are sensitive to activated macrophages but not to NK. The significance of this modulation of natural cell-mediated cytotoxicity following TLI is discussed.
INTRODUCTION Total lymphoid irradiation (TLI) is an immunomanipulative radiotherapy regimen used to induce tolerance to soluble protein antigens (1) and histoincompatible organ grafts (2-8) without subsequent development of lethal graft-versus-host disease (GVHD) (3, 5-8). The mechanism by which tolerance is achieved and the role of various cellular subsets in the generation of tolerance has not yet been elucidated. The immediate post TLI period is characterized by an almost absolute lymphocytopenia (2, 3) with profoundly depressed T and B lymphocyte functions both in vivo (3, 9) and in vitro (3). Additionally, spleen cells from TLI-treated mice are capable of potent nonspecific suppression of the mixed lymphocyte reaction (MLR) until approximately 30 days after irradiation ( 10, 11). Kinetic studies of the regenerating cellular subpopulations revealed an immediate slow linear increase in the number of circulating B cells as well as non-B, non-T (null) cells, while T cells began reappearing at only 3 weeks following radiotherapy (3). Morphologically, spleens from TLI-treated mice consist of a striking population of large mononuclear cells as can be seen by light microscopy and by scatter profiles in the fluorescence-activated cell sorter (unpublished observations). It seemed, therefore, that the altered immune status of the TLI-treated animal may be due to a combination of elimination of immunocompetent T and B lymphocytes on the one hand, and suppression of their functions by newly generated cellular subpopulations on ’ Address correspondence to: Dr. S. Slavin, Department of Medicine A, Hadassah University Hospital, Jerusalem, Israel. 188 OOOS-8749/82/090188-08SO2.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form rcscrvcd.
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the other. It has been previously suggested that natural killer (NK) cells may play an important role in the genetic resistance to allogeneic marrow grafts (12, 13). Murine NK cells, initially described by Herberman et al. (14) and Kiessling et al. (15) were found to be nonadherent, non B lymphocytes with a modest expression of Thy 1 antigen. These cells are cytotoxic for certain cultured lymphoma cells. More recently, another cell population designated natural cytotoxic (NC) cells were described by Stutman et al. (16). NC cells are cytotoxic for adherent tumor cell lines. In view of the possible role of natural killer mechanisms in allogeneic BM transplantation, we have investigated the kinetics of NK and NC activities in mice conditioned with TLI. MATERIALS
AND METHODS
Animals. Three- to six-month-old (BALB/c X C57BL/6)F1 (H-2”‘“) male mice (F,), obtained from the Weizmann Institute of Science (Rehovot, Israel), were housed in conventional animal facilities during all experiments. Total lymphoid irradiation. TLI consisted of eight daily fractions of 200 rads/ day directed at the major lymph nodes, thymus, and spleen, with shielding of the bone marrow, as previously described in detail (2, 3). Radiation was delivered by a Phillips X-ray unit (250 kV, 20 mA) at a rate of 52 rads/min using 0.2 mm Cu filter. The source to skin distance was 70 cm. Target cells for cytotoxicity assays. The RLd 1 cell line, originated from a radiation-induced BALB/c T-cell lymphoma, was kindly provided by Dr. R. Herberman, The National Cancer Institute, National Institutes of Health, Bethesda, Maryland. A Moloney virus-induced YAC lymphoma of A/J mice, kindly provided by Dr. D. Naor, The Lautenberg Center for General and Tumor Immunology, Jerusalem, Israel, was maintained by weekly intraperitoneal passagesin A/J mice. YAC targets were collected from peritoneal exudate on the day of experimentation. Adherent BALB/c methylcholanthrene-induced fibrosarcoma cells (FS) were obtained from a tumor-bearing animal and passagedin vitro. The P 8 15 target which originated from a DBA/2 mastocytoma was obtained from the Weizmann Institute of Science. RL$ 1, P 8 15, and FS cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and antibiotics and were used at log growth at the time of testing in the chromium release assays. RL6 1 and YAC lymphomas were employed to assessNK activity while FS cells were used to measure NC activity, although cytotoxic activity by NK on FS target cells was not excluded. P 815 was used as a target for activated macrophages. Preparation of efictor cells. Spleens were removed asceptically from untreated and from TLI mice and teased gently through a nylon mesh. The cells were washed twice and resuspended in RPMI-1640 containing 10% heat-inactivated FCS. Removal of plastic adherent spleen cells. Ten milliliters of 2 X lo6 spleen cells/ ml RPMI- 1640 containing 10% FCS were incubated for 1 hr on 90 X 15-mm plastic tissue culture petri dishes (Miniplast, Ein Shemer, Israel) at 37°C in 5% CO*. The nonadherent cells were harvested by gently rinsing the plates once with medium. Target cell labeling. RLb 1, P 8 15, and YAC cells were labeled in vitro with 200 &i 51Crin 0.2 ml medium containing 10% FCS and incubated with occasional shaking for at least 1 hr at 37°C in 5% Co* in air. FS cells were labeled overnight and collected by a brief trypsinization. After labeling, cells were washed three times in RPMI-1640 (5% FCS) and resuspended in RPMI-1640 ( 10% FCS).
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Cytotoxicity assay. Effector cells (0.1 ml) were added in triplicate to lo4 (0.1 ml) target cells at ratios of 20: 1 to 100:1 in Linbro 96-well microtest plates. Control wells for spontaneous release contained lo4 target cells in 0.2 ml medium. Total “Cr release was determined by adding 0.1 ml of 1% Triton X- 100 to 0.1 ml of target cells. The plates were centrifuged for 3 min at 1200 rpm and incubated at 37°C in 5% CO* in air for 4 hr (NK assay on YAC and RL$ 1 cells) or overnight (NC assay on FS cells). Cytotoxicity against P 8 15 was examined after incubations of4and18hr. Samples of 0.1 ml of the supernatants were transferred to small vials and radioactivity of the “Cr released into the supernatant was counted in a Beckman gamma counter. Percentage cytotoxicity was calculated as follows: Test release - control release 9%specific lysis = x 100. Total counts - control release Results are also expressed in lytic units. One lytic unit (LU) is defined as the number of effector cells needed to lyse 7.5-16% of lo4 target cells as calculated from the dose-response curve (17). These values were selected because they were in the linear part of the curves. LU were calculated per spleen and per lo6 spleen cells. In each experiment the ratio of LU in the TLI-treated spleens to LU in the control spleens was calculated in order to facilitate comparison between individual experiments. RESULTS NK and NC Activity in the Spleen of Mice Treated with TLl Spleen cells from 3- to 6-month-old TLI-treated (BALB/c X C57BL/6)F, mice were collected on Days l-l 5 after irradiation and their ability to kill RLB 1, YAC or FS cells was examined (Table 1). Splenocytes from unirradiated age-matched TABLE 1 Natural Killer (NK) and Natural Cytotoxic (NK) Activity of Splenocytes of TLI-Treated Mice as Compared to Untreated Controls Percentage specific lysis” Target cells
Untreated controls
RLb 1
lb
3
6
15
9.3 k 3.1’ (5.0-13.5)
0
18.1 + 3.7 ( 16.0-22.4)
19.1 f 2.0 (17.0-21.0)
10.7 f 5.3 (6.9-14.4) (2 exp.)
YAC
2.2 + 2.6 (O-5.7)
0
6.9 (1 ew.)
11.4 * 4.5 (8.4-16.6)
4.8 (1 exp.)
FS
2.0 A 0.6 (1.6-2.7)
0
8.7 (I em.)
9.1 k 5.2 (5.3-15.0)
2.9 (1 ew.)
’ Effecter/target cell ratio was 50/ 1 for all groups. Assay time was 4 hr with RL6 1 and YAC targets, and 18 hr with FS. b Days after TLI. c Except where otherwise noted, values represent the mean +_standard deviation of at least three separate experiments. Ranges are shown in parentheses.
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5
10 DAYS AFTER
1s TLI
FIG. 1. Kinetics of appearance of NK/NC activity in spleens of TLI-treated mice, expressed as lytic units (LU)/ 1O6spleen cells of TLI-treated mice as compared to untreated age-matched controls.
controls showed low to nearly undetectable cytotoxic reactivity at this age. At Day 1 after TLI, the total number of viable leukocytes in the spleen was very low (about 5% of untreated) and had no cytotoxic reactivity whatsoever against any of the target cells tested. Thereafter, the number of spleen cells as well as the cytotoxic activity gradually increased. At 3 days after irradiation, the NK assay against RL8 1 cells showed an average of 18.1% specific lysis at an effector to target ratio of 50: 1, compared with 9.3% in the unirradiated control mice. This activity was maintained at 6 days after TLI but fell to approximately the same level as control at 15 days after TLI. The same results were obtained when YAC cells were used as the NK target, i.e., chromium release was elevated at 3 and 6 days post-TLI but returned to normal levels by 15 days after TLI. Similar results were also obtained in the overnight NC assay directed against FS adherent cells. Because of the sharp decrease in spleen cell number in TLI-treated mice, these cytotoxic activities were also calculated to express lytic units (LU) per 1O6spleen cells and as LU per spleen. The maximum LU/ lo6 TLI spleen cells against YAC, RL8 1, and FS cells relative to normal F, was seen 6-7 days after irradiation (Fig. 1) with values reaching 193, 22, and 29 times greater than control, respectively. Similarly, the ratio of LU/ spleen (TLI):LU/spleen (control) (Table 2) showed values of 185 and 21 against YAC and FS, respectively, on Day 6, and 30 against RLB 1 on Day 7. By Day 15, however, no significant differences were noted between TLI and control mice. Natural Killing Activity of Nonadherent Spleen Cells When plastic adherent cells were removed from spleens of mice at 6 days after TLI (at the same time of the peak of NK activity), NK activity tested against RL$
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Natural Killer (NK) and Natural Cytotoxic (NC) Activity in TLI-Treated Mice as Compared to Untreated Normal Controls Expressed as Lytic Units (LU) per Spleen LU per spleen“ (ratio of TLI/normal) Target cells
lb
3
4
6
I
RLd 1 YAC FS
0 0 0
0.5 ND’ ND
1.7 39.2 1.8
ND 185.0 21.6
30.4 27.3 ND
‘Mean number of viable spleen cells recovered at 1, 3, 4, 6, and 7 days after TLI were 2.5, 10.0, 13.5, 18.0, and 42.0 X 106,respectively. Mean number of spleen cells recovered from untreated control was 50.0 X 106. b Days after TLI. ’ ND, not done.
1 target cells was significantly enhanced as compared to the unseparated spleen cells (Table 3). NK activity of nonadherent as compared to unseparated TLItreated spleen cells was also tested against RLB 1 and YAC target cells at 6,11, and 13 days after TLI and the results indicate that the cytotoxic effect was not due to adherent cells (data not shown). Natural Cytotoxic Activity against P 815 Target Cells Cytotoxicity against the NK-insensitive but activated macrophage-sensitive target cell P 8 15 (18) was tested at 11 and 13 days after irradiation in a 4-hr assay, and at 13 and 14 days after irradiation in an 18-hr assay. Spleen cells obtained from both TLI-treated and nontreated mice showed negligible cytotoxicity in both assays(0- 1.4% lysis at effector to target cell ratios of up to 100: 1) with no difference between them. Taken together, these results suggest that the cytotoxicity observed after TLI is effected by a nonadherent cell, and is to be distinguished from cytotoxicity by activated macrophages. TABLE 3 Natural Killer Activity of Plastic Nonadherent Splenocytes Obtained from TLI-Treated Mice as Compared to Untreated Controls Percentage specific lysis” Effector cells TLI’ TLI, plastic nonadherent’ Untreated controls Untreated controls, plastic nonadherent
50:lb 21.3 + 36.6 + 0.15 + 7.4 +
1.35” 3.4 0.1 1.1
0 RM 1 cells used as targets. ’ Effector to target cell ratio. ’ Spleen cells obtained on Day 6 post-TLI, at peak of NK activity. d Mean of two sets of different experiments k standard deviation.
25:1
12.5:l
23.5 f 3.5 30.1 + 2.8 0 2.9 -+ 0.3
16.5 k 2.1 19.1 + 2.3 0 0.5 + 0.2
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DISCUSSION There is increasing evidence that NK cells may play an important role in resistance to tumor growth and also in rejection of BM transplants (reviewed by Herberman et al., 19, 20). According to Stutman and Lattime (21), natural cellmediated cytotoxicity can be divided into at least two categories. The classical murine NK activity directed against T lymphoma-derived targets appears at approximately 3 weeks of age, peaks at 4-8 weeks, with a decline in activity beginning at lo- 12 weeks of age. NC activity directed against adherent targets derived from solid tumors is already apparent at birth and does not seem to decay with age. In fact, there may be various subpopulations of natural cell-mediated cytotoxic cells each directed at different target determinants (22, 23). The present study was undertaken in order to examine NK and NC activities after TLI and their possible roles in TLI-induced tolerance to BM allografts. Natural cell-mediated cytotoxicity can be affected by ionizing radiation, depending on the mode and dose of irradiation. Studies by Oehler and Herberman (24) have shown that NK activity in rats subjected to 500 rads whole body irradiation is only moderately if at all affected 18 hr later while 1000 rads of whole body irradiation causes a drop in NK activity. However, in both cases postirradiation induction of NK was easily accomplished by treatment with polyinosinic-polycytidilic acid suggesting a population of preNK cells resistant to X-irradiation. Split dose of total body irradiation in mice (175 rads weekly for 4 weeks) resulted in severe depression of NK cell activity (25). Spontaneous NK activity could be restored after syngeneic BM transfusion (25). In vitro irradiation of human peripheral blood leukocytes revealed a dosedependent effect on NK cell activity: loss of all NK activity after 3000 rads as opposed to enhanced activity at doses ranging from 500 to 2000 rads (26). In the present investigation, we have studied the cytotoxic ability of spleen cells from TLI-treated animals directed against YAC and RLB I lymphoid targets (NK) and against a BALB/c fibrosarcoma-derived adherent target (NC). Our results indicate that following disappearance of detectable cytotoxicity on Day 1 post-TLI, both types of activities were significantly elevated at 3 to 7 days, but returned to normal levels by 15 days postirradiation. Spleen cells from TLI-treated mice depleted of plastic adherent cells showed significantly increased NK activity at 6 days after irradiation. Additionally, no cytotoxicity was observed against P 8 15, a target cell which has been shown to be sensitive to activated macrophages but not to NK cells ( 18), thus diminishing the likelihood that macrophage-mediated cytotoxicity is involved. The transiently augmented NK seen after TLI includes activity against the in vivo carried YAC cell which is not a classically NK-sensitive target. The observed elevated activities might either be due to selective survival in the spleen of NK/NC precursor cells which then differentiate into fully mature effector cells or else due to de novo production from bone marrow-derived stem cells with rapid mobilization into the regenerating spleen tissues. Alternatively, enhanced cytotoxic activity seen up to Day 7 might also be explained by a selectively rapid generation of NK/NC cells prior to regeneration of radiation sensitive cells which may suppress NK activities (26). It is worth noting that the augmented activity after TLI occurs during a period when all other cellular immune functions tested are profoundly depressed,including cell-mediated proliferative responses to mitogens and allogeneic lymphocytes (3), killer T cell function (Slavin et al., unpublished observations), T-cell-dependent
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antibody responses(9), as well as the ability to reject a skin allograft (3). It is also interesting that during this time period an overwhelming population of non-B, nonT large mononuclear cells is apparent in the spleen of mice recovering from TLI (Slavin et al., in preparation). Such cells may be analogous to the large granular lymphocytes (LGL) recently described in human peripheral blood as the main source of NK cells (27, 28). A characteristic property of TLI treatment is the resulting ability to implant histoincompatible BM cells without subsequent GVHD or graft rejection (3-8). Two main hypotheses have been put forward to explain this phenomenon. One is that an active suppressor cell mechanism induced by TLI inhibits the alloreactivity against and by the foreign BM graft (6-8). Indeed, spleen cells isolated from TLItreated mice and TLI-conditioned BM transplanted mice are capable of profound suppression of the in vitro mixed lymphocyte reaction (10, 11). In this respect, it is interesting to note that NK cells may also act as nonspecific suppressor cells in some immune responses (20). It is thus possible that the large mononuclear cells appearing in the spleen may express both NK and suppressor activity. A second hypothesis addresses itself to the question of the specificity of tolerance observed in TLI-conditioned mice (1, 3). Elevated NK and/or NC cell activity as well as the suppressor cells generated by TLI may be responsible for elimination of antigenspecific alloreactive clones during the immediate postirradiation period. Thus, tolerance could be induced to histoincompatible grafts during the first few days following TLI but not to challenges of third party grafts at a later date. Since BM cells have been reported to be relatively NK sensitive (29), the complete absence of natural cytotoxicity 1 day following TLI, may offer an explanation for the fact that establishment of BM chimerism is most efficient 1 day post-TLI (30). We are presently involved in experiments designed to characterize the cellular subpopulations generated after TLI and their possible biological role in the induction and maintenance of the state of tolerance. ACKNOWLEDGMENTS This work was supported by NIH Grants AI- 15387, CA-303 13, the United States-Israel Science Foundation 1786/79, and by the Kroc Foundation.
Binational
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