CELLULAR
IMMUNOLOGY
Natural
43, 185-191 (1979)
SHORT COMMUNICATIONS Killer Cell Activity in Reticulum Cell Sarcomas (RCS) of SJL/J Mice1,2 KAREN L. FITZGERALD~ANDNICHOLAS
Department
M. PONZIO
of Microbiology-Immunology and the Cancer Center Northwestern and Dental Schools, Chicago, Illinois 60611
University
Medical
Received August 23, 1978
Two transplantable reticulum cell sarcomas (RCS) of SJL mice expressed marked levels of natural killer (NK) activity when tested against susceptible Yr-labeled tumor targets. In contrast, normal SJL lymph node and spleen cells demonstrated low levels of NK activity. Neither depletion of macrophages nor pretreatment with anti-Thy-l.2 sera and complement reduced the capacity of RCS cells to express NK activity. Systemic injection of irradiated RCS cells into SJL mice induced a transient increase in NK activity at 3 and 7 days after injection. However, the NK activity observed in recipients of irradiated RCS cells never reached levels comparable to those of control mice injected with viable tumor cells. These data suggest that the transplantable reticulum cell sarcomas of SJL mice may represent a tumor of natural killer cells and thus provide an enriched source of these effecters that may be useful for further characterization of natural cytotoxicity.
INTRODUCTION The expression of natural cell-mediated cytotoxicity against tumor cells has been well documented in mice (l-4), rats (5-7), and man (8- 10). Cells that mediate this natural tumor immunity differ from other effecters of cytoxicity in that they do not appear to require prior sensitization to, or antibody specific for, antigens expressed on the tumor cell target. Hence, these effecters have been termed natural killer (NK)4 cells (11). Various factors influence the expression of NK activity in mice, including age and genetic background (3, 4). Although primarily detected on the basis of cytotoxic assays, there is a high correlation between expression of NK activity in vitro and resistance to growth of NK sensitive tumor cells in vivo (12, 13). Considerable effort has been directed toward characterizing murine NK cells. NK activity is detectable in most lymphoid tissues (except thymus) with particularly high activity in peripheral lymph nodes, spleen, and peripheral blood (3,4). Based upon the presence of high NK activity in congenitally athymic “nude” mice and upon a lack of significant reduction of activity after pretreatment of ’ Supported in part by USPHS Grant CA22544 and Grant 78-6 from the American Cancer Society, Illinois Division. ’ Submitted in partial fulfillment for the Doctor of Philosophy degree from the Department of Microbiology-Immunology of Northwestern University Medical School. ” Supported in part by a fellowship from the Chicago Baseball Cancer Charities. 4 Abbreviations used: NK, natural killer: C, complement; LN, lymph node; RCS, reticulum cell sarcoma. 185
0008-8749/79/030185-07$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
186
SHORT
COMMUNICATIONS
effector cells with anti-Thy-l sera and complement (11, 14- 16), it was originally thought that murine NK cells were non-T in nature. More recent evidence (17, 18), however, suggests that NK cells express a low, but detectable, amount of Thy-l marker and should be categorized as a pre-T cell. Thus, NK cells in nude mice are also susceptible to pretreatment with anti-Thy-l sera and complement under optimal conditions (18). Further characterization of murine NK cells has also revealed the absence of surface Ig and the presence of F, receptors (11, 15, 19). Since NK cells appear in rather low frequency, even after augmentation with different stimuli, the majority of investigations concerning their nature have been performed by deletion of NK cells using various separation methods. Isolation of lymphoid populations that are enriched for NK effector cells is less well documented. The present brief communication provides evidence that the reticulum cell sarcomas (RCS) that occur with high incidence in SJL mice may represent a tumor of NK cells and thus provide a readily available enriched source of NK cells for further characterization. MATERIALS
AND METHODS
Mice and tumors. Female and male SJL/J, and female CBA/J, A/J, and BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, Me). The RCS tumors used in these studies were derived from reticulum cell sarcomas (RCS) that arose spontaneously in SJL mice and were established as RCS lines by serial iv transfer of 10’ tumorous lymph node cells at regular intervals (20-22). The NK susceptible target cells used in the cytotoxic assay included YAC-292 (an A-strain Moloney virus-induced lymphoma), RL 6-l (a BALB/c radiation-induced leukemia), and RBL-5 (a Rauscher virus-induced leukemia of C57B1/6 origin). All cell lines were maintained in vitro in RPM1 1640 + 10% fetal calf serum (FCS) (GIBCO, Grand Island, N.Y.). NK cytotoxic assay. One hundred microcuries of 51Cr (as sodium chromate) (Amersham, Arlington Heights, Ill.) was added to 5- 10 x lo6 target cells in 1 ml of RPM1 1640 + 10% FCS (GIBCO) and incubated at 37°C in an atmosphere of 5% CO, and 95% air for 1 hr. Following incubation, the labeled cells were washed and resuspended to a concentration of 4 x 105/ml. Effector cells were taken from the lymph nodes and spleen of tumor-bearing SJL mice or from normal donors. Effector cells were adjusted to various concentrations and added to fixed number of 51Cr-labeled target cells in order to test different effectorltarget cell ratios. One hundred microliters of effector cells and 100 ~1 of target cells were incubated in Linbro microtest plates (No. 76-11 l-05, Hamden, Conn.) at 37°C in 5% COz and 95% air. Two time periods (4 and 16 hr) were routinely tested with all effecter/target cell concentrations, in order to compare the different time intervals for optimal Wr release. The Titer-tech Supematant Collection System (Flow Laboratories, Rockville, Md.) was used to harvest the supematants from the microtiter wells. Each sample was counted in a gamma counter (Beckman Instruments, Fullerton, Calif.) and the percentage specific 51Cr release was calculated according to the following formula: counts per minute (experimental - spontaneous release) x loo Percentage 51Cr release = counts per minute (maximum release -spontaneous release)
187
SHORT COMMUNICATIONS
Maximum release (~80%) was determined by incubating 51Cr-labeled target cells in 1% NP40 (Bethesda Research Laboratories, Rockville, Md.) and spontaneous release (5-30%) was measured following incubation of labeled target cells either alone or in the presence of unlabeled target cells or normal thymus cells to compensate for cell density. Anti-Thy-l.2 serum, prepared by immunization of (A/Thy Antisera. 1.1 x AKR/H-2b) F, mice with the A strain leukemia ASL-1, (23) was a generous gift from Dr. G. J. Thorbecke (N. Y. U. Medical Center). Rabbit serum, agarose absorbed and selected for low toxicity against mouse lymphocytes, was used as a source of complement. Irradiation. In some experiments RCS cells were given 10,000 R y irradiation using a Philips Cobalt 60 Unit at a central dose rate of 140 rad/min. RESULTS Tables 1 and 2 provide a summary of experiments that demonstrate NK activity of RCS cells against two susceptible tumor target cell lines (RLcl-1, Table 2 and YAC-292, Table 1). Mean percentage 51Cr release, as well as the range of TABLE I Presence of NK Activity against YAC Target Cells in Lymphoid Tissues of SJL Mice Injected with Transplantable RCS Tumor Cells “‘0 release from YAC target cells in (%)’ 4 Hours Strain
Tissue”
SJL
RCS-5 LN
SJL
RCS-X LN
SJL
Normal LN
SJL
Normal spleen
CBA
Normal spleen
A/J
Normal spleen
E/T ratio* 100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 100 50 25
16 Hours
Mean
Range
Mean
Range
24 19 16 24 28 23 2
19-28 11-32 9-26 IO-38 13-37 12-35 o-3 o-4 -0.8-4 o-7 -3-6 -14-13 17-70 11-64 IO-23 d 2-9 o-4
56 56 71 47 66 73 2 3 3 2 9 6 4-l 52 50 4 9 11
51-60 40-83 62-77 20-74 26-86 36-85 o-4 - 15- 10 -16-12 o-3 - 16-20 -11-18 22-53 22-72 20-72 ,l -6-29 -5-22
0.5 4 2 4 40 39 33 1 4 3
a RCS effector cells were taken from SJL donors injected 7-10 days previously iv with IO7viable RCS tumor cells. Normal effector cells were derived from 7-g-week-old donors of the indicated strain. b Effecter/target cell ratios: 100 ~1 of Wr-labeled target cells (4 x 105/ml) were mixed with 100 ~1 of effector cells at concentrations adjusted to give the indicated ratios. e Data are calculated from a minimum of five separate experiments. Mean maximum release: 4 hr = 88%, 16 hr = 88%; mean spontaneous release: 4 hr = 14%, 16 hr = 33%. d Data from a single experiment.
188
SHORT COMMUNICATIONS TABLE 2 Presence of NK Activity against RLd-1 Target Cells in Lymphoid Tissues of SJL Mice Injected with Transplantable RCS Tumor Cells SLCrrelease from RLd-1 target cells in (%)’ 4 Hours
Strain
Tissues”
SJL
RCS-5 LN
SJL
RCS-X LN
SJL
Normal LN
SJL
Normal spleen
CBA
Normal spleen
A/J
Normal spleen
16 Hours
E/T ratio*
Mean
Range
Mean
Range
100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 100 50 25
20 20 12 21 25 17 0 1 2 1 3 4 38 27 20 -5 -1 -1
14-26 9-27 9-16 3-38 10-35 lo-24 d -16-10 -15-12 o-1 -14-13 -11-15 17-55 9-53 4-36 d -4-2 -2-4
49 59 66 45 70 76 2 5 4 14 3 5 70 44 43 -1 9 12
33-65 18-83 31-84 20-69 69-87 75-86 d -1-10 -1-10 8-20 -5-18 -1-7 40-92 22-81 13-68 d l-21 4-19
a As in Table 1. b As in Table 1. c Mean maximum release: 4 hr = 88%, 16 hr = 88%; mean spontaneous release: 4 hr = 16%, 16 hr = 32%. d Data from a single experiment.
percentage release, is given for both the 4- and the 16-hr assays. Effector cells, taken from the grossly enlarged lymph nodes of tumor-bearing SJL donors, caused significant lysis of these two NK susceptible target cells at 4 hr, and optimal cytotoxicity after 16hr of incubation. Similar results were also obtained with a third target cell, RBL-5 (data not shown). The percentage 51Crrelease obtained in the 4-hr assay using RCS lymphoid cell effecters is comparable, at all ratios tested, to the levels observed with effector cells from a high NK strain, CBA/J. With the longer incubation time, however, RCS tumor cells demonstrated greater cytotoxic activity than CBA effector cells. This was even more apparent at the lower effector to target cell ratios. The cytotoxicity obtained with a low NK strain, A/J, is shown for comparison. As has been demonstrated by others (H.T. Holden and R.B. Herberman, personal communication), normal SJL lymphoid cells fail to elicit significant cytotoxic potential against the target cells used in these studies. Thus, SJL mice are classified as a low NK strain. Two possible mechanisms might explain the observed results: (i) RCS tumor cells themselves mediate the cytotoxic activity observed, or (ii) RCS cells act as a powerful stimulus to augment NK activity in the low responder SJL recipient. In view of the marked proliferative response induced by RCS cells in SJL T cells (22, 24, 25) and the known ability of activated T cells to recruit the proliferation of bystander lymphocytes (26), the latter possibility warrants serious consideration.
189
SHORT COMMUNICATIONS TABLE 3 NK Activity against YAC Target Cells in LN Cells of Normal SJL Mice Injected with y-Irradiated RCS Cells” Wr release with LN cells removed after (%)b Recipient
RCS injection
3 Days
7 Days
11 Days
SJL/J SJL/J SJL/J CBA/J spleen
None RCS (10,000 R) Viable RCS None
6.4 21.3 53.2 32.6
11.8 24.8 47.7 48.2
15.6 9.5 57.6 79.2
” Normal SJL mice were injected iv with 10’ irradiated (10,ooOR) or viable RCS cells and LN cells were assayed for NK activity at the indicated times after injection. b Effector cells were incubated for 16 hr with 5LCr-labeled YAC target cells at a ratio of 50: 1.
Therefore, experiments designed to distinguish between these two alternatives were performed. SJL recipients were injected iv with 10’ y-irradiated (10,000 R) RCS cells and the lymph nodes and spleens of these recipient mice were tested for NK activity at various times after injection. SJL mice that received a similar number of viable RCS cells were tested in parallel for comparison. The results presented in Table 3 demonstrate a two- to threefold increase in NK activity in LN cells taken at early times after injection of irradiated RCS cells (Days 3 and 7) when compared to noninjected control SJL mice. In contrast, 11 days after injection of irradiated RCS cells there was no increase over noninjected SJL controls. At no time interval tested, however, did the levels of NK activity in LN taken from donors injected with irradiated RCS cells attain the degree of activity observed following transfer of viable RCS tumor cells. While these data suggest that irradiated RCS cells can induce precursors to express NK activity, it is possible that the injected, irradiated RCS cells are responsible for the observed activity. Indeed, preliminary data suggest a significant radioresistance of the NK activity present in the irradiated tumor cell preparation. (Fitzgerald and Ponzio, unpublished observations). In order to further characterize the cell(s) responsible for the NK activity in the RCS cell preparation, LN cells from SJL mice bearing the transplantable lymphoma, RCS-5, were treated with anti-Thy-l.2 or normal mouse serum and rabbit C prior to their use as effecters against 51Cr-labeled target cells. As shown in Table 4, no significant reduction in lytic activity was apparent following such pretreatment. Similar treatment of normal CBA/J spleen cells yielded identical results. The same concentrations of anti-Thy- 1.2 and C, however, were sufficient to abolish the cytotoxic activity of alloantigen sensitized killer T cells in a similar 51Cr-release assay (data not shown). DISCUSSION The results presented in Tables 1 and 2 provide evidence for NK activity in lymphoid tissues of RCS-bearing SJL donors. That RCS cells may represent a tumor of NK cells is suggested by the following observations. (i) Lymphoid cells from normal SJL donors elicit meager NK cytotoxic responses, even following attempts to augment NK activity with known inducers, such as BCG, C. parvum, or Poly 1:C (H. T. Holden and R. B. Herberman, personal communication).
190
SHORT COMMUNICATIONS TABLE 4 Effect of Anti-Thy-l.2
Effector cells
+ C Pretreatment on NK Activity in RCS Lymphoid Cells”
Pretreatment’
Wr release on YAC targets (%I*
RCS-5 LN
Anti-Thy-l.2 NMS+C None
+ C
80.8 86.2 73.0
CBA/J spleen
Anti-Thy-l.2 NMS+C None
+ C
41.7 30.1 28.5
a Cells were pretreated with anti-Thy-l.2 serum (1: 100) or normal mouse serum (NMS) (1: 100) and rabbit complement (C) (1: 12) prior to use as effecters. * Pretreated effector cells were incubated for 16hr with Wr-labeled YAC target cells at a ratio of 50: 1.
(ii) The observed cytotoxic activity is not mediated by killer T cells, since pretreatment of RCS-containing lymphoid cells with anti-Thy-l.2 sera and complement, at concentrations sufficient to abolish cytotoxic activity of sensitized T cells (25), did not diminish NK activity. Although RCS cells do not appear to express amounts of Thy-l .2 antigen necessary for lysis with anti-Thy-l .2 serum and complement (24), this does not exclude the possibility that RCS cells may express extremely low levels of this marker, as has recently been observed on NK cells present in normal lymphoid tissues of high NK strains (17, 18). Furthermore, RCS tumor cells do not elicit cytotoxic T-cell responses in SJL lymphoid cells, even after repeated immunizations in vivo or after primary sensitization in vitro (25). Consequently, it does not appear that the observed results are due to a nonspecific activation of cytotoxic effector T-cell precursors by RCS stimulation. (iii) Macrophages are not responsible for the NK activity in RCS lymphoid cell effector preparations, since removal of these cells by sequential treatments based on adherence and on phagocytosis of carbonyl iron did not diminish the cytotoxic potential of RCS cell preparations (Fitzgerald and Ponzio, unpublished observations) . Similar cytotoxic activity of RCS tumors against another NK-susceptible target cell, RBL-3, has also been reported by Chang (27). Therefore, the presence of NK activity in RCS-containing lymphoid cell preparations appears established. The effector cell within the RCS tumors that mediates the cytodestruction of NK-susceptible targets has not, as yet, been clearly identified. It is possible that the RCS tumor cell itself may represent either neoplastic expansion of NK effector cells or an entirely new class of cytotoxic effecters. On the other hand, RCS cells can induce marked proliferation of SJL T cells (22,24,25) and there is evidence (28) that RCS cells act as a polyclonal activator of Ly 1+, 2,3- T cells (29). Since NK cells appear to belong in the T-cell lineage (17, 18), it is conceivable that NK precursors may be included among the T cells activated via RCS stimulation. Alternatively, since interferon has been shown to play a significant role in the expression of NK cytotoxicity (30-32) the possibility that RCS cells stimulate NK cells via interferon production (either directly or indirectly) cannot be excluded. Attempts are in progress to define more precisely the nature of the effector cell and mechanism(s) of cytotoxicity apparent in RCS tumors. Thus, the reticulum
SHORT COMMUNICATIONS
191
cell sarcomas of SJL mice may provide a valuable model for further characterization of natural cytotoxicity. ACKNOWLEDGMENTS The authors wish to thank Dr. Myron Essex (Massachusetts General Hospital, Boston) for providing the YAC-292 cell line, Dr. Ronald B. Herberman (Laboratory of Immunodiagnosis, National Cancer Institute) for the generous gift of RLd-1 and RBL-5 tumor cells, and Dr. G. Jeanette Thorbecke (N. Y. U. Medical Center) for stimulating discussion and review of this manuscript. The provision of irradiation facilities by Dr. Pat Johnson and the Radiation Therapy Department, Northwestern Memorial Hospital, is gratefully acknowledged.
REFERENCES 1. Herberman, R. B., Nunn, M. E., Lavrin, D. H., and Asofsky, R., J. Nat. Cancer Inst. 51, 1509, 1973. 2. Herberman, R. B., Ting, C. C., Kirchner, H., Holden, H., Glasser, M., Bonnard, G. D., and Lavrin, D. H., In “Progress in Immunology” (L. Brent and J. Holborow, Eds.), Vol. II, pp. 285-295. North-Holland, Amsterdam, 1974. 3. Herberman, R. B., Nunn, M. E., and Lavrin, D. H., Inf. J. Cancer 16, 216, 1975. 4. Kiessling, R., Klein, E., and Wigzell, H., Eur. J. Immunol. 5, 112, 1975. 5. Holterman, 0. A., Klein, E., and Casale, G. P., Cell. Immunol. 9, 339, 1973. 6. Nunn, M. E., Djeu, J. Y., Glasser, M., Lavrin, D. H., and Herberman, R. B., J. Nat. Cancer Inst. 56, 393, 1976. 7. Shellam, G. R., and Hogg, N., Int. J. Cancer 19, 212, 1977. 8. Oldham, R. K., Siwarski, D., McCoy, J. L., Plata, E. J., and Herberman, R. B., Nat. Cancer Inst. Monograph
37, 49, 1973.
9. Takasugi, M., Micky, M. R., and Terasaki, P. I., Cancer Res. 33, 2898, 1973. 10. Rosenberg, E. B., McCoy, J. L., Green, S. S., Donnelly, F. C., Siwarski, D. F., Levine, P. H., and Herberman, R. B., J. Nat. Cancer Inst. 52, 345, 1974. 11. Kiessling, R., Klein, E., Pross, H., and Wigzell, H. Eur. J. fmmunol. 5, 117, 1975. 12. Kiessling, R., Petranyi, G., Klein, G., and Wigzell, H., Int. J. Cancer 15, 933, 1975. 13. Haller, O., Hansson, M., Kiessling, R., and Wigzell, H., Nature (London) 270, 609, 1977. 14. Gomard, E., Leclerc, J. C., and Levy, J., Nature (London) 250, 671, 1974. 15. Herberman, R. B., Nunn, M. E., Holden, H. T., and Lavrin, D. H., Int. .I. Cancer 16, 130, 1975. 16. Sendo, F., Aoki, T., Boyse, E. A., and Buafo, C. K., 1. Nat. Cancer Inst. 55, 603, 1975. 17. Herberman, R. B., Nunn, M. E., Holden, H. T.. Staal. S., and Djeu, J. Y., Int. J. Cancer 19, 555, 1977. 18. Herberman, R. B., Nunn, M. E., and Holden, H. T., J. Immunol. 121, 304, 1978. 19. Herberman, R. B., Bartram, S., Haskill, J. D., Nunn, M. E., Holden, H. T., and West, W. H.. J. Immunol. 119, 322, 1977. 20. Murphy, E. D., Proc. Amer. Ass. Cancer Res. 4, 46, 1963. 21. Siegler, R., and Rich, M. A., J. Nut. Cancer Inst. 41, 125, 1968. 22. Lerman, S. P., Chapman, J. M., Carswell, E. A., and Thorbecke, G. J.,fnt. J. Cancer 14,808, 1974. 23. Hammerling, U., Chen, A. F., Abbitt, J., and Sheid, M. P., J. fmmunol. 115, 1425. 24. Ponzio, N. M., David, C. S., Shreffler, D. C., and Thorbecke, G. J., J. Exp. Med. 146, 132. 1977. 25. Ponzio, N. M., Lerman, S. P., Chapman-Alexander, J. M., and Thorbecke, G. J., Cc//. [mmuno/. 32, 10, 1977. 26. Romano, T. J., Ponzio, N. M., and Thorbecke, G. J., J. Immunol. 116, 1618, 1976. 27. Chang, K. S., In “Advances in Comparative Leukemia Research 1977” (P. Bentvelzen, J. Hilgers and D. S. Yohn, Eds.), pp. 327-330. ElsevieriNorth-Holland, Amsterdam, 1978. 28. Ponzio, N. M., Chapman-Alexander, J. M. and Thorbecke, G. J., Ccl/. Immune/. 41, 157, 1978. 29. Lerman, S. P., Chapman-Alexander, J. M., Umetsu, D., Shcn, F. W.. and Thorbecke. G. J.. manuscript in preparation. 30. Herberman, R. B., and Holden, H. T., In “Advances in Cancer Research” (G. Klein and S. Weinhouse, Eds.), Vol. 27, pp. 305-377. Academic Press, New York, 1977. 31. Trinchieri, G., and Santoli, D., J. Exp. Med. 147, 1314, 1978. 32. Gidlund, M., Orn, A., Wigzell, H., Senik, A., and Gresser. I., Nature (London) 273, 759, 1978.
IMMUNOLOGY
Natural
43, 185-191 (1979)
SHORT COMMUNICATIONS Killer Cell Activity in Reticulum Cell Sarcomas (RCS) of SJL/J Mice1,2 KAREN L. FITZGERALD~ANDNICHOLAS
Department
M. PONZIO
of Microbiology-Immunology and the Cancer Center Northwestern and Dental Schools, Chicago, Illinois 60611
University
Medical
Received August 23, 1978
Two transplantable reticulum cell sarcomas (RCS) of SJL mice expressed marked levels of natural killer (NK) activity when tested against susceptible Yr-labeled tumor targets. In contrast, normal SJL lymph node and spleen cells demonstrated low levels of NK activity. Neither depletion of macrophages nor pretreatment with anti-Thy-l.2 sera and complement reduced the capacity of RCS cells to express NK activity. Systemic injection of irradiated RCS cells into SJL mice induced a transient increase in NK activity at 3 and 7 days after injection. However, the NK activity observed in recipients of irradiated RCS cells never reached levels comparable to those of control mice injected with viable tumor cells. These data suggest that the transplantable reticulum cell sarcomas of SJL mice may represent a tumor of natural killer cells and thus provide an enriched source of these effecters that may be useful for further characterization of natural cytotoxicity.
INTRODUCTION The expression of natural cell-mediated cytotoxicity against tumor cells has been well documented in mice (l-4), rats (5-7), and man (8- 10). Cells that mediate this natural tumor immunity differ from other effecters of cytoxicity in that they do not appear to require prior sensitization to, or antibody specific for, antigens expressed on the tumor cell target. Hence, these effecters have been termed natural killer (NK)4 cells (11). Various factors influence the expression of NK activity in mice, including age and genetic background (3, 4). Although primarily detected on the basis of cytotoxic assays, there is a high correlation between expression of NK activity in vitro and resistance to growth of NK sensitive tumor cells in vivo (12, 13). Considerable effort has been directed toward characterizing murine NK cells. NK activity is detectable in most lymphoid tissues (except thymus) with particularly high activity in peripheral lymph nodes, spleen, and peripheral blood (3,4). Based upon the presence of high NK activity in congenitally athymic “nude” mice and upon a lack of significant reduction of activity after pretreatment of ’ Supported in part by USPHS Grant CA22544 and Grant 78-6 from the American Cancer Society, Illinois Division. ’ Submitted in partial fulfillment for the Doctor of Philosophy degree from the Department of Microbiology-Immunology of Northwestern University Medical School. ” Supported in part by a fellowship from the Chicago Baseball Cancer Charities. 4 Abbreviations used: NK, natural killer: C, complement; LN, lymph node; RCS, reticulum cell sarcoma. 185
0008-8749/79/030185-07$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
186
SHORT
COMMUNICATIONS
effector cells with anti-Thy-l sera and complement (11, 14- 16), it was originally thought that murine NK cells were non-T in nature. More recent evidence (17, 18), however, suggests that NK cells express a low, but detectable, amount of Thy-l marker and should be categorized as a pre-T cell. Thus, NK cells in nude mice are also susceptible to pretreatment with anti-Thy-l sera and complement under optimal conditions (18). Further characterization of murine NK cells has also revealed the absence of surface Ig and the presence of F, receptors (11, 15, 19). Since NK cells appear in rather low frequency, even after augmentation with different stimuli, the majority of investigations concerning their nature have been performed by deletion of NK cells using various separation methods. Isolation of lymphoid populations that are enriched for NK effector cells is less well documented. The present brief communication provides evidence that the reticulum cell sarcomas (RCS) that occur with high incidence in SJL mice may represent a tumor of NK cells and thus provide a readily available enriched source of NK cells for further characterization. MATERIALS
AND METHODS
Mice and tumors. Female and male SJL/J, and female CBA/J, A/J, and BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, Me). The RCS tumors used in these studies were derived from reticulum cell sarcomas (RCS) that arose spontaneously in SJL mice and were established as RCS lines by serial iv transfer of 10’ tumorous lymph node cells at regular intervals (20-22). The NK susceptible target cells used in the cytotoxic assay included YAC-292 (an A-strain Moloney virus-induced lymphoma), RL 6-l (a BALB/c radiation-induced leukemia), and RBL-5 (a Rauscher virus-induced leukemia of C57B1/6 origin). All cell lines were maintained in vitro in RPM1 1640 + 10% fetal calf serum (FCS) (GIBCO, Grand Island, N.Y.). NK cytotoxic assay. One hundred microcuries of 51Cr (as sodium chromate) (Amersham, Arlington Heights, Ill.) was added to 5- 10 x lo6 target cells in 1 ml of RPM1 1640 + 10% FCS (GIBCO) and incubated at 37°C in an atmosphere of 5% CO, and 95% air for 1 hr. Following incubation, the labeled cells were washed and resuspended to a concentration of 4 x 105/ml. Effector cells were taken from the lymph nodes and spleen of tumor-bearing SJL mice or from normal donors. Effector cells were adjusted to various concentrations and added to fixed number of 51Cr-labeled target cells in order to test different effectorltarget cell ratios. One hundred microliters of effector cells and 100 ~1 of target cells were incubated in Linbro microtest plates (No. 76-11 l-05, Hamden, Conn.) at 37°C in 5% COz and 95% air. Two time periods (4 and 16 hr) were routinely tested with all effecter/target cell concentrations, in order to compare the different time intervals for optimal Wr release. The Titer-tech Supematant Collection System (Flow Laboratories, Rockville, Md.) was used to harvest the supematants from the microtiter wells. Each sample was counted in a gamma counter (Beckman Instruments, Fullerton, Calif.) and the percentage specific 51Cr release was calculated according to the following formula: counts per minute (experimental - spontaneous release) x loo Percentage 51Cr release = counts per minute (maximum release -spontaneous release)
187
SHORT COMMUNICATIONS
Maximum release (~80%) was determined by incubating 51Cr-labeled target cells in 1% NP40 (Bethesda Research Laboratories, Rockville, Md.) and spontaneous release (5-30%) was measured following incubation of labeled target cells either alone or in the presence of unlabeled target cells or normal thymus cells to compensate for cell density. Anti-Thy-l.2 serum, prepared by immunization of (A/Thy Antisera. 1.1 x AKR/H-2b) F, mice with the A strain leukemia ASL-1, (23) was a generous gift from Dr. G. J. Thorbecke (N. Y. U. Medical Center). Rabbit serum, agarose absorbed and selected for low toxicity against mouse lymphocytes, was used as a source of complement. Irradiation. In some experiments RCS cells were given 10,000 R y irradiation using a Philips Cobalt 60 Unit at a central dose rate of 140 rad/min. RESULTS Tables 1 and 2 provide a summary of experiments that demonstrate NK activity of RCS cells against two susceptible tumor target cell lines (RLcl-1, Table 2 and YAC-292, Table 1). Mean percentage 51Cr release, as well as the range of TABLE I Presence of NK Activity against YAC Target Cells in Lymphoid Tissues of SJL Mice Injected with Transplantable RCS Tumor Cells “‘0 release from YAC target cells in (%)’ 4 Hours Strain
Tissue”
SJL
RCS-5 LN
SJL
RCS-X LN
SJL
Normal LN
SJL
Normal spleen
CBA
Normal spleen
A/J
Normal spleen
E/T ratio* 100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 100 50 25
16 Hours
Mean
Range
Mean
Range
24 19 16 24 28 23 2
19-28 11-32 9-26 IO-38 13-37 12-35 o-3 o-4 -0.8-4 o-7 -3-6 -14-13 17-70 11-64 IO-23 d 2-9 o-4
56 56 71 47 66 73 2 3 3 2 9 6 4-l 52 50 4 9 11
51-60 40-83 62-77 20-74 26-86 36-85 o-4 - 15- 10 -16-12 o-3 - 16-20 -11-18 22-53 22-72 20-72 ,l -6-29 -5-22
0.5 4 2 4 40 39 33 1 4 3
a RCS effector cells were taken from SJL donors injected 7-10 days previously iv with IO7viable RCS tumor cells. Normal effector cells were derived from 7-g-week-old donors of the indicated strain. b Effecter/target cell ratios: 100 ~1 of Wr-labeled target cells (4 x 105/ml) were mixed with 100 ~1 of effector cells at concentrations adjusted to give the indicated ratios. e Data are calculated from a minimum of five separate experiments. Mean maximum release: 4 hr = 88%, 16 hr = 88%; mean spontaneous release: 4 hr = 14%, 16 hr = 33%. d Data from a single experiment.
188
SHORT COMMUNICATIONS TABLE 2 Presence of NK Activity against RLd-1 Target Cells in Lymphoid Tissues of SJL Mice Injected with Transplantable RCS Tumor Cells SLCrrelease from RLd-1 target cells in (%)’ 4 Hours
Strain
Tissues”
SJL
RCS-5 LN
SJL
RCS-X LN
SJL
Normal LN
SJL
Normal spleen
CBA
Normal spleen
A/J
Normal spleen
16 Hours
E/T ratio*
Mean
Range
Mean
Range
100 50 25 100 50 25 100 50 25 100 50 25 100 50 25 100 50 25
20 20 12 21 25 17 0 1 2 1 3 4 38 27 20 -5 -1 -1
14-26 9-27 9-16 3-38 10-35 lo-24 d -16-10 -15-12 o-1 -14-13 -11-15 17-55 9-53 4-36 d -4-2 -2-4
49 59 66 45 70 76 2 5 4 14 3 5 70 44 43 -1 9 12
33-65 18-83 31-84 20-69 69-87 75-86 d -1-10 -1-10 8-20 -5-18 -1-7 40-92 22-81 13-68 d l-21 4-19
a As in Table 1. b As in Table 1. c Mean maximum release: 4 hr = 88%, 16 hr = 88%; mean spontaneous release: 4 hr = 16%, 16 hr = 32%. d Data from a single experiment.
percentage release, is given for both the 4- and the 16-hr assays. Effector cells, taken from the grossly enlarged lymph nodes of tumor-bearing SJL donors, caused significant lysis of these two NK susceptible target cells at 4 hr, and optimal cytotoxicity after 16hr of incubation. Similar results were also obtained with a third target cell, RBL-5 (data not shown). The percentage 51Crrelease obtained in the 4-hr assay using RCS lymphoid cell effecters is comparable, at all ratios tested, to the levels observed with effector cells from a high NK strain, CBA/J. With the longer incubation time, however, RCS tumor cells demonstrated greater cytotoxic activity than CBA effector cells. This was even more apparent at the lower effector to target cell ratios. The cytotoxicity obtained with a low NK strain, A/J, is shown for comparison. As has been demonstrated by others (H.T. Holden and R.B. Herberman, personal communication), normal SJL lymphoid cells fail to elicit significant cytotoxic potential against the target cells used in these studies. Thus, SJL mice are classified as a low NK strain. Two possible mechanisms might explain the observed results: (i) RCS tumor cells themselves mediate the cytotoxic activity observed, or (ii) RCS cells act as a powerful stimulus to augment NK activity in the low responder SJL recipient. In view of the marked proliferative response induced by RCS cells in SJL T cells (22, 24, 25) and the known ability of activated T cells to recruit the proliferation of bystander lymphocytes (26), the latter possibility warrants serious consideration.
189
SHORT COMMUNICATIONS TABLE 3 NK Activity against YAC Target Cells in LN Cells of Normal SJL Mice Injected with y-Irradiated RCS Cells” Wr release with LN cells removed after (%)b Recipient
RCS injection
3 Days
7 Days
11 Days
SJL/J SJL/J SJL/J CBA/J spleen
None RCS (10,000 R) Viable RCS None
6.4 21.3 53.2 32.6
11.8 24.8 47.7 48.2
15.6 9.5 57.6 79.2
” Normal SJL mice were injected iv with 10’ irradiated (10,ooOR) or viable RCS cells and LN cells were assayed for NK activity at the indicated times after injection. b Effector cells were incubated for 16 hr with 5LCr-labeled YAC target cells at a ratio of 50: 1.
Therefore, experiments designed to distinguish between these two alternatives were performed. SJL recipients were injected iv with 10’ y-irradiated (10,000 R) RCS cells and the lymph nodes and spleens of these recipient mice were tested for NK activity at various times after injection. SJL mice that received a similar number of viable RCS cells were tested in parallel for comparison. The results presented in Table 3 demonstrate a two- to threefold increase in NK activity in LN cells taken at early times after injection of irradiated RCS cells (Days 3 and 7) when compared to noninjected control SJL mice. In contrast, 11 days after injection of irradiated RCS cells there was no increase over noninjected SJL controls. At no time interval tested, however, did the levels of NK activity in LN taken from donors injected with irradiated RCS cells attain the degree of activity observed following transfer of viable RCS tumor cells. While these data suggest that irradiated RCS cells can induce precursors to express NK activity, it is possible that the injected, irradiated RCS cells are responsible for the observed activity. Indeed, preliminary data suggest a significant radioresistance of the NK activity present in the irradiated tumor cell preparation. (Fitzgerald and Ponzio, unpublished observations). In order to further characterize the cell(s) responsible for the NK activity in the RCS cell preparation, LN cells from SJL mice bearing the transplantable lymphoma, RCS-5, were treated with anti-Thy-l.2 or normal mouse serum and rabbit C prior to their use as effecters against 51Cr-labeled target cells. As shown in Table 4, no significant reduction in lytic activity was apparent following such pretreatment. Similar treatment of normal CBA/J spleen cells yielded identical results. The same concentrations of anti-Thy- 1.2 and C, however, were sufficient to abolish the cytotoxic activity of alloantigen sensitized killer T cells in a similar 51Cr-release assay (data not shown). DISCUSSION The results presented in Tables 1 and 2 provide evidence for NK activity in lymphoid tissues of RCS-bearing SJL donors. That RCS cells may represent a tumor of NK cells is suggested by the following observations. (i) Lymphoid cells from normal SJL donors elicit meager NK cytotoxic responses, even following attempts to augment NK activity with known inducers, such as BCG, C. parvum, or Poly 1:C (H. T. Holden and R. B. Herberman, personal communication).
190
SHORT COMMUNICATIONS TABLE 4 Effect of Anti-Thy-l.2
Effector cells
+ C Pretreatment on NK Activity in RCS Lymphoid Cells”
Pretreatment’
Wr release on YAC targets (%I*
RCS-5 LN
Anti-Thy-l.2 NMS+C None
+ C
80.8 86.2 73.0
CBA/J spleen
Anti-Thy-l.2 NMS+C None
+ C
41.7 30.1 28.5
a Cells were pretreated with anti-Thy-l.2 serum (1: 100) or normal mouse serum (NMS) (1: 100) and rabbit complement (C) (1: 12) prior to use as effecters. * Pretreated effector cells were incubated for 16hr with Wr-labeled YAC target cells at a ratio of 50: 1.
(ii) The observed cytotoxic activity is not mediated by killer T cells, since pretreatment of RCS-containing lymphoid cells with anti-Thy-l.2 sera and complement, at concentrations sufficient to abolish cytotoxic activity of sensitized T cells (25), did not diminish NK activity. Although RCS cells do not appear to express amounts of Thy-l .2 antigen necessary for lysis with anti-Thy-l .2 serum and complement (24), this does not exclude the possibility that RCS cells may express extremely low levels of this marker, as has recently been observed on NK cells present in normal lymphoid tissues of high NK strains (17, 18). Furthermore, RCS tumor cells do not elicit cytotoxic T-cell responses in SJL lymphoid cells, even after repeated immunizations in vivo or after primary sensitization in vitro (25). Consequently, it does not appear that the observed results are due to a nonspecific activation of cytotoxic effector T-cell precursors by RCS stimulation. (iii) Macrophages are not responsible for the NK activity in RCS lymphoid cell effector preparations, since removal of these cells by sequential treatments based on adherence and on phagocytosis of carbonyl iron did not diminish the cytotoxic potential of RCS cell preparations (Fitzgerald and Ponzio, unpublished observations) . Similar cytotoxic activity of RCS tumors against another NK-susceptible target cell, RBL-3, has also been reported by Chang (27). Therefore, the presence of NK activity in RCS-containing lymphoid cell preparations appears established. The effector cell within the RCS tumors that mediates the cytodestruction of NK-susceptible targets has not, as yet, been clearly identified. It is possible that the RCS tumor cell itself may represent either neoplastic expansion of NK effector cells or an entirely new class of cytotoxic effecters. On the other hand, RCS cells can induce marked proliferation of SJL T cells (22,24,25) and there is evidence (28) that RCS cells act as a polyclonal activator of Ly 1+, 2,3- T cells (29). Since NK cells appear to belong in the T-cell lineage (17, 18), it is conceivable that NK precursors may be included among the T cells activated via RCS stimulation. Alternatively, since interferon has been shown to play a significant role in the expression of NK cytotoxicity (30-32) the possibility that RCS cells stimulate NK cells via interferon production (either directly or indirectly) cannot be excluded. Attempts are in progress to define more precisely the nature of the effector cell and mechanism(s) of cytotoxicity apparent in RCS tumors. Thus, the reticulum
SHORT COMMUNICATIONS
191
cell sarcomas of SJL mice may provide a valuable model for further characterization of natural cytotoxicity. ACKNOWLEDGMENTS The authors wish to thank Dr. Myron Essex (Massachusetts General Hospital, Boston) for providing the YAC-292 cell line, Dr. Ronald B. Herberman (Laboratory of Immunodiagnosis, National Cancer Institute) for the generous gift of RLd-1 and RBL-5 tumor cells, and Dr. G. Jeanette Thorbecke (N. Y. U. Medical Center) for stimulating discussion and review of this manuscript. The provision of irradiation facilities by Dr. Pat Johnson and the Radiation Therapy Department, Northwestern Memorial Hospital, is gratefully acknowledged.
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