Comparison of Natural Killer Cells Induced by Kunjin Virus and Corynebacterium parvum with Those Occurring Naturally

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1 INFECTION AND IMMUNITY, Dec. 1979, p Vol. 26, No /79/ /05$02.00/0 Comparison of Natural Killer Cells Induced by Kunjin Virus and Corynebacterium parvum with Those Occurring Naturally in Nude Micet RODERICK I. Department ofmicrobiology, University ofmelbourne, Parkville, Victoria 3052, Australia,1 and the Walter and Eliza Hall Institute ofmedical Research, Parkville, Victoria 3052, Australia2 MACFARLAN,1 RHODRI CEREDIG,2 AND DAVID 0. WHITE`* Received for publication 18 September 1979 Natural killer (NK) cells are rapidly elicited in the spleen and peritoneal cavity of mice inoculated intravenously or intraperitoneally with live Kunjin virus, and more slowly in the peritoneal cavity of mice inoculated intraperitoneally with Formalin-inactivated Corynebacteriumparvum. NK cells induced by either agent display cytotoxicity for a similar spectrum of syngeneic, allogeneic, and xenogeneic cultured cell lines. By contrast, the cells occurring naturally in the spleen of congenitally athymic (nude) mice show substantially lower NK activity and are cytotoxic for a more restricted range of target cell lines. The distinction suggests that there may be more than one type of NK cell or that activation enhances the cytotoxicity and perhaps broadens the range of target specificity of endogenous NK cells. Natural killer (NK) cells cytotoxic for a wide range of tumor cells were first recognized in spleen, lymph node, and peripheral blood lymphoid populations of unmanipulated mice, rats, and humans (6, 10). More recently, a cytotoxic cell with similar physical characteristics has been shown to be induced in the peritoneal cavity and spleen shortly after inoculation of mice with viruses (11, 19; R. I. Macfarlan and D. 0. White, submitted for publication), bacteria (BCG or Corynebacterium parvum) (13, 22), tumor cells (7), interferon (2, 4, ), or interferon inducers (2, 4). In previous papers (11, 15; Macfarlan and White, submitted for publication), we have described the characteristics of the NK cell appearing in the peritoneal cavity and spleen 1 to 2 days after infection of normal or congenitally athymic BALB/c mice with the togavirus Kunjin. Briefly, this NK cell is nonadherent, nonphagocytic, Thy 1.2 deficient, ethylenediaminetetraacetic acid sensitive, trypsin resistant, Fc receptor bearing, but antibody independent, labile upon cultivation in vitro, and sensitive to X irradiation before infection, and it kills continuous cell lines as well as some normal mouse cells. In this paper we compare the tissue distribution and target cell specificity of Kunjin virus-induced NK cells with those induced by Corynebacterium parvum, which is of particular interest because of its known antitumor effects (7, 12, 13, 23). In addition, both types of "induced" NK t Walter & Eliza Hall Institute publication no cells are contrasted with those "endogenous" NK cells occurring naturally in the spleens of normal and congenitally athymic mice. MATERIALS AND METHODS Mice. BALB/c An Bradley Wehi (BALB/c) mice were maintained from stocks originally supplied by the Walter and Eliza Hall Institute, Melbourne. Specific pathogen-free congenitally athymic BALB/c.nu mice were obtained as required from the same source. Tumor cell lines. All cell lines were maintained in culture by using RPMI 1640 with % inactivated fetal calf serum as growth medium. The origins of the cell lines used are summarized in Table 1. Effector cells. Cytotoxic effector cells were generally obtained from BALB/c mice 2 days after intraperitoneal (i.p.) inoculation of 5 x 105 plaque-forming units of live Kunjin virus (11), or 4 days after i.p. inoculation with 350,ug of Formalin-killed Corynebacterium parvum (Coparvax, Wellcome) (23). Spleen and peritoneal cell (PC) suspensions were prepared by standard techniques (15); final cell suspensions were in RPMI with 10% inactivated fetal calf serum. Nylon wool columns. Removal of adherent cells by nylon wool columns has been previously described (11). Briefly, 5 x 107 to 10 x 107 cells in 4 ml of RPMI with % fetal calf serum were applied to the column (0.6 g of Fenwall Leukopak nylon wool packed to 7.5 ml in a 10-ml plastic syringe) and incubated at 370C for 5 min, and the nonadherent cells were eluted with 30 ml of the above medium. A proportion of the nylon wool-adherent cells could be recovered by elution with 0.2% ethylenediaminetetraacetic acid in isotonic phosphate-buffered saline. We have previously shown that these cells are phagocytic and can mediate antibody-dependent cytotoxicity (11; 832

2 VOL. 26, 1979 TABLE 1. NK CELLS INDUCED BY KUNJIN VIRUS AND C. PARVUM 833 Cell lines used as target cells in cytotoxicity assays Cell line Species Cell type WR-9 Rat (ACI) Fibrosarcoma W/FuG-1.16 Rat (W/Fu) Lymphoma HeLa SIII Human Carcinoma WEHI-l1 Mouse (BALB/c) Fibrosarcoma WEHI-164 Mouse (BALB/c) Fibrosarcoma W54 Mouse (CBA) Mammary tumor MPC- Mouse (BALB/c) Plasma cell tumor C1.18 Mouse (C3H) Plasma cell tumor EL-4.1 Mouse (C57BL/6) Thymoma HC.3 Mouse (A/J x Mastocytoma C57/L)F, P-815X-2.1 Mouse (DBA/2) Mastocytoma L929 Mouse (C3H) Connective tissue Macfarlan, unpublished data). However, they may represent a subpopulation of moderately adherent PC. Cytotoxicity assay. Effector cell populations were titrated for cytotoxicity against 5'Cr-labeled tumor cells in a 9-h chromium release assay (11). Briefly, effector and target cells (15,000/well) were incubated in Linbro FB TC 96 plates in a total volume of 0.25 ml. At the conclusion of the assay, 100 p1 of the supernatant was sampled for released 5"Cr, and the specific chromium release was calculated as a percentage of the maximum 5'Cr released by 0.5% Nonidet P- 40 after subtracting the spontaneous release in medium alone. Inhibition of 5'Cr release was sometimes observed at very high effector-target ratios. Results are expressed as the mean of triplicate samples. The standard error of the mean rarely exceeded 2%. RESULTS Induction of NK cytotoxicity by C. parvum and Kunjin virus. After i.p. inoculation into BALB/c mice, both Formalin-inactivated C. parvum and live Kunjin virus rapidly induce the appearance in the peritoneal cavity of cells cytotoxic for the tumor cell line P815 (Fig. 1). Cytotoxic cells appear and disappear earlier after Kunjin virus infection, peaking at 2 days (c.f. 4 days after C. parvum). These Kunjin virus-induced PC have been well characterized as NK cells in our previous papers (11; Macfarlan and White, submitted for publication). They may be defined as nonadherent, non-thymus-dependent cells cytotoxic for tumor cells. The C. parvum-induced cytotoxic PC are shown here also to be NK cells by the same criteria of nonadherence to nylon wool (Fig. 2) and occurrence in congenitally athymic (nude) BALB/c mice (Fig. 1 and 3). The endogenous NK cell found in the spleens of normal or nude mice is also nonadherent and Thy 1.2 deficient (1, 6, 10). Effect of route of inoculation on distribution of cytotoxic NK cells. Kunjin virus and C. parvum were inoculated into BALB/c mice by the i.p. or intravenous routes. At the time of maximum NK response (2 days after Kunjin virus; 4 days after C. parvum) PC and spleen cells were tested for cytotoxicity against P815 cells (Fig. 4). In the case of both Kunjin virus and C. parvum i.p. inoculation elicited greater cytotoxic activity than intravenous, and PC were considerably more active than spleen cells regardless of the route of inoculation. In contrast, normal DAYS AFTER INOCULATION FIG. 1. Time of appearance of cytotoxic PC. At various times before assay, mice were inoculated ip. with killed C. parvum or live Kunjin virus. PC were tested for cytotoxicity against 5"Cr-labeled P815 cells at an effector-target ratio of50:1. Symbols: 0, BALBE c mice inoculated with 5 x 105 plaque-forming units ofkunjin virus;, BALB/c mice inoculated with 350 pg of C. parvum; 0, nude BALB/c mice inoculated with 350 pg of C. parvum A B C 3 6 I EECTOR/TARET RATIO FIG. 2. Effect of removal of adherent cells on cytotoxicity. (A) PC from BALB/c mice infected 2 days earlier with Kunjin virus, assayed against P815 cells. (B) PC from BALBIc mice inoculated 4 days earlier with C. parvum, assayed against P815 cells. (C) Spleen cells from nude BALB/c mice, assayed against C1.18 cells. Symbols:, unfractionated effector cells; *, nylon wool-nonadherent effector cells; 0, nylon wool-adherent effector cells.

3 834 MACFARLAN, CEREDIG, AND WHITE ~ FIG. 3. Induction of NK cytotoxicity in athymic mice. PC from normal BALBIc mice (A) and PC from BALBIc (-) or nude BALB/c (0) mice 4 days after i.p. inoculation with 350,ug of C. parvum were assayed for cytotoxicity against a panel of target cells. 30 KUNJIN S~~~~~~~~1 C. PARVUM FIG. 4. Effect of route of inoculation on the distribution of cytotoxic NK cells. Spleens (open symbols) and PC (closed symbols) from BALB/c mice inoculated 4 days earlier with C. parvum or 2 days earlier with Kunjin virus were titrated for cytotoxicity against P815 cells. Symbols: A, A, normal mice; U, Ol intravenous inoculation; 0, 0, i.p. inoculation. PC and spleen cells were inactive against this particular target. Target specificity of NK cells induced by Kunjin virus and C. parvum compared with those from uninfected athymic mice. PC from BALB/c mice inoculated 2 days earlier with Kunjin virus, or 4 days earlier with C. parvum, were compared with spleen cells from uninoculated nude BALB/c mice for cytotoxicity against a range of syngeneic, allogeneic, and xenogeneic cell lines (Fig. 5). C. parvum-induced NK cells displayed cytotoxicity for all the cell lines tested, as did those induced by Kunjin virus. Furthermore, the rank order of susceptibility was similar. In contrast, BALB/c nude spleen cells were considerably less cytotoxic than either Kunjin- or C. parvuminduced PC and lysed only a restricted range of INFECT. IMMUN. lines, with P815, HeLa, W54, L929, and HC.3, all totally resistant to lysis. These resistant target cells were also resistant to spleen cells from normal BALB/c mice and to spleen cells from normal or nude BALB/c mice after enrichment for NK cells. It will be observed that PC from normal BALB/c mice are somewhat cytotoxic for certain cell lines (especially EL4); there is no correlation between the levels of cytotoxicity displayed by nude spleen and normal PC against particular targets. This cytotoxicity by normal PC against certain tumor targets is mainly attributable to macrophages and will be the subject of a forthcoming publication (Macfarlan and White, manuscript in preparation). DISCUSSION It is now well established that many agents, including viruses, bacteria, and tumor cells, are capable of generating an NK cell cytotoxic response very shortly after inoculation (6, 10, 11, 19). The common factor may be interferon, the synthesis of which can be induced by all these agents. Interferon or interferon inducers elicit cytotoxic NK cells in vivo (2, 4, ) or in vitro (2, 18), and induction of NK cytotoxicity in vivo can be completely abrogated by anti-interferon (4) Ṅot yet understood, however, is the relationship of such induced NK cells to the endogenous NK cells present in unmanipulated mice. Although the two share many properties in common, notably that they are nonadherent, nonphagocytic, non-thymus dependent, and non-antibody dependent (6, 7, 10, 11, 19; Macfarlan and White, submitted for publication), they also differ in certain key respects investigated in this paper. Whereas the endogenous NK cells of untreated normal or nude mice are found principally in the spleen and not in the peritoneal cavity (1, 8, 9), those elicited by C. parvum and Kunjin virus are found predominantly in PC, although significant activity is also induced in the spleen after both intravenous and i.p. inoculation. In contrast, intravenous inoculation of either agent was markedly less effective than i.p. inoculation in the induction of peritoneal NK activity. This suggests that the NK response tends to be localized to the sites of distribution of the agent. Since macrophages appear to be important as accessory cells in the induction of NK cytotoxicity (at least in the case of polyinosinic:polycytidylic acid) (3), it may not be surprising that the peritoneal cavity is a particularly favorable site for NK cell induction, even though PC are not a good source of endogenous NK cells. Little is yet known of the ontogeny of NK

4 VOL. 26, 1979 NK CELLS INDUCED BY KUNJIN VIRUS AND C. PARVUM L929 HC.3 P815 W/FuG 40 ~~~~~~~~~~~~~~~~~~~~~~~~~~- 60 MPC-11 W54 HeLa / WR ' ' ' O FIG. 5. Specificity of NK cells for tumor cell lines. PC from normal BALB/c mice (0), PC from BALBIc mice infected i.p. 2 days earlier with Kunjin virus (U), PC from BALBIc mice inoculated ip. 4 days earlier with C. parvum (E), spleen cells from nude BALBIc mice (0). cells; evidence has been presented to support the view that NK precursors may originate from bone marrow (5, ). We have recently presented data which indicate that cell proliferation is necessary for optimum induction of NK cytotoxicity by Kunjin virus (Macfarlan and White, submitted for publication), but it is not clear whether endogenous NK cells, NK cell precursors, or accessory cells must divide, or which if any of these migrate to the peritoneal cavity after infection. It has been shown that inoculation of tilorone by the i.p. route can enhance NK cytotoxicity of spleen cells without a concomitant increase in the number of cells capable of binding to targets (16). NK cells generated in the peritoneal cavity or spleen of BALB/c mice inoculated i.p. with Kunjin virus or C. parvum displayed cytotoxicity for all of 36 continuous cell lines tested (Fig. 5; and Macfarlan, unpublished data); indeed the rank order of sensitivity of various target cells was almost the same in the two cases. In addition, C. parvum-induced NK cells killed normal thymocytes with the same selectivity for target cell genotype (R. Ceredig, unpublished data) as found for Kunjin virus-induced NK cells (Macfarlan and White, submitted for publication). This strongly suggests that the NK cells elicited by the two agents are identical. However, the endogenous NK cells occurring naturally in the spleens of nude BALB/c mice killed only a restricted range of cell lines (even after enrichment for NK cells), and even against these targets were considerably less cytotoxic than Kunjin virus- or C. parvum-induced NK cells from the peritoneal cavity or spleens of BALB/c mice. It is clear that virus-induced NK cells differ from naturally occurring NK cells. What has yet to be established, however, is whether infection leads, via the mediation of interferon, to activation of endogenous NK cells (or a common precursor cell), thereby enhancing their cytotoxicity and range of demonstrable target specificities (7, 10, 12, 13), or whether the two represent inherently different NK cell subclasses with distinct specificities for different ranges of target structures. Evidence has been presented to suggest that there may be distinct subclasses of "naturally cytotoxic" cells with different spectra of "lytic

5 836 MACFARLAN, CEREDIG, AND WHITE specificity" and slightly different physical properties (14, 17). It has also been postulated that lymphocytic choriomeningitis virus-induced NK cell populations may operate via two different recognition systems (19, 21). ACKNOWLEDGMENTS This work was supported by grants from the National Health and Medical Research Council of Australia. We are particularly indebted to R. C. Burton and M. F. A. Woodruff for valuable discussions during the early stage of this work and to G. Shellam for supplying the rat cell lines. C. Adeney and L. Vogels provided capable technical assistance. LITERATURE CITED 1. Burton, R. C., D. Grail, and N. L. Warner Natural cytotoxicity of haemopoietic cell populations against murine lymphoid tumours. Br. J. Cancer 37: Djeu, J. Y., J. A. Heinbaugh, H. T. Holden, and R. B. Herberman Augmentation of mouse natural killer cell activity by interferon and interferon inducers. J. Immunol. 122: Djeu, J. Y., J. A. Heinbaugh, H. T. Holden, and R. B. Herberman Role of macrophages in the augmentation of mouse natural killer cell activity by poly I:C and interferon. J. Immunol. 122: Gidlund, M., A. Orn, H. Wigzell, A. Senik, and I. Gresser Enhanced NK cell activity in mice injected with interferon and interferon inducers. Nature (London) 273: Haller, O., R. Kiessling, A. mrn, and H. Wigzell Generation of natural killer cells: an autonomous function of the bone marrow. J. Exp. Med. 145: Herberman, R. B., and H. T. Holden Natural cell-mediated immunity. Adv. Cancer Res. 27: Herberman, R. B., M. E. Nunn, H. T. Holden, S. Staal, and J. Y. Djeu Augmentation of natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic target cells. Int. J. Cancer 19: Herberman, R. B., M. E. Nunn, and D. H. Lavrin Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumours. I. Distribution of reactivity and specificity. Int. J. Cancer 16: Kiessling, R., E. Klein, and H. Wigzeil Natural killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur. J. Immunol. 5: Kiessling, R., and H. Wigzell An analysis of the INFECTr. IMMUN. murine NK cell as to structure, function, and biological relevance. Immunol. Rev. 44: Maefarlan, R. L, W. H. Burns, and D. 0. White Two cytotoxic cells in peritoneal cavity of virus-infected mice: antibody-dependent macrophages and non-specific killer cells. J. Immunol. 119: Ojo, E., 0. Hailer, A. Kimura, and H. Wigzell An analysis of conditions allowing Corynebacterium parvum to cause either augmentation or inhibition of natural killer cell activity against tumour cells in mice. Int. J. Cancer 21: Ojo, E., 0. Hailer, and H. Wigzell Corynebacterium parvum-induced peritoneal exudate cells with rapid cytolytic activity against tumour cells are nonphagocytic cells with characteristics of natural killer cells. Scand. J. Immunol. 8: Paige, C. J., E. F. Figareila, M. J. Cuttito, A. Cahan, and 0. Stutman Natural cytotoxic cells against solid tumors in mice. II. Some characteristics of the effector cells. J. Immunol. 121: Rodda, S. J., and D. 0. White Cytotoxic macrophages: a rapid non-specific response to viral infection. J. Immunol. 117: Roder, J. C., R. Kiessling, P. Biberfeld, and B. Andersson Target-effector interaction in the natural killer (NK) cell system. II. The isolation of NK cells and studies on the mechanism of killing. J. Immunol. 121: Stutman, O., C. J. Paige, and E. F. Figareila Natural cytotoxic cells against solid tumors in mice. I. Strain and age distribution and target cell susceptibility. J. Immunol. 121: Trinchieri, G., and D. Santoli Antiviral activity induced by culturing lymphocytes with tumor-derived or virus-transformed cells. Enhancement of human natural killer cell activity by interferon and antagonistic inhibition of susceptibility of target cells to lysis. J. Exp. Med. 147: Welsh, R. M Mouse natural killer cells: induction, specificity, and function. J. Immunol. 121: Welsh, R. M Cytotoxic cells induced during lymphocytic choriomeningitis virus infection of mice. I. Characterization of natural killer cell induction. J. Exp. Med. 148: Welsh, R. M., R. M. Zinkernagel, and L. A. Hallenbeck Cytotoxic cells induced during lymphocytic choriomeningitis virus infection in mice. II. "Specificities" of the natural killer cells. J. Immunol. 122: Wolfe, S. A., D. E. Tracey, and C. S. Henney Induction of "natural killer" cells by BCG. Nature (London) 262: Woodruff, M. F. A., and N. L. Warner Effect of Corynebacterium parvum on tumor growth in normal and athymic (nude) mice. J. Natl. Cancer Inst. 58: