Feline cytotoxic immune mechanisms against virus-associated leukemia and fibrosarcoma

81,

CELLULARIMMUNOLOGY

Feline Cytotoxic

157-168(1983)

Immune Mechanisms against Virus-Associated Leukemia and Fibrosarcoma

JANET M. MCCARTY Department

of Cancer

Biology, Received

Harvard April

School

AND CHRIS K. GRANT of Public

5, 1983; accepted

Health,

Boston,

Massachusetts

02115

June 5, I983

Humoral and cellular cytotoxic immune mechanisms of cats were compared against feline leukemia virus (FeLV)- and feline sarcoma virus (Fe%+transformed cells. The groups of animals studied were nonexposed control cats; FeLV-infected immune or viremic tumor-bearing cats; FeSV-inoculated tumor progressor or regressor cats, and cats immunized with FeSV-transformed autochthonous fibroblasts (ATF). Sera containing complement-dependent antibodies (CDA), which lysed FeLV-producer lymphoma lines, had no cytotoxic effectswhen tested against FeLVproducer FeSV-transformed fibroblasts. Sent with lytic CDA activity were also tested for antibodydependent cellular cytotoxic (ADCC) effects with peripheral blood lymphocytes (PBL) from nonimmune cats. No ADCC activity was detected against either lymphoid or fibroblast target lines. To demonstrate that cat PBL contained ADCC effector cells, antibody-coated murine target cells were employed and positive results obtained. Natural killer (NK) assayswere performed using PBL from normal and tumor-bearing cats. Cytotoxic effects were only detectable to FeLVproducer lymphomas, and comparable levels of NK activity were found in normal and lymphoid tumor-bearing animals. In cats immunized with ATF, a population of effector cells was found in peripheral blood which had functional characteristics of cytotoxic T lymphocytes (CTL). The killing of ATF by CTL-like cells was not inhibited by FeLV/FeSV immune sera or by sera from autochthonous immune cats. The comparative importance of humoral and cellular cytotoxic mechanisms against FeLV- and FeSV-induced tumors is discussed.

INTRODUCTION Cats persistently infected with feline leukemia virus (FeLV) are susceptible to a variety of hematopoietic neoplastic diseases including erythroleukemia, granulocytic leukemias, and, most commonly, lymphoma (1, 2). The mechanism by which FeLV induces lymphoid malignancies in viremic animals is unknown, but tumors associated with FeLV infection arise following a long latent period. FeLV is a replication competent retrovirus which, in rare instances, is believed to have undergone genetic recombination with normal cellular sequences, resulting in the appearance of acute transforming feline sarcoma viruses (FeSV) (3-5). The genetic material transduced from normal cells, in the context of the FeSV genome, functions as a specific transforming oncogene (6, 7). FeSV is replication deficient as a result of replacement of viral structural genes by the oncogene, and therefore FeSV replicates only in the presence of a helper virus (FeLV). FeSV has been regularly isolated from cats bearing multicentric fibrosarcomas (8), and this virus also causes malignant melanomas when injected intraocularly (9). Thus, the induction periods, the oncogenic mechanisms, 157 0008-8749/83

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Copyright Q 1983 by Academw Press. Inc All rights ofreproductson in any form reserved

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the target cells, and the classes of malignant disease associated with infections by FeLV and FeSV are different. The purpose of this study was to examine feline cytotoxic immune responses to tumor cells induced by either FeLV or FeSV and to identify effector mechanisms that mediate tumor protection. The humoral immune response to FeLV infection has been studied in detail (10, 11). Following FeLV exposure, cats which do not develop lymphomas exhibit high titers of cytotoxic complement-dependent antibodies (CDA) which can be titrated against FeLV-producer lymphoma lines (12). These findings have been extrapolated to suggest that antibodies also protect against FeSVassociated fibrosarcomas (6, 13). We have examined this conclusion by comparing the capacity of CDA-positive sera to lyse FeLV-producer iymphoma cells and FeSVtransformed fibroblasts. The possibility that antibodies also function in antibodydependent cellular cytotoxicity (ADCC) reactions to protect against FeLV- and FeSVassociated tumors has been studied. In some animal systems, nonimmune cells with natural killer (NK) activity have been shown to be important in tumor immunity (14). Peripheral blood lymphocytes (PBL) from normal cats were tested for NK activity, and these levels were compared with NK activities detected in tumor-bearing animals. Tumor resistance in sensitized animals is generally correlated with high cytotoxic T-lymphocyte (CTL) activity ( 15, 16). In cats immunized with autochthonous-transformed fibroblasts (ATF) high levels of CTL activity have been described ( 17), and in most cases this activity was specifically restricted by histocompatibility barriers. These results have led us to conclude that CDA is an effective mechanism to protect cats against FeLV infection and associated lymphomas, but that cellular immune mechanisms are responsible for fibrosarcoma immunity in FeSV-infected animals. MATERIALS

AND

METHODS

Cats Specific pathogen-free (SPF) cats were bred and housed within the isolation facility at Cornell University, Ithaca, New York. Some adult SPF cats were kindly provided by Dr. F. de Noronha, School of Veterinary Medicine, Cornell University, and were then housed with normal cats in the facility at Harvard School of Public Health. Cats bearing naturally occurring tumors were presented at Angel1 Memorial Animal Hospital, Boston, Massachusetts, and diagnoses were made after clinical, pathological, and histological examinations; these cats were obtained following informed-owner consent for donation. Autochthonous

Transformed Fibroblasts

Isolation and transformation of feline fibroblasts by FeSV have been described in detail elsewhere (17). Briefly, fibroblasts were harvested from cat testicular tissue. Aliquots of normal cells were cryopreserved for use in microcytotoxicity assays while others were infected and transformed with FeSV. After transformation had occurred, as judged by phenotypic and biochemical criteria, the FeSV-transformed fibroblasts were established in cell culture for use in immunizations and in microcytotoxicity assays.Cats were inoculated sc with 2 to 5 X 10’ autochthonous transformed fibroblasts. At this dose tumor development did not occur, although occasionally small nodules were detected at the injection site which regressed after a few days.

FELINE CYTOTOXIC

RESPONSES TO FeLV or FeSV

159

Antisera Cat blood was obtained by venipuncture; sera were recovered l-4 hr after clotting and stored at -70°C prior to assay. Sera were obtained from normal healthy cats, from cats which were naturally FeLV exposed and which developed high titers of complement-dependent antibody to FeLV-producer lymphomas and from cats immunized at least two times with ATF. Feline and mm-me antisera to the murine tumor cell lines P8 15 and EL4 were obtained following three subcutaneous and intraperitoneal injections of 1 X 10’ to 1 X lo8 cells. Goat antiserum to disrupted FeLV was provided through the division of Research Resources of the National Cancer Institute. Complement Sera from normal SPF or other healthy cats, isolated from FeLV contact, were used as a source of cat complement in CDA assays. Low toxicity rabbit complement was purchased from Accurate Chemical and Scientific Corporation (Hicksville, N.Y.). The final concentrations of normal serum complement used in lytic assays were 15% for cat and 7.5% for rabbit. Target Cells The FeLV-producer lymphoma cell lines FL74, 328 1, 3272, and F422 were established as cell lines from feline lymphomas (18). All cell lines were maintained in culture medium supplemented with 10% fetal calf serum, 1% antibiotic-antimycotic mixture (Gibco Laboratories, Grand Island, N.Y.), and 1% L-glutamine (200 mM) (Gibco Laboratories). FL74 and 3281 had been maintained in McCoy’s 5A, F422 and 3272 were grown in a mixture of McCoy’s 5A and Leibovits L- 15 medium ( 1: I), murine mastocytoma cells (P8 15) and thymoma cells (EL4) and all FeSV-transformed fibroblast lines were maintained in culture in Dulbecco’s modified essential medium (DMEM). Determination

of FeL V Infection

FeLV viremia was determined by the fixed-cell immunofluorescence by Hardy et al. ( 19).

test as described

Eflector Cells Lymphocytes were isolated from 1O-30 ml of venous blood containing panheparin (Abbot Labs) (20 units/ml). Plasma was diluted 1: 1 with Hanks’ balanced salt solution (HBSS) and layered onto lymphocyte separation medium (Bionetics Laboratory Products, Kensington, Md.). Interface mononuclear cells (3 to 8 X 10’) were washed three times with HBSS and depleted of adherent cells by passage through a 50-ml nylon wool column. ADCC and NK effector cells were isolated from normal healthy cats unless otherwise indicated. Immune lymphocytes were isolated from cats previously immunized at least three times with ATF. Mcrocytotoxicity

Assays

All target cell lines for microcytotoxicity assays were labeled by incubating 5 X lo6 to 1 X 10’ cells with 250 &i of sodium “Cr-chromate solution (New England Nuclear Corp.) for 90 min at 37°C with occasional shaking; subsequently the cells

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were washed three times with growth medium. Labeled target cells were plated in Linbro microcytotoxicity plates at a concentration of 104 cells/well. The percentage specific release for each class of assay (detailed below) was calculated as % specific release

experimental cpm - control cpm x 100. total releasable cpm - control cpm

Percentage specific release shown represents the arithmetic mean of results from triplicate wells (SD < 10%). Control release varied from 10 to 30% of the total releasable figure (according to the incubation period); total release was obtained following detergent lysis of lo4 target cells and removal of cellular debris by centrifugation. Complement-dependent antibody assays. Serial twofold dilutions of antisera were made in 50 ~1 of target cell growth medium without FCS in 96-well microcytotoxicity plates. To each well 25 ~1 of either cat or rabbit complement (C) and 100 CLof labeled target cells were added. For assays with cat C the plates were incubated for 18 hr (37°C 5% CO,); for assays containing rabbit C a 2-h incubation period was used. After incubation the plates were centrifuged and 50 ~1 of cell-free supematant was counted for released activity. Control wells contained nonimmune sera and complement. Antibody-dependent cellular cytotoxicity assays. Assays were performed in two ways: (A) Target cells were presensitized with a specific dilution of antiserum and washed prior to addition of effector cells or (B) target cells, serum dilutions, and effector cells were mixed and incubated. To each well, 50 ~1 of PBL-to establish an appropriate effector to target cell (E:T) ratio-and 100 ~1 of labeled target cells in growth medium containing 5% heat-inactivated (56°C 90 min) FCS (HI-FCS) were added. Following a 2-hr incubation period for mouse target cells, or an 18-hr incubation period (37’C, 5% COZ) for cat target cells, the plates were centrifuged and 50 ~1 of cell-free supematant was counted for released activity. Control wells contained target cells, effector cells, and nonimmune serum dilutions. Natural killer cell assays and cytotoxic T-lymphocyte assays. PBL, at appropriate E:T ratios, were plated in 50 ~1 of target cell growth medium containing 5% HI-FCS for NK assays and 2% HI-FCS for CTL assays. To this 100 ~1 of labeled target cells was added. Following an 18-h assay period (37°C 5% CO& the plates were centrifuged and 504 of cell-free supematant was counted for released activity. Control wells contained target cells alone. Cold target inhibition assays. Unlabeled (cold) cells were added to wells containing labeled (hot) target cells at cold to hot cell ratios ranging from 16: 1 to 0: 1. In these assays, PBL in 50 ~1 of target cell growth medium and 2% HI-FCS were added to 50 ~1 of cold cells or to 504 of medium alone. Labeled target cells (50 ~1) were then added and incubated for 18 hr (37°C 5% C02). The percentage reduction of CTL lolling was calculated from the percentage specific release of PBL against target cells with no cold cells added at an E:T ratio of 50: 1. RESULTS Antibody- and Complement-Mediated sarcomas

Cytotoxicity

against Lymphomas

and Fibro-

Grant et al. (20) have shown that FeLV-producer lymphoma lines were lolled in the presence of cat complement (C) and CDA in sera from FeLV-exposed cats.

FELINE CYTOTOXIC

161

RESPONSES TO FeLV or FeSV

Furthermore, the presence of high titers of antibodies, including CDA, in sera from FeLV-exposed cats protected them from FeLV-associated lymphoma growth (lo12). Sera from cats inoculated with FeSV, and whose fibrosarcomas regressed, also contained high titers of antibodies as measured on lymphoma cells, and a protective effect for these antibodies in FeSV immunity has been postulated (13). We tested FeSV tumor regressor sera with high CDA titers to feline lymphomas (FL74 and 328 1) for lytic activity against FeSV-transformed fibroblasts (FTF), but were unable to detect cytotoxic antibody against this class of target cells in the presence of cat or rabbit C. An example of the cytotoxic effects of this class of immune regressor serum is shown in Table 1. Sera from cats immunized with autochthonous FeSV-transformed fibroblasts contained antibodies to ATF as shown by indirect immunofluoresence assays and also by immunoprecipitation of viral products from FeSV-transformed cells. These sera were also tested for CDA activity to FL74 and 328 1 and were shown to have high levels of lytic antibody ( 1: 10 to 1:60) to both target cell lines (Table 1). In order to determine if these antibodies might protect from FeSV-induced fibrosarcomas, we tested these sera against a variety of FTF targets. No lysis was obtained when sera from ATF-immunized cats were tested, at various intervals following immunization, on ATF and on six unrelated FTF lines. In addition, we have tested 34 sera derived from FeSV-inoculated tumor regressor cats whose lytic titers on FL74 varied from 1:8 to 15 12, but no lysis was ever detected when FfF were substituted for lymphoma targets (Table 1). As a positive control for antibody- and C-mediated lysis of FTF, we utilized goat

TABLE

1

Lytic Effects of Feline Sera in CDA Assays Lytic CL50 with target cell” Complement source

Serum

FIFb

FL74

3281

FeLV/FeSV nonexposed

Cat’ Rabbitd

0 0

0 0

0 0

FeLV immune

Cat Rabbit

0 0

I:57 I:148

152 1:lOO

FeSV tumor regressor

Cat Rabbit

0 0

I:120 I:220

I:1 16 1:189

ATF immunized No. I

Cat Rabbit

0 0

I:27 I:12

1:29 I:30

ATF-immunized

Cat Rabbit

0 0

1:56 I :30

1:59 1:56

Cat Rabbit

0

I:8 1:12,800

1:2 1:10,600

Goat anti-FeLV

No. 2

1:505

’ Serum dilution which resulted in 50% specific “Cr release. b FeSV-transformed fibroblasts. Serum from ATF-immunized No. 1 is autochthonous for FIF and serum from ATF immunized No. 2 is allogeneic for FTF. ’ 18-hr incubation. d 2-hr incubation.

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serum raised to disrupted FeLV; it was effective in lysing FTF in the presence of rabbit C, but was not cytotoxic when assayed with cat C (Table 1). The results show that the lytic effects of CDA are restricted to FeLV-producer lymphoma lines, and that under identical conditions no lysis is obtained when the sera are assayed against FeSV-transformed fibroblast targets. Lack ofAntibody-Dependent

Cellular Cytotoxicity

against Feline Tumor Target Cells

As CDA plays a major role in FeLV immunity and protection (12, 18, 20, 2 l), we examined the possibility that immune sera might also mediate ADCC against feline lymphomas and/or fibrosarcomas. Sera from nonimmune, FeLV immune tumor regressor, FeSV-inoculated tumor regressor, and ATF-immunized cats were heat inactivated and used to sensitize labeled target cells in the presence of normal cat PBL to test for ADCC activity. In order to detect ADCC effects, serum dilutions were varied from 1:4 to 1:4000, E:T ratios were varied from 5: 1 to 200: 1, and incubation periods were varied from 4 to 18 hr. No specific cytotoxicity was measured (against either FeLV-producer lymphomas or FTF) in 5‘Cr release assays which involved either presensitization of target cells prior to assay or assays where immune sera, target cells, and effector cells were mixed and incubated (Table 2). To determine that cat blood contained cells with ADCC effector function, we utilized a xenogeneic system involving murine tumor target cells (P815 and EL4) of known sensitivity to ADCC-mediated lysis (14,22, 23). In the presence of feline antiP815 or anti-EL4 sera, cat PBL were effective at mediating ADCC effects (Table 2). The ADCC response was dependent both on serum dilution and on the E:T ratio. Specific antiserum to murine target cells was most effective as a sensitizing agent, but there was cross-reactivity between feline sera to P815 and EL4, presumably due to the broad spectrum cat anti-mouse reactivity. We have, therefore, measured ADCC activity using feline effector cells in assays involving murine target cells, but we have been unable to identify any specific ADCC effects against feline lymphoma cells or transformed fibroblasts in the presence of a variety of different FeLV/FeSV immune sera. TABLE 2 ADCC Assays with Feline Effector Cells % Specific “0

release from target cells*

Murine Serum” source Mouse Cat

Goat ’ Sera dilutions = 1: 10. *E:T = 5O:l. ’ -, not determined.

Feline

Anti-

EL4

P815

FL74

Fl-F

EL4 P815 EL4 FL74 mF FeLV

33 31 66 0 0 0

0 57 38 0 0 0

-c 0 0 0

0 0 0

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RESPONSES TO FeLV or FeSV

Detection of Natural Killer Cells to Feline Lymphoma

Cell Lines

In many animal systems the cytotoxic effects of nonimmune lymphocytes (NK) to certain lysis-sensitive target cells is considered relevant to tumor immunity (14). We have detected NK cells among PBL isolated from normal cats (i.e., no known exposure to FeLV) which were cytotoxic for FL74 (24). Nonimmune PBL isolated from three cats killed three different FeLV-producer lymphoma lines but caused no lysis of FTF (Table 3). We were able to measure NK activity in PBL from all 18 normal cats tested, but there was variation in activity, both among individuals and occasionally within the same animal tested at different times. The results in Table 4 demonstrate this point; PBL from 5 cats were tested against a single target cell line and showed cytotoxic effects at an E:T ratio of 100: 1 ranging from 58 to 12% specific “Cr release. In order to determine if the variability of NK activity among cats was affected by environmental conditions, we compared levels of NK activity among animals living in a normal environment with levels found among cats raised and maintained in a specific pathogen-free facility. None of the 19 SPF cats selected had demonstrable NK activity when tested on several occasions against different lymphoma targets. Three of these cats were removed from the SPF colony and placed in a normal, nonrestricted, facility for 6 months but they did not develop NK activity during this time (Table 5). The possibility, that a function of feline NK cells is to protect animals from FeLVassociated tumor development was tested by comparing levels of NK activity in healthy cats and in pet cats bearing naturally occurring tumors. Blood was removed from 5 tumor-bearing cats with leukemia or lymphoma, and PBL were assayed for NK cytotoxic activity. The range of NK effects detected in tumor-bearing animals was slightly lower than that found in a group of 10 normal cats, but cytotoxicity was extremely variable among animals in both groups (Table 5). Cytotoxic T Lymphocytes Cytotoxic T lymphocytes (CTL) have been detected in the blood of cats immunized with autochthonous FeSV-transformed fibroblasts ( 17). These cytotoxic cells remain in the circulation for approximately 30 days after each immunization and, in each of five cats previously reported, the CTL have demonstrated complete restriction of target cell killing to ATF, i.e., allogeneic FeSV-transformed fibroblasts and autochTABLE 3 Naturally Occurring Cytotoxicity of PBL to FeLV-Producer Lymphoma and FeSV-Transformed Cell Lines % Specific “Cr release (from target cells)” Cat

FL74

F422

3272

FTF

1 2 3

36 49 30

48 43 32

39 60 67

0 0 0

’ E:T ratio = 200:1, 18-hr, incubation period, SD on triplicate wells
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TABLE 4 NK Activity in PBL from Normal Cats 70 Specific release from FL74” E:T

Cat No.:

400: 1 200: 1 1OO:l

1

2

3

4

5

-b 57 58

86 23

81 59 12

48 22 26

69 52 33

’ 18-hr incubation period, SD from triplicate wells ~8%. b -, not determined.

thonous nontransformed fibroblasts were not killed ( 17). Results of further experiments designed to explore CTL-like killing are shown in Table 6. Cats B, C, G, and J were each immunized with ATF and PBL were isolated between 14 and 21 days postimmunization. The time course for appearance of CTL in these cats was similar to that for the animals reported earlier ( 17). PBL from cats C and G demonstrated complete restriction of target cell killing such that CTL from cats C and G were cytotoxic only for ATF and not for autochthonous normal fibroblasts or for unrelated FTF targets. CTL from cats B and J, however, exhibited both specific and limited cross-reactive cytotoxic activity. PBL from cat B (B-CTL) caused 88% specific 5’Cr release from ATF, they were not cytotoxic to FTF from cats C and G, but they were cytotoxic for FTF from cat J (60%). In the reciprocal experiment using effector cells from cat J (J-CTL) and target cells from cats J, B, C, and G, similar results were obtained; JCTL were specifically cytotoxic for ATF and for B-FTF, though they had no effect on FIEF from cats C or G (Table 6). In order to investigate whether the determinants expressed by cells from cats B and J were similar, we performed cold target inhibition studies for specific and cross-reactive CTL activity. Unlabeled (cold) cells and labeled (hot) target cells were mixed at a variety of ratios to test for blocking of cytotoxicity to both ATF and cross-reactive FTF target cells. At a PBL effector to hot target cell ratio of 50: 1 and a cold to hot target cell ratio of 16: 1, the cytotoxic effect of B-CTL on 51Cr-labeled ATF was inhibited 67% by cold B cells and 25% by cold J cells. Reciprocally, the cytotoxic effect of J-CTL on “Cr-labeled ATF was reduced 50 and 22% by cold J and B cells, respectively. In all combinations of B and J cells, the TABLE 5 The NK Activity of PBL from SPF, Normal Healthy, and Tumor-Bearing

Cats

% Specific 5’Cr release from FL74 targer Cat

No. cats tested

SPF Normal Tumor bearing

19 10 5

a 18-hr incubation. b Mean (range).

E:T:

400: 1 0 58 (1 1-86)b 40 (11-71)

200: 1 0 34 (10-66) 26 (4-44)

1OO:l 0 20 (8-52) 15 (6-36)

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RESPONSES TO FeLV or FeSV TABLE 6

Serum Blocking of Cytotoxic T-Lymphocyte Activity % Specific “Cr release from target cellsb Cat

Serum”

B

C

G

J

B

+

88 76

6 0

3 2

60 56

+

0 2

71 70

3 3

9 6

+

0 0

8 5

69 60

0 0

+

40 41

11 8

6 3

51 53

C G J

’ Sera tested included nonimmune, ATF-immune

and FeLV/FeSV tumor regressor CDA-positive sera

at a variety of dilutions, results from 1:4 serum dilution are shown. b Cats were bled between Day 14 and 21 following immunization with ATF. E:T ratio = 50: 1. Cats B. C. G, and J are autochthonous with target cells B. C, G, and J, respectively.

extent of blocking was increased if the effector to hot target cell ratio was decreased or if the cold to hot target cell ratio was increased; reduced blocking was obtained by reversing these manipulations. In both combinations cold autochthonous cells were the more efficient inhibitors of ATF killing by CTL, but the cold nonautochthonous cells also inhibited lysis. Irrespective of cell ratios employed, other unrelated FTF lines did not block the cytotoxic effects of CTL from cats B and J. These results are consistent with the possibility that cats B and J share histocompatibility determinants. As sera from ATF-immune cats contained antibodies that bound to, but were not cytotoxic for, ATF (Table I) we tested to determine if such antibodies would block CTL effects against autochthonous transformed target cells. Sera from nonimmune control cats, from ATF-immunized cats, and from FeLV/FeSV tumor regressor animals were each mixed with target cells and CTL assays were performed. No blocking or inhibition of CTL-mediated cytotoxicity was detected against ATF after the addition of immune serum (Table 6). Therefore we have tested a total of eight ATF-immunized cats which have developed CTL in the blood. In six cats cytotoxicity was completely restricted to autochthonous targets, and in two cats cytotoxicity was restricted to autochthonous and one other target cell. CTL activity was not inhibited by the addition of immune serum containing antibodies to ATF, irrespective of the serum dilution employed (range 1:4 to 1:40). DISCUSSION In cats a humoral immune response to FeLV-induced antigens protects against development of leukemia/lymphoma, and the mechanism responsible for this protection is CDA ( 12, 18,20,2 1). Many cats whose FeSV-induced fibrosarcomas regressed also developed antibodies, including CDA, detectable on FeLV-producer lymphoma lines (13, 25). The role these antibodies play in FeSV immunity was postulated to be analogous to that involved with FeLV immunity (11, 13). We have used FeSV-

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transformed fibroblasts as targets in assays containing cat sera with high CDA titers and have shown that FTF were not susceptible to complement-mediated lysis with either cat or rabbit complement. Further, sera from cats immunized with ATF were cytotoxic for lymphoma targets but were not cytotoxic for ATF, though specific antibodies were demonstrated in these sera by immunofluorescence and by immunoprecipitation of viral proteins from disrupted ATF. Given extrapolation to the in vivo situation, it appears that CDA plays a major role in protecting cats from FeLVassociated lymphomas, but that the antibody probably has little effect in mediating FeSV-associated fibrosarcoma regression. These findings are consistent with other studies showing that FeSV tumor immunity sometimes developed in the absence of CDA (25), and that fibrosarcoma regression sometimes occurred in persistent FeLV viremic cats that lacked detectable CDA (26). Rojko et al. (27) have demonstrated that a mixture of normal cat PBL and sera from animals with self-limiting FeLV infections exhibited ADCC activity to nontransformed virus-infected target cells. We have tested a variety of immune sera for ADCC against both lymphoid and fibroblastic targets, but no cytotoxic effects were detected. ADCC effector cells were detected in cat PBL by utilizing murine tumor target cells and cat anti-mouse immune sera. In appropriate circumstances, therefore, cats will produce antibodies with ADCC function, and as ADCC effector cells are present in feline blood, we conclude that either the feline target cells used are refractory to ADCC-mediated lysis, or that FeLV/FeSV infections do not stimulate the appearance of antibodies with ADCC characteristics. In either case it seems unlikely that ADCCassociated immunity protects cats from FeLV-/FeSV-related tumors. Feline NK activity in healthy nonimmune cats has been reported briefly (24), and here that work is extended. NK cells were cytotoxic for several FeLV-producer lymphoma lines but not to a variety of FeSV-transformed fibroblast lines. In normal cats the levels of NK activity varied, but cytotoxicity was reproducibly detected. Paradoxically, we were not able to detect NK cytotoxicity in PBL from 19 cats which had been bred and maintained in a SPF colony; moreover, NK activity did not appear after removal of these cats from the controlled SPF environment and placement under normal conditions for a period of 6 months. The finding that the group of SPF cats lacked NK cells differs from a finding by Koren et al. (28), in which SPF swine were shown to have normal levels of NK activity. Our SPF cats, however, are relatively inbred, so it is possible that this lack of NK activity has a genetic basis. NK cells have been reported to function in tumor immune surveillance (14). Evidence supporting this concept is derived from nude mice that lack cytotoxic T effector cells but have normal levels of spontaneous tumors (14). Truesdale et al. (29) inoculated mice with Moloney murine sarcoma virus (M-MSV) and then compared the properties of tumors in normal animals and in NK-deficient beige mutants of the same strain. The beige mice were found to be somewhat less susceptible to M-MSV-induced tumor growth than were mice with normal NK activity. In this study, we found normal levels of NK activity in five tumor-bearing cats. As PBL from these animals exhibited NK activity to lymphoma cell lines at times when the autochthonous tumors were progressing inexorably, and because NK cells were not cytotoxic for FTF, we must question the biological relevance of these cytotoxic cells in terms of protection against FeLV-/FeSV-associated tumors of cats. Cats immunized with autochthonous FeSV-transformed fibroblasts developed circulating lymphocytes which killed ATF, but did not kill unrelated FTF, autochthonous

FELINE CYTOTOXIC

RESPONSES TO FeLV or FeSV

167

nontransformed fibroblasts, or FeLV-producer lymphoma lines ( 17). Although cell surface antigens which define feline T-cell subsets have not been described, we feel that these effector lymphocytes behave as histocompatibility restricted CTL. Two ATF immunized cats, however, were found whose CTL exhibited limited cross-reactivity, but the data were consistent with the concept that the pair share histocompatibility determinants analogous to murine H-2K and H-2D products. Demonstration of CTL whose activity is restricted to self-/virus-induced determinants is strong evidence to indicate that cats exhibit markers of histocompatibility and is at odds with a recent report suggesting the lack of a conventional histocompatibility system in this species (30). The finding also helps to explain the lack of alloreactive CTL in cats immunized with live unrelated FeLV-producing feline lymphomas (24) because following immunization a transient viremia is frequently observed (25). Presumably viremia results from FeLV shed from the injected foreign lymphoma cells. FeLV then infects recipient lymphocytes, and therefore the appropriate CTL target is not the injected lymphoma cell line but rather the FeLV-infected cells from the recipient. In conclusion, we believe that immune mechanisms play major roles in protecting cats from FeLV- and FeSV-associated tumors, but just as the classes of tumors induced by these viruses are different, the immune mechanisms that limit tumor progression are different. Lytic CDA and complement, which kill FeLV-producing tumor cells of lymphoid origin, appear to be the primary mechanism in FeLV-related leukemia/ lymphoma immunity, the cytotoxic T lymphocytes, which are specific for “altered self,” are probably the major defense mechanism against fibroblastic tumors induced by FeSV. ACKNOWLEDGMENTS We would like to express our gratitude to Mike Michalek, Barbara Emisse, Dr. Fernando de Noronha, and Dr. Sue Cotter for their invaluable assistance. This work was supported by PHS Grants CA 24608 and T32 CA09031 awarded by the National Cancer Institute, DHHS. C.K.G. is a Scholar of the Leukemia Society of America and of the American Cancer Society (Massachusetts Division).

REFERENCES 1. Hardy, W. D., Jr., Zuckerman, E. E., MacEwen, E. G., Hayes, A. A., and Essex, M., Nature (London) 270,249, 1977. 2. Hardy, W. D., Jr., in “Feline Leukemia Virus” (W. D. Hardy, Jr., M. Essex, and A. J. McClelland, Eds.), pp. 3-78. Elsevier/North-Holland, New York, 1980. 3. Snyder, S. P., and Theilen, G. H. Nature (London) 221, 1074, 1969. 4. Gardner, M. B., Rongey, R. W., Amstein, P., Estes, J. D., Sartna, P., Huebner, R. J., and Rickard, C. G., Nature

(London)

226,801,

1970.

5. McDonough, S. K., Larsen, S., Brodey, R. S., Stock, M. D., and Hardy, W. D., Jr., Cancer Res. 31, 952, 1971. 6. Frankel, A. E., Gilbert, H. J., Pot-zig, K. J., Scolnick, E. M., and Aaronson, S. A., J. Viral. 30, 821, 1979. 7. Franchini, G., Even, J., Sherr, C. J., and Wong-Staal, F., Nature (London) 290, 154, 198 1. 8. Hardy, W. D., Jr., In “Feline Leukemia Virus” (W. D. Hardy, Jr., M. Essex, and A. J. McClelland, Eds.), pp. 79-l 18. Elsevier/North Holland, New York, 1980. 9. Chen, A. P., Essex, M., Shadduck, J. A., Niederkom, J. Y., and Albert, D., Proc. Natl. Acad. Sci. USA 78,3915, 1981. 10. Essex, M., Sliski, A. H., Cotter, S. M., Jakowski, R. M., and Hardy, W. D., Jr., Science 190, 790, 1975.

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I I. Essex, M., In “Contemporary Topics in Immunobiology: Immunobiology of Oncogenic Viruses” (M. G. Hanna, Jr., and F. Rapp, Ed%), Vol. 6, pp. 71-106. Plenum, New York, 1977. 12. Grant, C. K., Harris, D., Essex, M. E., Pickard, D. K., Hardy, W. D., Jr., and de Noronha, F., 1. Nufl. Cancer Inst. 64, 1527, 1980. 13. Essex, M., Klein, G., Snyder, S. P., and Harrold, J. B., Nature (London) 233, 195, 1971. 14. Herberman, R., and Holden, H. T., Advan. Cancer Rex 27, 305, 1978. IS. Collavo, D., Colombatti, A., Chieco-Bianchi, L., and Davies, A. J. S., Nafure (London) 249, 169, 1974. 16. Glaser, M., Lavrin, D. H., and Herberman, R. B., J. fmmunol. 116,430, 1976. 17. McCarty, J. M., and Grant, C. K., Inc. J. Cancer 31,627, 1983. 18. Grant, C. K., and Michalek, M., Inf. J. Cancer 28, 209, 1981. 19. Hardy, W. D., Jr., Hirshaut, Y., and Hess, P., In “Unifying Concepts of Leukemia, Bibl. Haemat.” (R. M. Dutcher and L. Chieco-Bianchi, Eds.) Vol. 39, pp. 778-799. Karger, Basel. 1973. 20. Grant, C. K. DeBoer, D. J., Essex, M., Worley, M. B., and Higgens, J., J. Immunol. 119, 401, 1977. 21. Grant, C. K., Pickard, D. K., Ramaika, C., Madewell, B. R., and Essex, M., Cancer Res. 39, 75, 1979. 22. Press, H. F., and Jondal, M., Clin. Exp. Immunol. 21, 226, 1975. 23. Grant, C. K., Adams, E., and Miller, H. R. P., Eur. J. Immunol. 5, 324, 1975. 24. McCarty, J. M., and Grant, C. K., ln “Feline Leukemia Virus” (W. D. Hardy, Jr., M. Essex, and A. J. McClelland, Eds.), pp. 203-210. Elsevier/North-Holland, New York, 1980. 25. Grant, C. K., de Noronha, F., Tusch, C., Michalek, M. T., and McLane, M. F., J. Null. Cancer Inst. 65, 1285, 1980. 26. delrloronha, F., Grant, C. K., Lutz, H., Keyes, A., and Rowston, W., Cancer Res. 43, 1663, 1983. 27. Rojko, J. L., Hoover, E. A., Quackenbush, S. L., and Olsen, R. G., Nature (London) 298, 385, 1982. 28. Koren, H. S., Amos, D. B., and Kim, Y. B., Proc. Null. Acad. Sci. USA 75, 5127, 1978. 29. Truesdale, A. T., Johnson, D. A., Bed&an, H. G., Outzen, H. C., and Carlson, G. A., Cell. Immunol. 74, 120, 1982. 30. Pollack, M. S., Mastrata, F., Chin-Louis, J., Mooney, S., Hayes, A., Immunogenetics 16, 339, 1982.