Journal of Immunological Methods, 140 (1991) 269-279

269

© 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100220F JIM05980

Identification of unique murine tumor associated antigens by tumor infiltrating lymphocytes using tumor specific secretion of interferon-T and tumor necrosis factor R i c h a r d J. Barth, Jr., J a m e s J. Mul6, A n t h o n y L. Asher, Martin G. S a n d a a n d Steven A. R o s e n b e r g Surgery Branch, National Cancer Institute, Building 10, Room 2B46, National Institutes of Heahh. Bethesda, MD 20892, U.S.A.

(Received27 February 1991. accepted 15 March 1991)

Stimulation of multiple CD8 ÷ murine tumor infiltrating lymphocyte (TIL) lines and one TIL clone with the tumor of origin of the TIL induced at least three-fold more secretion of T N F a n d / o r INF-y than was elicited by other syngeneic, methylcholanthrene (MCA) induced sarcomas. TIL which specifically secreted lymphokines were generated from three different sarcomas. Specific iymphokine secretion was a stable characteristic of the lines over time. IL-2 was necessary for maximal lymphokine secretion by TIL. These investigations demonstrate that lymphokine secretion by CD8 + lymphocytes derived from tumor bearing mice can be used to define unique tumor associated antigens on at least three different sarcomas and may be valuable in studies of the biologic nature of these antigens and of the adoptive imtnullotilerapy of cancer. Key words: Tumor infiltratinglymphocyte;Lymphokinesecretion: Interferon-y: Tumor necrosis factor; Adoptiv,:immunotherapy;

Tumor antigens

Introduction Murine class 1 MHC restricted CD8 ÷ lymphocytes were defined on the basis of their in vitro cytotoxic capabilities (Shiku et al., 1975). Subsequent investigations have revealed that CD8 ÷ lymphocytes may also have a helper function mediated by the secretion of lymphokines. For ex-

Correspondence to: J.J. Mul6, Surgery Branch. National Cancer Institute, Building 10. Room 2B46. National Institutes of Health, Bethesda, MD 20892. U.S.A. Abbret'iations: CM. complete media: GM-CSF. granulocyte/maerophage colony stimulating factor; MCA. 3-methyleholanthrene; TIL, tumor infiltrating lymphocyte.

ample, non specific stimulation of CD8 + lymphocyte clones with mitogens has induced the secretion of IL-2, IFN-y, lymphotoxin, IL-3 and GMCSF (Prystowski et al., 1982; Roopenian et al., 1983; Guerne et al., 1984; Kelso and Glasebrook, 1984; Fong and Mosmann, 1990). Stimulation of the T cell receptor of CD8 + clones with anti-CD3 mAb has been shown to induce secretion of IFNy, TNF-a, IL-3 and GM-CSF (Gajewski and Fitch, 1990). In addition, alloantigens have stimulated secretion of IL-2 and IFN-y by some CD8 ÷ lymphocytes (Keiso and Glasebrook, 1984; Mizuochi et al., 1985; Fong and Mossmann, 1990). However, there has been little investigation of the effect of tumor antigens as stimulants for lymphokine secretion by CD8 + lymphocytes.

2711 We have previously shown that CD8 + tumor infiltrating lymphocytes (TIL), derived from tumor bearing mice, could be used in cytotoxicity assays to define unique tumor associated antigens on four different sarcomas (Barth et al., 1990). The current study investigates whether CD8 + TIL also specifically secrete iymphokines in response to stimulation with a group of syngeneic sarcomas. Lymphokine release by a TIL that is specific for the tumor of origin might serve as a sensitive assay for studies of the nature of murine tumor antigens. Furthermore, since many of the lymphokines secreted by lymphocytes in response to mitogen stimulation have potent immunomodulatory and/or anti-tumor effects, the specific secretion of lymphokines at the site of a tumor may be important in the mechanism of tumor regressions mediated by adoptively transferred TIL.

203 (but not from challenge with MCA 105, 205 or 207); therefore, MCA 203 and 233 appear to share common tumor antigens. MC 38 is an immunogenic colon adenocarcinoma of B6 origin (Spiess et ai., 1987). P815 mastocytoma was kindly provided by Dr. Judith Pace (University of Kansas).

Depletion of macrophages from tumors Single cell suspensions were depleted of macrophages as follows (Wong and Varesio, 1984): 107 tumor cells/ml were suspended in complete medium (CM) with 15 mg/ml carbonyl-iron beads (4.5.5.2 /.tm particle diameter, Sigma, St. Louis, MO) and incubated at 37 ° C, 5% COz on a rocker platform for 1 h. Phagocytic macrophages were then removed with a magnet and the remaining cells were washed three times with HBSS (Biofluids, Rockville, MD) prior to use in lymphokine release assays or FACS analyses.

Materials and methods

Mice Female C57BL/6 mice (denoted B6) were obtained from the Animal Production Colonies of the National Cancer Institute, NIH (Frederick Facility), Frederick, MD, and from the Charles River Laboratories, Wilmington, MA. All mice were used at age 10 weeks or older. Tumors The MCA 105, 203, 205, 207 and 233 tumors are weakly immunogenic, 3-methylcholanthrene (MCA) induced fibrosarcomas of B6 origin (Shu and Rosenberg, 1985). These tumors were generated in our laboratory and were passaged subcutaneously for ten generations, at which time a cryopreserved vial from the first generation was thawed and transplanted. The subcutaneous injection of MCA 203, 205 and 207 with C part'urn has been shown to be capable of eliciting protective immunity to subsequent tumor challenge which is specific to the tumor used for the immunization (Barth et al., 1990). Similar immunization/challenge experiments using MCA 105 have shown that MCA 105, 203, 205 and 207 do not share antigens. However, immunization with MCA 233 will protect mice from challenge with

TIL culture conditions TIL were cultured following immunoselection from freshly digested tumors using anti-Thy 1.2 mAb coated magnetic beads as described previously (Barth et al., 1990; Yang et al., 1990). TIL were then stimulated with irradiated tumor cells and B6 splenocytes on day 1 of culture and maintained in CM supplemented with 10-20 U / m l of recombinant 1L-2 (Cetus Corp., Emeryville, CA). Fresh CM containing rIL-2 was added to the cultures every 3 days. Every 10 days the cultures were stimulated with irradiated (3000 tad) tumor cells at 5 × 106 tumor cells/plate (Costar, Cambridge, MA). TIL clone 205-87 was generated as follows. The MCA 205 tumor was cloned at 0.3 cells/well; one clone, designated WP4, was subsequently passaged in vivo. TIL generated from the WP4 tumor were placed in 96 well, flat bottom microtiter plates (Costar) in CM + 20 U / m l rIL-2 at 0.3 ceils/well. Irradiated (3000 tad) tumor cells and lethally irradiated (3000 rad) TIL feeders were added to each well at 2 × 104 cells/well and 105 cells/well, respectively. (In multiple experiments we have not observed proliferation of TIL after 3000 rad irradiation.) The medium was changed every 4 days; at day 10 the culture was restimulated with irradiated tumor

271 and TIL. On day 16, 3 of 192 wells were observed to have TIL colonies and these were expanded in CM + 20 U rlL-2/ml with restimulation with irradiated tumor every 10 days.

Fluorescence actit'ated cell sorter analysis Flow cytometry analysis of lymphocyte surface phenotypes was carried out by direct immunofluorescenee using a FACS 440 flow microfluorometer (Becton Dickinson and Co., Mountain View, CA). Pelleted single cell suspensions of l0 n cells were resuspended in 20 #1 of appropriately titered, fluorescein (FITC) conjugated, anti-Lyt 2 (mAb 2.43, American Type Culture Collection (ATCC), Rockville, MD) or anti-L3T4 (mAb GK1.5, ATCC) mAbs (Becton Dickinson and Co.). The cells were then washed and analyzed by FACS. Flow cytometry analysis of tumor cells and macrophage depleted tumor cells was performed as follows. The samples were first blocked for non-specific antibody binding by incubation of 5 × 105 cells with 1 mg/ml murine IgG (Jackson Immunoresearch Laboratories, West Grove, PA) in HBSS with 2.5% heat inactivated FCS for 31) min at 4°C. Cells were washed once and incubated with F4/80, a rat mAb directed specifically against C57BL/6 macrophages (Austyn and Gordon, 1981), at a 1/10 dilution at 4 ° C for 30 min (mAb kindly provided by Dr. Kevan Roberts, NIH). All cells were then resuspended in a solution of 50 p.g/ml FITC conjugated Fab fragments of mouse anti-rat IgG (Jackson lmmunoresearch Laboratories) at 4 ° C for 30 min. Cells were then washed and analyzed by FACS. Lymphokine secretion and detection TIL were harvested, centrifuged, resuspc,,ded in CM plus 20 U / m l rlL-2 and plated at 5 × 105 cells/well in a 2 ml volume in 24 well plates (Costar). 106 freshly digested and washed tumor cells were added in 100 #1 of CM to each well. The TIL plus tumor mixture was incubated at 37°C for 12-18 h and then the superuatant was aspirated, centrifuged at 1800 rpm to rcmove any cells, decanted and frozen at - 7 0 ° C . Aliquots were then thawed and tested in duplicate in an IFN-y ELISA (Amgen, Thousand Oaks, CA) and a TNF ELISA (Genzymc Corp., Boston, MA), The TNF ELISA employed a solid phase ham-

ster mAb specific for murine TNF. A goat polyclonal anti-murine TNF second antibody was then used, followed by a horseradish peroxidase conjugated donkey anti-goat lg. The antibodies detected both mouse TNF-a and TNF-/3, but the assay was specifically calibrated for TNF-a. This ELISA has been determined not to detect rll.,-l, rlL-2, rlL-3, rGM-CSF or rlFN-y. The IFN-y ELISA utilized a solid phase, specific anti-murine 1FN-y mAb and then a biotinylated second antimurine IFN-y mAb was added. The tumors used for TIL stimulation were also plated and their supernatants were assayed for the presence of lymphokines in each experiment. On multiple determinations, 1FN-y concentrations in the tumor supernatants were negligible (< 2 U/ml). TNF concentrations in the tumor supernatants were variable and ranged from 0 to 100 pg/ml. These baseline values were subtracted from the concentrations of TNF observed in the TIL plus tumor wells to determine the amount of TNF secreted by the TIL.

Statistics Student's t test was used for the comparisons of means in Table V. Two sided p values are presented in all experiments.

Results

Specific secretion of TNF and IFN-y by CD8 ÷ TIL Multiple TIL lines and one TIL clone were generated from the MCA sarcomas 105, 203. 205 and 233 and were found to be CD4-, CD8 ~ by FACS analysis. The specificity of tumor-induced lymphokine secretion by these TIL was tested by adding I x ll) h freshly harvested and digested tumor cells to 5 × 105 TIL in CM containing 20 U / m l rlL-2. The supernatants were assayed 1418 h later for the presence of lymphokines using ELISAs specific for murine TNF and IFN-y. Eight different TIL cultures derived from three different sarcomas, and one TIL clone (205-87) from the WP4 subline of MCA 205 were tested for TNF secretion. Three characteristic experiments are presented in Table I. All TIL lines that were tested in each experiment are shown in

272 Table I. Stimulation with the tumor of origin of the TIL (the relevant tumor) is highlighted with a box. Fol purposes of analysis, we have arbitrarily considered secretion of T N F (and IFN-y) to be specific if the amount secreted in response to relevant tumor stimulation was > three times that secreted in response to at least two other syngeneic, M C A induced irrelevant tumors in the same experiment. As shown in Table l, in the absence of tumor stimulation, there was little constitutive secretion of TNF. In experiment 1, stimulation with the relevant tumor induced secretion of > 3-fold more T N F than that secreted in response to irrelevant tumor stimulation in seven of eight cultures. T I L line 205-21C did not secrete T N F in response to tumor stimulation. When these same TIL were tested at a later date (experiment 2), specific secretion of T N F in response to relevant tumor stimulation could still be observed in three T I L lines (203-72, 203-23B and 205-21B) and TIL clone 205-87. TIL lines 203-23A, 203-23C and

205-21A no longer specificaliy secreted TNF; TIL line 205-21C remained non responsive to stimulation. In experiment 3, ~.pecific secretion of T N F in response to M C A 105, as well as M C A 203, was observed. Specific secretion of T N F in response to MCA 105 has been observed in multiple experiments (Table liD. The supernatants from these experiments were also assayed for the presence of IFN-y (Table II). In all experiments, in the absence of tumor stimulation, there was little constitutive secretion of IFN-y. In experiment 1, stimulation with the relevant tumor induced secretion of 3-10-fold more IFN-y than that secreted in response to irrelevant tumor stimulation in five of the eight cultures tested (203-23C, 205-87, 205-21A, 205-21B and 205-21C). Three lines (203-72, 203-23A and 203-23B) secreted at least twice as much IFN-y upon relevant compared with irrelevant tumor stimulation. In experiment 2, six cultures specifically secreted IFN-y in response to relevant tumor stimulation; two lines (203-23A and 205-21C)

TABLE 1 TUMOR-SPECIFIC SECRETION OF TNF BY CD8 + TIL ExpI. #

TIL culture

Tumor of origin of TIL

(1)

203-72 203-23A 203-23B 203-23C 205-87 205-21A 205-21B 205-21C

203 203 203 203 205 205 205 205

TNF a MCA tumor stimulator b None 105 15 0 35 0 15 0 0 0 65 30 t00 65 25 0 45 0

203-72 203-23A 203-23B 203-23C 205-87 205-21A 205-21B 205-21C

203 203 203 203 205 205 205 205

25 35 40 20 50 115 40 , 25

105 75 5 35 85 205 0 70

105-53 203-72

105 203

0 20

~ 140

(2)

(3)

203 ~ ~

205 40 25 0 0

35 65 0 0 ~ ~

95 95 35 0 45 105 0 25 110

~

NT NT

a pg/2.5 × 105 "rlL/ml. hiT = not tested. Stimulation with the tumor of origin of the TIL is highlightedwith a box. b All 'IIL cultures at 5 × 10s cells/well were stimulated simultaneouslywith 106 tumor cells/well. Supernatants were collected at 14 h and assayed by ELISA.

273 TABLE I1 TUMOR-SPECIFIC SECRETION OF IFN-y BY CD8 ~" T1L Expt. #

TIL culture

Tumor of origin of TIL

IFN-y ~ MCA tumor stimulator h 105

203

205

(I)

203-72 203-23A 203-23B 203-23C 205-87 205-2 IA 205-21B 205-21C

203 203 203 203 205 205 205 205

0 2 0 0 0 0 0 0

39 17 23 7 7 8 9 9

lTrff'] 1~7~j

41 24 42 17

203-72 203-23A 203-23B 203-23C 205-87 205-21A 205-21B 205-21C

203 203 203 203 205 205 205 205

1 4 l 0 5 I0 3 2

9 7 l0 0 9 10 5 4

105-53 203-72

105 203

3 l

[:~ 26

None

(2)

(3)

10 12 II 6 ~ ~

10 4 l0 0 6 8 3 l 14

~1 [

NT NT

1

a U/2.5 × 105 TIL/ml. Stimulation with the tumor of origin of the TIL is highlighted with a box. NT = not tested. h All TIL cultures at 5 × 105 cells/well were stimulated with 10t' tumor cells/well Supernatants were collected at 14 h and assayed by ELISA.

did not specifically secrete I F N - y . In e x p e r i m e n t 3, specific secretion of I F N - y in r e s p o n s e to M C A 105 was seen; this has been observed in multiple e x p e r i m e n t s (Table Ill).

A l t h o u g h lymphokine secretion by some T I L cultures may change over time (e.g., line 203-23C, Table 1), most T I L cultures that exhibited a pattern of specific lymphokine secretion continued

TABLE III SPECIFICITY OF LYMPHOKINE SECRETION IS A STABLE CHARACTERISTIC OF T1L CULTURES OVER TIME T1L Cx

Culture Age (days)

MCA tumor stimulator " IFN-y secretion b None

Rel.

105-53

49 82 94 135

I 2 2 1

~ ~

205-87

99 202 229

4 4 6

['~ ~

TNF secretion b Irr.

Irr.

None

Irr.

lrr.

5 8 3 6

20 19 6 17

5 10 NT 2o

~ [ ~9~8!I

110 0 NT 140

65 0 NT 200

21 17 12

NT

2(111 51) 25

~ [1~220 I

300 45 185

NT 40 50

11

7

Rel.

~' The relevant (Rel.) tumor is the tumor of origin of the TIE: stimulation with relevant tumor is highlighted with a box. Irrelevant (lrr.) tumors are other syngeneic, weakly immunogenic MCA induced sarcomas. b Secretion of lymphokines was measured in response to tumor stimulators as described in the materials and methods section. IFN-'/ units are U/2.5 × l05 eells/ml" TNF units are pg/2.5 × 10~ cells/ml. NT = not tested.

274 to specifically secrete T N F a n d / o r IFN-~ over time (e.g., TIL lines 203-72, 203-23B, 205-21A, 205-21B and T1L clone 205-87, Tables I and II). Two TIL lines (105-53 and 233-92B) and one TIL clone (205-87) were assayed multiple times over a 3 month period to determine if specific lymphokine secretion was a stable, reproducible property. As shown in Table III, secretion of IFN-~, and T N F by TIL line 105-53 and TIL clone 205-87 remained specific over a three month period. After 6 months in culture, lymphokine secretion by line 105-53 became non-specific. Secretion of IFN-~/ by TIL line 233-92B was also specific in multiple assays during a 3 month period, although this culture did not secrete ~ignificant amounts of T N F (data not shown). These experiments demonstrate that multiple CD8 + TIL cultures can be generated which respond to the tumor from which the lymphocytes were derived with at least three-fold more T N F a n d / o r IFN-~, secretion than that elicited by other syngeneic, MCA induced sarcomas. TIL that specifically secreted lymphokines were generated from three different sarcomas.

Frequency of lymphokine secreting TIL To assess the frequency of generation of TIL which specifically secrete lymphokines, M C A 203, 205 and 233 were each grown subcutaneously in several mice and then tumors from individual mice were separately harvested and used to initiate TIL cultures. Established TIL cultures were assayed for specific lymphokine secretion after 3-5 weeks of growth. As shown in Table IV, nine of 15 cultures specifically secreted IFN-~/and five of 15 cultures specifically secreted T N F upon relevant tumor stimulation. In most cases, the lack of specific lymphokine secretion was due to a failure to secrete substantial amounts of iymphokine rather than high iymphokine secretion in response to irrelevant tumors. All cultures were proliferating well at the time of the assays. These studies indicate that TIL which specifically secrete lymphokines can be relatively frequently generated from these MCA sarcomas. Furthermore, although TIL commonly secrete both T N F and IFN-y in response to tumor stimulation (e.g., TIL 203-72, 203-23B, 205-87, 205-21 B, 105-53 in Tables I, II and III), some TIL may

TABLE IV FREQUENCY OF GENERATION OF TUMOR SPECIFIC, LYMPHOKINESECRETING CD8+ T1L Tumor

# of TIL Cultures tested

# of cultures Specific a secretion of 1FN-y TNF

233 203 205

9 3 3

5 2 2

0 3 2

Total

15

9

5

Secretion of 1FN-~/(andTNF) was considered specific if the amount secreted in response to relevant tumor stimulation was > 3 times the amount secreted by two irrelevant syngeneic, weakly immunogenic.MCA induced tumors.

respond to antigen stimulation with specific secretion of only one of these lymphokines (e.g., TIL from MCA 233 in Table IV).

Lymphokine secretion assay sensitivity The optimal concentrations of TIL and tumor for the detection of specific secretion of IFN-~/ and T N F by TIL were determined by plating 203-23B TIL at three different concentrations in 24 well plates and stimulating with multiple concentrations of relevant (203) and irrelevant (205) MCA tumor. Supernatants were assayed 12 hours after stimulation. As shown in Fig. 1, secretion of IFN-y and T N F was specific at all T I L and tumor concentrations that generated detectable lymphokine secretion. (In a simultaneous experiment, this M C A 205 tumor induced IFN-y and T N F secretion from a 205 TIL culture.) The lower limits and optimal concentrations for iymphokine detection were similar for both IFN-~, and TNF. Specific iymphokine secretion could be observed with as few as 1.2 × 105 T I L / w e l l when 5 - 1 0 × 105 tumor stimulators were added. Increasing the TIL concentration from 1.2 × 105 T I L / w e l l to 5 × 105 T I L / w e l i greatly increased the lymphokine secretion induced by the relevant stimulator; a further increase to 20 × 105 T I L / w e l l did not result in greater iymphokine secretion. For each TIL concentration, increasing tumor concentrations led to higher lymphokine secretion. Tumor concentrations of at least 2 × 105

275

IFN-.y 80 7o ~ 60 I50 ~-

5x10 m

TNF 3oo

Stimulator

H MCA 203 o---o MCA 205

250 r 200 -

10

50 0

70

250 r"

06 5O

200 --

s 40 3O

,soiO0 '--

20 .

5O

70 60

250 ~--

so

200

20 x 105 40

~so

30 20-

tO0 ~ 1.0

1o

o

5xlO"

50

o

2xlo~ 5xlo s lO6 2xlo ~

5 lO

2 lO

s o

o 2 o

Number of Tumor Cells/Well Fig. 1. Secretion of TNF and 1FN-y by T | L is a function of the concentration of TIL and tumor used as stimulators. T I L 2 0 3 - 2 3 B , at three different concentrations, were stimulated with multiple concentrations of relevant 1203) and irrelevant (2051 M C A tumor. The supentatants were assayed 12 h later for TNF and I F N - % T N F units arc p g / m l , IFN-~, units are U / m l .

cells/well were necessary to generate significant lymphokine secretion. At the highest tumor concentrations, IFN-y release on stimulation with irrelevant tumor became notable; this was not observed when T N F secretion was assayed. TIL culture 203-72 was tested in an analogous manner with similar results (data not shown). Based on these experiments, we chose 5 x l05 T I L / w e l l and 106 tumor cells/well as the optimal concentratiotts for detection of specific lymphokine release by TIL. Kinetics of lymphokinesecretion The kinetics of lymphokine secretion were determined using four different TIL cultures generated from three different sarcomas; a representative experiment using TIL 203-72 is shown in Fig. 2. At all times examined from 2 to 24 h, secretion of IFN-y and TNF was specific. The secretion of both lymphokines rapidly reached a plateau. By 12 h, IFN-y secretion was 90% of the maximum amount secreted. TNF secretion was more rapid: 90% of the maximum level was reached by 6 h after stimulation with tumor. The optimal times

,00s'7.~% ~ ~

_--4

o - o 2o5

8O ~o

20

",~ _.0~ . . . . . .

0

2

4

6

8

.°" . . . . . . . . . . .

o,. . . . . . . . . . .

o,

12

18

24

I/ '°°I// T,me (hfs)

Fig. 3. Kinetic,'; o f secretion of T N F and IFN-'), by T I E T I L 203-72. at 5 x 1() ~ cells/well, were stimulated with 106 relevant (203) or irrelevant (205) M C A tumor ceils/v::¢ll aad the supernatants were assayed at various times for TNF and IFN-),. T N F units are p g / 2 . 5 x l0 s cells/ml: IFN-"/ units arc U/2.5 × 10 5 cells/ml.

276 for supernatant assay for specific IFN-y and T N F secretion are between 12-18 h and 6 - 1 8 h, respectively. The plateau in the iymphokine secretion after 12 h appears to be due to the lack of continued secretion of lymphokine rather than to an equilibrium between secretion and utilization. Two TIL cultures which had been stimulated 24 h previously were extensively washed and then replated and the supernatant was assayed 24 h later. In both cultures, the IFN-y concentration was only 10% of that found 24 h after the initial stimulation. These TIL were restimulated at 48 h after their initial stimulation and were found to secrete > 75% of the IFN-y they secreted during the first stimulation, indicating that the TIL do not appear to become refractory to repeated stimulation.

1L-2 dependence of lymphokine secretion We investigated the influence of IL-2 on lymphokine secretion by adding tumor stimulators to TIL in the presence or absence of 20 U / m l rlL-2. The supernatant was assayed for iymphokine secretion 8 hours after the stimulation to minimize any bias due to TIL proliferation fn the presence of IL-2. The results of three separate experiments performed on two different T~L cultures are shown in Table V. The presence of IL-2 made a significant difference in the ~mount of IFN-y secreted in response to tumor ,,timulation. TIL in rlL-2 containing media secreted approximately twice as much IFN-y as did TIL in media without rlL-2

Lack of influe,.ce of macrophages in the stimulating tumor on lymphokine secretion In all experiments, the tumors used for stimulation were plated in the absence of TIL and were also assayed for iymphokine secretion. On multiple determinations, IFN-y concentrations in the tumor supernatants were negligible ( < 2 U / m l ) . T N F concentrations in the tumor superna.:ants were variable and ranged from 0 to 100 pg./ml. These baseline values were subtracted from the concentrations of T N F observed in the TIL plus tumor wells to determine the amount of T N F secreted by the TIL. However, it was possible that IFN-y or other factors secreted by TIL

TABLE V IL-2 IS NECESSARY FOR MAXIMAL SECRETION OF IFN-y BY TIL IN RESPONSE TO TUMOR STIMULATION TIL Cx

IL-2 a in media

105-53

+

IFN-y secretion h No Tumor Tumor stimulator stimulator c 0 (0) 33 (6) o 2 (0.7) 51 (6) e

203-72

+

0 (0) 1 (0)

26 (5) c 54 (5) c

20 U/ml rlL-2. h 2.5 × 105 cells/ml, measured by ELISA at 8 h after stimulation. Values presented are the mean (SEM) of three separate experiments. c 106 relevant tumor stimulators added to 5 x 105 TIL/well. u Stimulated 105-53 culture without rlL-2 vs. with rlL-2, p = 0.028. e Stim~Jlated203-72 culture without rlL-2 vs. with rIL-2, p = 0.002.

might induce T N F secretion by macrophages present in the fresh tumor preparation used to stimulate the TIL; thus, part or all of the T N F detected in the supernatants might be secreted by these macrophages rather than the TIL. We investigated this possibility in two different sets of experiments. Firstly, we compared the concentrations of T N F detected in the supernatants of tumors incubated alone in the presence or absence of 80 U / m l IFN-T (the highest IFN-y concentration detected in our TIL supernatants). In two experiments, IFN-y did not increase the amount of T N F detected in the tumor supernatants (data not shown). Secondly, we stimulated TIL with tumor that had been depleted of macrophages using the carbonyl-iron magnet technique (Wong and Varesio, 1984). Macrophages in the fresh tumor suspensions were detected by FACS using the mAb F 4 / 8 0 , which is specific for murine macrophages (Austyn and Gordo0, 1981). As shown in Table VI, carbonyl iron be~d treatment of MC38 tumor preparations enabled us to obtain a three-fold depletion of macrophages (28-9%); treatment of the WP4 tumor (a clone of M C A 205) led to a ten-fold depletion (4-0.4%). In the absence of tumor stimulation, there was no detectable T N F in the

277 TABLE VI DEPLETION OF MACROPHAGES FROM THE TUMORS USED TO STIMULATE TIL DOES NOT AFFECT THE CONCENTRATION OF LYMFHOKINES IN THE SUPERNATANT Tumor stimulator a

Macro- %F4/80 + phage deple-

TIL

tion b

None MC38 MC38

+

None WP4 WP4

+

[TNF] in supernatant of stimulated TIL ¢

28 9 4 0.4

MC38-1 MC38-1 MC38-1

210 240

205-87 205-87 205-87

0 370 290

0

a WP4 is a clone of MCA 205. b Fresh tumor suspensions were depleted of macrophages using carbonyl iron beads as described in the materials and methods section. c Tumor stimulators (106) were added to 5 × 105 TIL derived from that tumor and the supernatant was assayed at 14 h. for TNF. TNF units are pg/2.5 × 105 cells/ml.

TIL supernatants. Stimulation of T1L with either macrophage depleted or non depleted tumor led to equivalent increases in the amount of T N F detected in the supernatant (Table VI). This experiment was repeated with similar results. Specific stimulation of T N F secretion by TIL could also be achieved by relevant culturea tumor cell lines devoid of macrophages (data not shown). These experimental approaches indicated that the majority of the T N F detected in the fresh tumor stimulations was not secreted by macrophages in the tumor preparations.

Discussion

It has been known for some time that unfractionated splenocytes from mice immunized with tumor, when cultured in vitro with tumor cells, will secrete a factor that will activate macrophages to become cytotoxic (Evans and Alexander, 1971; Kripke et al., 1977). However, to our knowledge, only two previous reports have measured lymphokines secreted by a CD8 +, C D 4 - murine

lymphocyte population in response to tumor stimulation. Matis et al. (Matis et al., 1985) generated a CD8 + lymphocyte clone that secreted IFN-~/in response to stimulation with the leukemia FBL-3; the specificity of IFNq, secretion by these cells was not reported. Yamasaki et al. (1984) described a CD8 + clone that secreted IFN-y in response to a glioma. However, IFN-~/secretion by this clone was non-specific; both a M C A induced glioma and a viraUy induced glioma stimulated secretion. In contrast, in the experiments reported here we have shown that multiple CD8 ÷ lymphocyte cultures can be generated that respond to the tumor from which the lymphocytes were derived with at least three-fold more T N F a n d / o r IFN-~/ secretion than that induced by other syngeneic, M C A induced sarcomas. TIL that specifically secreted iymphokines were generated from three different sarcomas. The amount of 1FN-~, secreted by TIL in response to relevant tumor stimulation is of the same order of magnitude that others have described being secreted by CD8 + lymphocytes in response to mitogen stimulation (Kelso et al., 1982; Guerne et ai., 1984; Kelso and Glasebrook, 1984; Yamasaki et ai., 1984). In addition, the kinetics of release of IFN-~, in response to tumor stimulation is similar to that observed upon stimulation with concanavalin A or alloantigen; IFN-y levels rapidly peak by 12 h after stimulation and then plateau (Kelso et al., 1982; Kelso and Glasebrook, 1984; Fong and Mosmann, 1990). These observations suggest that tumor antigens are a potent stimulus for IFN-y secretion. The concentrations of IFN-~, (i.e., > 20 U / m l ) observed in the supernatants of stimulated TIL have been shown to be sufficient in vitro to activate tumoricidal macrophages (Pace et al., 1983). We confirmed the biologic activity of the IFN-y detected in the supernatants of our TIL by demonstrating that supernatants from TIL stimulated with tumor are capable of activating macrophages to iyse the mastocytoma P815. This activation was completely inhibited by anti-lFN-,/ mAb (data not shown). Although we have observed specific secretion of I F N - , / a n d T N F in response to relevant tumor at 3-50-fold the amount induced by irrelevant

278 tumor stimulation, there was, generally, somewhat more lymphokine secreted in response to irrelevant tumor than that seen by control TIL cultured in rIL-2 alone (Tables I, It and III). This response to irrelevant tumor stimulators may be due, in part, to the polyclonal nature of the TIL lines used in these investigations. However, increased !ymphokine secretion in response to irrelevant tumor stimulation was observed even when the TIL clone 205-87 was stimulated (Table lII). For example, at culture day 202, the secretion of IFN-T increases from 4 U / m l with no tumor stimulation to 11 and 17 U / m i with irrelevant tumor stimulators. Although a smaller non specific lymphokine response might be seen if irrelevant tumor clones were used to stimulate TIL clones, the possibility exists that the determinants on MCA tumors responsible for induction of lymphokine secretion represent a family of antigens with both shared and unique epitopes. Nevertheless, the 3-50.fold difference between levels of lymphokines secreted in response to relevant vs irrelevant tumor stimulation, the observation that specific lymphokine release is a stable characteristic of the TIL culture over time, and the rapidity and sensitivity of the assay all make this an attractive method to use for the molecular ~tudy of murine tumor antigens. A potential advantage of this assay over cytotoxicity assays is that viable intact tumor stimulator cells may not be required to induce lymphokine secretion. The use of tumor membrane ,reparations for TIL stimulation is currently being investigated. The relatively high frequency of TIL cultures that specifically secrete lymphokines under the culture conditions employed (Table IV) suggests that lymphokine secreting CD8 + lymphocytes constitute a significant proportion of the tumor reactive CD8 + iymphocytes infiltrating these murine sarcomas. A better estimate of the frequency of lymphokine secreting, tumor reactive CD8 + lymphocytes in a tumor might be obtained by a clonal analysis of initial TIL cultures. For instance, clonal analysis of alloreactive murine splenocytes indicated that IL-2 (Heeg et al., 1987) and IFN-~/ (Kelso et al., 1984) secreting CD8 + lymphocyte precursors are as frequent as lymphokine secreting CD4 + precursors.

Although stimulation of murine CD8 + iymphocytes with mitogens has induced secretion of IL-2, IL-3 and GM-CSF, as well as TNF and 1FN-T (Prystowski et al., 1982; Roopeneian et al., 1983; Guerne et al., 1984; Kelso and Glasebrook, 1984; Fong and Mosmann, 1990), we have not yet systematically evaluated TIL supernatants for cytokines other than IFN-y and TNF. However, it is unlikely that the lymphokine secreting TIL reported herein also secreted significant levels of IL-2. All TIL were initiated and maintained in culture media containing 10-20 U / m l rIL-2 and promptly lost their viability unless fresh culture media containing rIL-2 was added every 3-4 days. Moreover, the presence of exogenous IL-2 was necessary to obtain maximal lymphokine secretion by stimulated TIL (Table V). Others have also observed that IL-2 markedly enhanced the amount of IFN-~/secreted by CD8 + lymphocytes in response to concanavalin A or alloantigen stimulation (Kelso et al., 1984). It is difficult to dissociate the effect of IL-2 on IFN-~, secretion from its role as a growth factor for IL-2-dependent lymphocytes. However, the detection of increased IFN-y secretion as early as 4 h (Kelso et al., 1984) and 8 h (Table V) after stimulation, at which time there has been little opportunity for an increase in cell number, suggests that IL-2 directly facilitates IFN-y secretion. The finding that maximal iymphokine secretion by T1L in response to tumor is dependent on the presence of IL-2 has important implications for the use of TIL in the adoptive immunotherapy of tumors. IL-2 may enhance the anti-tumor effectiveness of TIL in vivo (Greenberg, 1986; Spiess et al., 1987) not only by supporting T1L proliferation, but also by maximizing the secr~tion of lymphokines important in the anti-tumor response. We, and others, have shown that the adoptive transfer of CD8 + lymphocyte lines and clones may very effectively cause the regression of established tumors (Rosenstein and Rosenberg, 1984; Yamasaki et al., 1984; Greenberg, 1986; Rosenberg et al., 1986; Spiess et al., 1987; Barth et al., 1990; Yang et al., 1990). The mechanism by which these lymphocytes exert their anti-tumor effects is currently unknown. In a separate report, we demonstrate that specific lymphokine secretion

279 by adoptively transferred T1L plays a major role in t u m o r r e g r e s s i o n s in vivo ( B a r t h e t al,, 19901.

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