IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6

CELLULAR

IMMUNOLOGY

133,327-341 (1991)

IL-4-Supported

MONICA

M. BERTAGNOLLI,*

Induction of Cytolytic T Lymphocytes Requires IL-2 and IL-6’ YASUYUKI

TAKAI,t,’

AND STEVEN H. HERRMANN$T~

*Department of Surgery, Brigham and Women 3 Hospital, Boston, Massachusetts: *Department c$ Pathology, Harvard Medical School, Boston, Massachusetts; and fDivision of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 Received July 18, 1990; accepted November 5, 1990

Previous work indicated that a CTL responsecan be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Becauseof the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence ofthese lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presenceof IL-4. The addition of IL-2 to limiting dosesof IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-Cmediated responsewe found: (a) that mAb to IL-2 significantly reduced the CTL responsegenerated in the presenceof IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presenceof IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presenceof IL-2 + IL-6 but readily blocked the CTL responseto IL-4. Theseresultsindicate that, for thymocyte responders,the CD8+ CTL generated in the presenceof IL-4 require both IL-2 and IL-6. o 1991 Academic

Press. Inc.

INTRODUCTION Cytotoxic T lymphocytes (CTL) are immune effector cells able to recognize short peptide fragments tightly associatedwith self-MHC proteins on the surface of target cells (I, 2). CTL bind to and directly lyse the antigen-bearing target cells. Since CTL are able to recognize a range of pathogenic cells, including virus-infected cells and malignant cells, the ability to specifically enhance the CTL responsewould be clinically quite beneficial. Generation of CTL requires signals delivered through the antigen-specific T cell receptor (TCR)-CD3 complex, and signals delivered in response to lymphokines (36). In vitro studies indicated that the generation of CTL may involve one or more of ’ This work was supported by grants from the National Institute of Health CA 4783 1 (S.H.), the National Cancer Institute 2T32CA09535-05 (M.B.), and Ethicon Inc., Society of University Surgeons(M.B.). * Present address:Dept. of Oncogenesis,Osaka University Medical School, Osaka, 553, Japan. 3 Present address:Genetics Institute, Cambridge MA 02 140. 327 000%8749/9 I $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

328

BERTAGNOLLI,

TAKAI,

AND

HERRMANN

the following lymphokines: IL- 1, IL-2, IL-4, IL-6, IFN-7, and TNF (7- 10).Our previous work indicated that, individually, IL-2, IL-6, and IFN-y were not able to support the generation of a CTL response; however, the combination of these three lymphokines resulted in a maximal CTL response(7,8). The involvement of multiple lymphokines in the generation of active CTL may indicate that a lymphokine network exists, whereby the action of one lymphokine is tied to the presence of other lymphokines. A problem inherent in determining the lymphokine requirements for CTL generation is the difficulty in obtaining responder populations devoid of lymphokine-producing cells. We and others have attempted to circumvent this problem by using thymocytes as responder cells, and a short-term 48- and 72-hr culture. When compared to spleen cells, PBLs, or lymph node cells, thymic cultures appear to have a much lower endogenous factor production. Cell populations within the thymus, however, are capable of producing IL-2, IL-6, IL-4, and possibly other lymphokines depending upon culture conditions and methods of isolation ( 11, 12).One must therefore consider the possibility that the CTL response observed is the result of both exogenous recombinant lymphokines added to culture and endogenous lymphokines produced during culture. In this report, we asked whether IL-4 was able to directly support the cytotoxic activation of concanavalin A (Con A)-triggered murine thymocytes. To control endogenous lymphokines, we used monoclonal antibodies (mAb) reactive with IL-2, IL-4, or IL-6 to block the action of these lymphokines. Our results indicated that the IL-4-supported generation of CD8+ CTL from murine thymocytes required the presence of endogenously generatedIL-2 and IL-6. CTL generation in responseto exogenousrecombinant IL-2 and IL-6, however, did not appear to require endogenously generated IL-4. MATERIALS AND METHODS Animals and cell lines. C57BL/6 (H-2b), and (BALB/c X DBA/2) Fi (H-2d) female mice, 6 to 8 weeks of age, were purchased from the Jackson Laboratory (Bar Harbor, ME). P8 15 (H-2d) was maintained by weekly passageas ascites in (BALB/c X DBA/ 2) Fl mice. The CTLL20 IL-2-dependent cell line was obtained from American Type Cell Culture Collection (Rockville, MD), and the B9 IL-6-dependent cell line was the generous gift of L. Aarden ( 13). These cell lines were maintained in complete culture medium (RPM1 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 pg/ml streptomycin, 2 mM glutamine, 5 X 10e5M 2-mercaptoethanol) plus human IL-2 (1-3 U/ml) and human IL-6 (2-5 rig/ml). Lymphokines. Recombinant human IL-2 was purchased from Amgen Biologicals (Thousand Oaks, CA). Recombinant murine IL-2 and IL-4 were purchased from Genzyme Corp. (Boston, MA). Different lots of murine IL-4 varied considerably in their ability to stimulate thymocytes. Although the units reported here are based upon the manufacturer’s stated potency, the amount of IL-4 used for each experiment was titered to give maximal CTL or proliferative response. Recombinant murine and human IL-6 were provided by Steve Clark (Genetics Institute, Cambridge, MA). Levels of IL-6 used in the assayswere also chosen to give maximal augmentation of CTL and proliferative responses. Antibodies. The 11B 11 cell line producing anti-murine IL-4 monoclonal antibody was the kind gift of W. Paul (14) and the S4B6 cell line producing the anti-mm-me IL-2 antibody was the generousgift of T. Mossman ( 15).J. Van Snick (Ludwig Institute, Brussels, Belgium) kindly provided us with a cell line producing the anti-murine IL-

INTERDEPENDENCE

OF IL-2, IL-4, AND

IL-6

329

6 monoclonal antibody, 6B4 ( 16). Supernatants from 11B 11 and S4B6 were purified using a mouse anti-rat affinity column (17). Culture supernatant from 6B4 cells was concentrated five times by Amicon filtration (Danvers, MA) and used as a source of anti-IL-6 antibody. Activities of these mAbs were determined by the ability to inhibit lymphokine-induced proliferation using CTLL20 for IL-2- and IL-Cdriven proliferation and B9 (13) for IL-6-driven proliferation. The antibodies were shown to block by binding to the appropriate murine lymphokines becauseeither addition of excess concentrations of recombinant murine lymphokines overcame the inhibition, or the addition of human lymphokines bypassed the inhibition. AMT- 13 monoclonal antibody against murine TAC-like IL-2 receptor was the generousgift of T. Diamantstein. Anti-mouse K (187.1) was obtained from American Type Culture Collection (Rockville, MD) (18) and used as an isotype control antibody for 11B 11 and 6B4. All antibodies were added to culture in amounts in excessof that required to block endogenous lymphokine levels (11). Assaysfir CTL activity. To detect soluble factors involved in the generation of CTL, we used unfractionated thymocytes as responders as described previously (7, 8). For most experiments, 8 X 1O5whole thymocytes from 6- to 8-week-old mice were cultured in 96-well round-bottom microtiter plates in the presence of 3 pg/ml Con A (Sigma Chemicals, St. Louis, MO) and purified lymphokines or monoclonal antibodies. Total volume was 200 &per well of complete culture medium (RPM1 1640 supplemented with 10%heat-inactivated FCS, 100 U/ml penicillin, 100 pg/ml streptomycin, 2 mA4 glutamine, 5 X 10e5A4 2-mercaptoethanol). After 48-72 hr at 37°C and 5% COz, microtiter plates were centrifuged at 1000 rpm for 5 min and culture media decanted. To determine cytotoxicity at a known E/T ratio, replicate wells were harvested and cells counted and adjusted to give the desired number of effector cells per well. “Cr-labeled P815 target cells were added to responder cells together with 10 pg/ml PHA-P (Difco). After a 4-hr incubation, the remaining cells were sedimented and 50% of the supernatant was removed and counted in a y counter. Percentage of specific lysis was calculated as follows: 100 X [(“Cr experimental release - spontaneous release)/(maximum 5’Cr release - spontaneous release)]. Postculture fractionation of thymocyte subsets.Thymocytes from C57BL/6 mice 68 weeks of age were cultured as described previously for generation of CTL in 3 pg/ ml Con A and 500 U/ml murine IL-4. The effector cell populations present after 68 hr of culture were then harvested, counted, and divided into four groups: (i) unfractionated cells, no further treatment, (ii) unfractionated cells incubated at 4°C for 1 hr with magnetic beadscoated with sheepanti-rat antibody (Dynal Inc. Great Neck, NJ) at a ratio of 1:l beads to cells, (iii) cells treated at 4°C for 1 hr with 5 pug/ml GKl.5 rat anti-murine CD4 mAb, washed twice followed by extraction of CD4+ cells by incubation for 60 min at 4°C with magnetic beads as above, or (iv) cells treated with 7 pg/ml 53.6 rat anti-murine CD8 mAb washed twice followed by magnetic bead extraction of CD8+ cells by incubation for 60 min at 4°C with magnetic beads as above. Following removal of bead bound cells the remaining cells were analyzed for cytotoxicity at effector-to-target ratios of 30: 1 and 10:1 against 51Cr-labeledP8 15 cells; and CD4, CD8 phenotype by two-color flow cytometry. Thymocyteproliferation. Whole thymocytes from 6- to 8-week-old C57BL/6 female mice were prepared asdescribedpreviously (7). For lectin-dependent assays,thymocytes (8 X lo5 per well) were cultured for 48-60 hr in 96-well flat-bottom microtiter plates in the presence of 3 pg/ml concanavalin A (Sigma Chemical Co., St. Louis, MO).

330

BERTAGNOLLI,

TAKAI,

AND

HERRMANN

Where indicated lymphokines or blocking antibodies were added either singly or in combinations as indicated at the onset of the 60- to 65hr culture period. Culture media consisted of RPM1 1640 supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM glutamine, and 10% fetal calf serum. After completion of the culture interval, proliferation wasassessedby the incorporation of [3H]thymidine during a final 4-hr pulse with 1 &i [3H]thymidine per well. Assay for IL-2 production. Unfractionated murine thymocytes were cultured in vitro as described above and supernatants harvested after 69 hr at 37°C. CTLL20 cells (8 X 103)per well were then cultured in 96-well flat-bottom microtiter plates in standard culture media with the addition of 50%test supernatant or test supernatant plus 11B 11 antibody in an amount shown to neutralize 2000 U/ml murine IL-4 in a CTLL20 assay.This amount of antibody was chosen such that it would neutralize the effect of the IL-4 added at the start of the culture, in addition to any endogenous IL-4 produced during the culture interval. Cells were cultured at 37” for 12 hr, followed by a 4-hr pulse with 1 &i [3H]thymidine per well. Immunofluorescence andflow cytometry analysis. CTL effecters induced under the same culture conditions as the microtiter plate CTL assay were collected, placed in 50 ~1of medium with 0.02% azide, and incubated for 40 min at 4°C with 5 pg/ml of anti-IL-2 receptor mAb (AMT- 13). Samples were then washed three times and incubated with fluorescein-conjugated goat anti-rat IgM (Tago, Burlingame, CA) for another 30 min on ice. Cells were then washed three times and fixed with 1% paraformaldehyde, followed by analysis with a Coulter Epics V cell sorter. For two-color analysis, cells were incubated with 5 pg/ml each of directly conjugated anti-CD4 (phycoerythrin) and anti-CD8 (fluorescein) (Becton-Dickinson, Mountain View, CA) for 40 min on ice. Cells were then washed three times, fixed, and analyzed with a Coulter Epics V cell sorter. RESULTS Multiple studies now show that IL-6 in combination with IL-2 supports the generation of active CTL (7- 10). In order to initially demonstrate the IL-6 requirement, the system used had to be free of cells that produce this lymphokine. By using a system producing low amounts of endogenous IL-2 and IL-6, a 2-day in vitro culture of unfractionated, Con A-stimulated murine thymocytes, we clearly demonstrated the IL-6 requirement (7, 8). Extending the culture to 4-5 days, however, resulted in the ability of IL-2 alone to induce a CTL response.We found that this longer culture time resulted in the production of IL-6 by the thymocytes (11). While others have reported that IL-6 alone can support the generation of a CTL response from peripheral lymphocytes (19) it has been our experience that even high doses of IL-6 alone do not elicit a CTL response from thymocytes over a 2-day incubation (8). IL-2 is required for IL-l-induced CTL generation. Previous work indicated that IL-4 was a lymphokine involved in the induction of a CTL response (16,20-23). We addressedthe question of whether IL-4 is acting alone or in combination with other lymphokines. We reasoned that if IL-4 required the presenceof IL-2, this dependence would become apparent with lower concentrations of IL-4. Since IL-2 gave only a minimal CTL response in our assaysystem, we asked if the addition of IL-2 to lower concentrations of IL-4 would enhance the response above that found for IL-4 alone. As shown in Fig. 1, the dose-response curve for IL-4 was shifted by the addition of

INTERDEPENDENCE

331

OF IL-2, IL-4, AND IL-6

60

50

40 % I Y s I s

30

20

10

0 1

--A-

IL-4

i

IL-2

100

10 IL-4

Cont. -fi

(20 U/ml)

1000

(U/ml)

4

IL-4

+ IL-2

(20 U/ml)

Con A SN (12.5%)

FIG. I. IL-2 enhances IL-Cinduced CTL. Thymocytes from C57BL/6 mice (8 X 10’) were cultured in 96-well round-bottomed microtiter plates with 3 &ml Con A and serial twofold dilutions of IL-4 Maximum CTL response was observed with addition of culture supernatant from Con A stimulated rat spleen cells (Con A SN) added at 12.5%.Cytotoxicity against “Cr-labeled P8 15 tumor cells was determined after 50 hr of culture.

low levels of IL-2 (20 U/ml). Half-maximum responsewas found at a concentration of 250 U/ml IL-4 in the presence of IL-2, versus 1000 U/ml in the absence of IL-2. At the highest concentrations of IL-4, however, IL-2 did not enhance the responseto IL-4 (Fig. 1). Previously, we demonstrated that the CTL generated in the presence of IL-2 plus IL-6 were CD8+ cells (7, 8). To determine whether the cells responding to IL-4 were also CD8+, thymocytes were fractionated following culture in the presence of Con A and 500 U/ml of IL-4. The fractionation was accomplished using magnetic beads plus: no antibody; anti-CD4; or anti-CD8. Cells not removed by this bead selection were used as effecters in a cytotoxic assay. Phenotypic analysis showed that control cells cultured in IL-4 for 68 hr were 6.9% CD4+, 12.5%CD8+, 78.8% double negative, and only 1.8%double positive (Table 1). For comparison, fresh, uncultured thymocytes from a 6- to 8-week-old C57BL/6 mouse were 8% CD4+, 4% CD8+, 9% double negative, and 78% double positive. This relative loss in double positive and gain in double

332

BERTAGNOLLI,

TAKAI,

AND

TABLE Phenotype of Thymocytes Groups

CD4+ CD8-

HERRMANN

1

After Postculture Fractionation CD4- CD8+

CD4+ CD8+

CD4

CD8-

Percent positive cells after fractionation Beads only Beads + anti-CD4 Beads + anti-CD8 No fractionation

6.4 0.1 7.8 6.9

12.8 14.4 0.5 12.5

1.8 0.3 0.2 1.8

79.0 85.3 91.4 78.8

Note. Thymocytes from C57BL/6 mice 6-8 weeks of age were cultured in 3 &ml Con A and 500 U/ml murine IL-4. The effector cell populations present after 68 hr of culture were harvested and divided into four groups: No fractionation, unfractionated cells analyzed without further treatment; beads only, cells incubated at 4°C with magnetic beads coated with sheep anti-rat antibody; beads + anti-CD4, cells treated with GKl.5 rat anti-murine CD4 mAb followed by extraction of CD4+ cells with sheep-anti-rat magnetic beads; beads + anti-CD8 cells treated with 53.6 rat anti-murine CD8 mAb followed by extraction of CD8+ cells with sheep-anti-rat magnetic beads. Cells left after bead removal were stained with directly conjugated anti-CD4 (phycoerythrin, y-axis) and anti-CD8 (fluorescein, x-axis). And analyzed by two-color fluorescence flow cytometry. Results are given as percentage of positive cells for the four quadrants.

negative thymocytes is a consistent finding whenever thymocytes are placed in in vitro culture for 60-70 hr (MB and SH, unpublished data), and is likely due to lack of proliferative ability in the double-positive cells. Analysis of cells remaining after CD8+ depletion revealed only 0.5% CD4-CD8+ and 0.2% CD4+CD8+ contaminating cells, and 7.8% CD4+CD8- thymocytes. The cells remaining after anti-CD8 bead selection had a greatly diminished level of lysis, indicating that the CD8+ thymocytes were the subset responsible for the majority of cytotoxic activity generated by IL-4. However since cytotoxic activity was not zero this leaves open the possibility that some other cell type might be responsible for a low level of cytolysis (Fig. 2). Thymocytes remaining after anti-CD4 bead selection contained only 0.1% CD4+CD8- and 0.3% CD4+CD8+ contaminating cells, and 14.4% CD4-CD8+ thymocytes. These cells had a somewhat increased level of lysis compared to the control groups (Fig. 2). This increase likely resulted from the removal of the noncytotoxic CD4+ population, which left a greater percentage of the remaining cells that were CD8+ and therefore able to lyse more target cells at a given E:T ratio. In the absenceof PHA, these cells were not cytotoxic for P8 15 cells (data not shown), indicating that triggering through cell surface proteins was required and suggesting that these were not LAK-like cells. Thus, thymocytes cultured in the presence of IL-4 gave rise to cytotoxic cells of the CD8+ phenotype consistent with classic CTL. We next sought to determine whether IL-4 would induce CTL differentiation independent of IL-2. We used S4B6, a mAb specific for murine IL-2, to block the action of endogenously generated IL-2 in our in vitro thymocyte assay.Adding 500 U/ml of IL-4, a CTL response of about 47% was generated at an E:T of 25: 1. When this level of IL-4 was added to the thymocytes along with enough anti-IL-2 mAb to block 50 U/ml of murine IL-2, the responsewas reduced to about 4% (Table 2). When human IL-2 was added along with the anti-IL-2 mAb, the inhibition was reversed and a CTL response comparable to that for IL-4 alone was generated (-48% lysis Table 2), indicating that the inhibition was not due to nonspecific blocking by the mAb.

INTERDEPENDENCE

OF IL-2, IL-4, AND

333

IL-6

No Beads t

Beads

only

r

anti-CD4

plus beads

F anti-CD8

plus beads r 0

5

10

15

20

25

30

% LYSIS FIG. 2. CTL generated by IL-4 are CDS+. Thymocytes from C57BL/6 mice were cultured as described previously in 3 &ml Con A and 500 U/ml murine IL-4. After 68 hr of culture, cells were harvested and fractionated using magnetic-core polystyrene beads and GK1.5 (anti-murine CD4) or 53.6 (anti-murine CD8) monoclonal antibodies to specifically remove either CD4+ or CD8+ cells from the effector population. Cytotoxicity against *‘Cr-labeled P8 15 tumor cells was determined for E:T ratios of 30: I and 10: 1. Values are expressed as SEM where n = 3.

If IL-4 required the presence of IL-2 to mediate thymocyte proliferation and differentiation, then perhaps one of the actions of IL-4 in the thymocyte culture was to induce the production of IL-2. Culture supematant from thymocytes incubated with Con A and IL-4 was therefore tested for the presence of IL-2. We incubated the IL-2 responsive line CTLL20 with the IL-4 culture supernatants and enough anti-IL-4 (1 lB11) to block the activity of the added IL-4, This demonstrated that culture of thymocytes with IL-4 resulted in the generation of IL-2 at levels of approximately 30 U/ml (Fig. 3). The level of IL-2 found in supernatants from thymocytes cultured in IL-4 was greater than the level of IL-2 found in supematants from thymocytes initially cultured in IL-2 alone, or IL-2 plus IL-6, which were only slightly above background (Fig. 3). If IL-4 increased the level of IL-2 production in thymocyte cultures, then this should result in the expression of the TAC-like IL-2 receptor (24). Thymocytes cultured under the same conditions as for generation of a CTL response were therefore examined for expression of IL-2 receptor. Culture in 500 U/ml IL-4 induced 18% expression of the TAC IL-2 receptor expression compared to 6.2% expression of IL-2 receptor for thymocytes cultured in the absence of added lymphokine. The levels of IL-2 receptor expressed following culture in the presence of IL-2 or IL-2 + IL-6 were about 17 and 16%, respectively (Fig. 4). When S4B6, anti-murine-11-2, was added to thymocytes

334

BERTAGNOLLI,

TAKAI, AND HERRMANN TABLE 2

Anti-IL-2 Blocks IL4-Generated CTL Response Effector-to-target ratio 50: I No lymphokine IL-4 IL-4 + anti-IL-2 IL-4 + anti-IL-2 + hlL-2

9.6 f 68.9 -t 7.2 + 74.2 +

0.6 1.5 0.2 0.7

25:l

1O:l

7.0 + 0.3 46.6 f 2.1 3.6 f 0.1 47.5 AI 2.1

0 21.9 + 0.6 1.6 + 0.1 23.9 zk0.1

Note. Thymocytes from C57BL/6 mice were cultured with 3 &ml Con A +/-lymphokines for 66 hr at 37°C. Anti-IL-2 mAb (S4B6) was added as indicated in an amount able to block expected endogenously produced levels of IL-2 (see Fig. 3). Replicate wells were harvested, counted, and cytotoxicity as chromium release was determined at the indicated E:T ratios following a 4-hr culture with 5’Cr-labeled P8 15 tumor cells. Values are expressedas % specific killing + SEM where n = 3. The recovery of cells following culture was: (a) no lymphokine: 53%, (b) culture with 500 U/ml murine IL-4: 98%, (c) culture with 500 U/ml murine IL-4 plus enough anti-IL-2 mAb (S4B6) to neutralize 50 U/ml murine B-2: 70%, and (d) culture with 400 U/ml murine IL-4, anti-IL-2 mAb able to neutralize 50 U/ml murine IL-2, and 40 U/ml human IL-2: 85%.

cultured in IL-4, however, receptor expression fell to 0.2%. Although the percentage of thymocytes expressing IL-2 receptor varied from experiment to experiment, enhanced expression of IL-2 receptor following culture of thymocytes in IL-4 was a consistent finding in multiple experiments. This suggestedthat the enhanced expression of IL-2 receptor was due to the ability of added IL-4 to induce IL-2 production by the thymocytes. IL-4 is known to cause proliferation of T lymphocytes (20, 21, 25, 26). Therefore, we determined whether the anti-IL-2 mAb would block IL-Cmediated proliferation.

N i

lymphokine

in culture

FIG. 3. IL-4 causesincreased IL-2 production in thymocytes. Unfractionated murine thymocytes (8 X 10’ cells/well) were incubated in 200 ~1 media containing 3 &ml of Con A for 69 hr. IL-2 (20 U/ml), IL-6 (1 U/ml), and IL-4 (500 U/ml) were added to the cultures as indicated. Serial twofold dilutions of supernatants from these cultures, beginning at 50% concentration, were added to 200 ~1wells containing 8 X IO3CTLL20 cells. All wells contained an amount of 1lB1 I anti-114 mAb in excessof that required to block 2000 U/ ml of IL-4. After 12 hr of proliferation [3H]thymidine was added and incorporation measured after an additional 4 hr incubation.

INTERDEPENDENCE

d

OF IL-2, IL-4, AND IL-6

20

10

IL-2 receptor

(percent

335

3c

positive)

FIG. 4. IL-2R expression. Thymocytes from C57BL/6 mice were cultured with 3 &ml Con A and the following lymphokines as indicated: murine IL-2 20 U/ml; murine IL-6 1 U/ml; murine IL-4 500 U/ml. S4B6 anti-murine IL-2 mAb was added as indicated in an amount able to block 50 U/ml murine IL-2. After 66 hr of culture at 37°C cells were stained using AMT-I 3 anti-TAC IL-2 receptor antibody and fluoresceinconjugated goat anti-rat IgM.

Culture of thymocytes in the presenceof IL-4 generated a strong proliferative response that was blocked by approximately 50% with the addition of anti-IL-2 in an amount sufficient to block endogenously produced IL-2 (Fig. 5). IL-6 is required for IL-4- and IL-2-induced CTL generation. After finding that IL-2 was involved in the action of IL-4, we determined whether IL-6 was also required. The CTL responsegenerated by 20 U/ml IL-2 plus 1 U/ml IL-6 was similar in magnitude to the responsegenerated by IL-4. The cytotoxicity generated by IL-4 was not enhanced by the addition of 1 U/ml murine IL-6 (43 vs 40% at 5O:l E:T, Table 3) nor was the response to an optimal level of IL-4 enhanced by addition of IL-2 plus

log serial

dilution

of antibody

FIG. 5. Anti-murine IL-2 inhibits IL-4 supported proliferation. Murine thymocytes (8 X lo5 cells/well + 3 &ml of Con A) were incubated in 200 ~1media containing 500 U/ml of murine IL-4 and anti-murine IL-2 monoclonal antibody S4B6. The S4B6 was started at a concentration that would block 42 U/ml of IL2 in the CTLLZO assayand serially diluted by twofold dilutions. [‘Hlthymidine incorporation was determined by a 4-hr pulse at 64 hr of culture. Background proliferation was 4 1,000 f 7200 cpm, bars indicate +SEM where n = 3.

336

BERTAGNOLLI,

TAKAI,

AND HERRMANN

TABLE 3 Comparison of CTL Activity Generated by IL-2, IL4, and IL-6 Effector-to-target ratio (cell recovery) 5O:l Experiment A. No lympbokine IL-2 IL-2 + anti-IL-6 IL-6 IL-2 + IL-6 IL-4 IL-4 + IL-6 IL-4 + anti-IL-6 Anti-IL-6 Experiment B. No lymphokine Anti-IL-6 IL-2 IL-4 IL-4 + anti-IL-6 Experiment C. No lymphokine IL-4 IL-4 + anti-IL-6 IL-4 + anti-IL-6 + human IL-6

0.6 + 17.0 f 2.9 + 1.1 f 31.3 + 43.2 + 40.4 2 5.2 f 0.3 f

25:l

IO:1

0.1 0.7 0.5 0.1 1.3 4.9 5.6 1.2 0.1

0.2 2 10.7 f 4.9 f 1.5 f 18.8 f 33.4 f 28.7 k 3.6 + 0

0.1 0.3 2.3 0.3 2.5 5.7 2.1 0.6

0 2.8 + 1.4 f 0 7.6 + 15.5 + 17.2 + 0.3 + 0

9.6 EL 0.6 5.2 + 0.3 6.9 f 0.3 68.9 f 1.7 24.2 + 24.2

7.0 + 1.3 + 1.4 f 46.6 + 11.5 +

0.3 0.1 0.1 1.2 0.8

0 0 0 21.9 f 1.1 2.6 I! 0.1

1.6 + 71.1 + 17.8 f 57.7 +

1.3 + 61.4 f 8.9 + 45.9 +

0.1 1.8 0.8 1.9

0 30.7 + 2.0 3.2 f 0.4 17.9 f 0.5

0.2 2.1 2.2 3.3

0.1 0.3 0.4 0.6 8.4 0.1

Note. 8 X 10’ thymocytes from C57BL/6 mice were cultured in 96-well round-bottomed microtiter plates with 3 &ml Con A and the indicated lymphokines. Lymphokine levels were: human IL-2 20 U/ml; murine IL-6 I U/ml; murine IL-4 2000 U/ml. Where indicated, 6B4 anti-murine IL-6 antibody was added in an amount able to block 300 U/ml IL-6. This amount ofantibody was chosento be adequate to block previously determined levels of endogenous IL-6 production (1 I). Controls for the 6B4 (IgGla) antibody included: (a) addition of 187.I anti-K antibody (IgGla) as an &type-matched control and (b) addition of human IL-6 (100 t&ml) to illustrate that blocking was specific for the removal of murine IL-6. At the end of 68 hr of culture, cells were harvested,counted, and cytotoxicity against 5’Cr-labeled P815 tumor cells was determined. Values are expressedas % specific killing f SEM where n = 3.

IL-6 (data not shown). The amount of IL-6 used for these studies was previously determined to give maximal augmentation of the CTL response in the presence of IL-2. Minimal cytotoxicity was found for thymocytes cultured with IL-2 alone (17 + 1%)and no cytotoxic responsewas generated in the presenceof IL-6 alone ( 1 f l%), in agreement with previous work (7,8). As IL-6 was likely to be present in the culture in low levels, we asked whether adding an anti-IL-6 mAb, 6B4, would alter the ability of IL-4 to support the induction of a CTL response. By adding a level of 6B4 (antiIL-6) mAb able to block 300 U/ml of IL-6, the generation of a CTL response by IL2 + IL-6 was blocked completely (Table 3). Thymocytes cultured in the presenceof Con A and 500 U/ml of recombinant murine IL-4 resulted in 40 to 70% lysis (E:T of 50: I), depending upon the experiment. The anti-IL-6 mAb consistently inhibited the CTL response as represented in Table 3. This inhibition was approximately 90% for the lower levels of lysis and approximately 75% for the higher levels of lysis (Table 3). Addition of human IL-6 reversed approximately 70% of this inhibition. The failure

INTERDEPENDENCE

OF IL-2, IL-4, AND

IL-6

331

of human IL-6 to completely overcome inhibition may indicate a need for a constant endogenous source of IL-6 to maintain CTL activity. The ability of anti-IL-6 to block the response found in the presence of IL-2 alone indicated, as we have suggested previously (1 l), that this CTL response required endogenous IL-6. We tested supernatants from thymocytes cultured in IL-4 for IL-6 using the IL-6-dependent cell line, B9. We were, however, unable to show that culture of thymocytes in IL-4 resulted in increased endogenous production of IL-6, which ranged from 10 to 30 U/ml for control and experimental cultures at 72 hr (data not shown). The data obtained with antiIL-6 mAb, therefore, indicated a requirement for IL-6 in the generation of an IL-4supported CTL response for at least the majority of the CTL generated. To determine whether the effect of IL-6 as a cocytotoxic factor with IL-4 involved lysis only or also involved proliferation, we asked whether the addition of the antiIL-6 mAb would effect IL4-mediated proliferation. The proliferation to 500 U/ml of IL-4 was blocked greater than 70% by the addition of the anti-IL-6 mAb, 6B4 (Fig. 6). The specificity of the antibody blocking was shown by the ability of human IL-6 to overcome the 6BCmediated inhibition and restore proliferation found in the presence of IL-4 to near control levels. The proliferation of thymocytes to 20 U/ml of murine IL-2 was also blocked by about 60% by the addition of the anti-IL-6 mAb. This proliferation was also restored by the addition of human IL-6. (Fig. 6). We found, as have others, that addition of IL-6 enhanced the proliferation found in the presence of IL-2 (19,27-29) (Herrmann et al., unpublished data). To examine the effect of IL-6 on the proliferative response to IL-4, we compared the proliferative response found with IL-4 to that found with IL-4 plus IL-6. As shown in Fig. 7, the proliferation to the combination of these two lymphokines was greater than the proliferation of either alone, and somewhat more than an additive response. Thus, IL-6 also enhanced the IL-4-mediated proliferation of thymocytes. Anti-IL-4 does not block CTL generation found with IL-2 plus IL-6. To determine whether the CTL response generated in the presence of IL-2 plus IL-6 required the

n 0 vi

E

120 100

60

2

FIG. 6. Anti-IL-6 inhibits IL-4-mediated proliferation. Thymocytes (8 X 10s cells/well + 3 &ml of Con A) were cultured in the presence of the indicated lymphokines: 500 U/ml of IL-4; 20 U/ml of murine IL-2; 100 ngJm1 of human IL-6. Where indicated the anti-murine IL-6 mAb, 6B4, was added at a concentration that would block 300 U/ml of IL-6 as assayed using an IL-6-responsive cell line.

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IL-4

IL-6

IL-4+IL-6

FIG. 7. IL-6 enhances the proliferative response to IL-4. Thymocytes (8 X IO5cells/well + 3 &ml of Con A) were cultured in the absence(-) or presence of the indicated lymphokines: 800 U/ml of IL-4; 2 U/ ml of murine IL-6, either alone or in combination. The cells were harvested at 65 hr of culture duration with t3H]thymidine added during the final 4 hr of culture. Bars indicate fSEM where n = 3.

presence of IL-4, we assessedwhether an anti-murine IL-4 mAb ( 11B 11) would block the action of IL-6. We found that the polyclonal CTL responsegeneratedin the presence of IL-2 plus IL-6 was not blocked by addition of 11Bl 1 (Table 4). The amount of 11B 11 antibody added was sufficient to completely block the CTL responsegenerated in the presence of 1000 U/ml of IL-4. An isotype-matched control rat mAb did not block the ability of either IL-6 or IL-4 to support the generation of CTL (Table 4). Thus, IL-4 appeared to require both IL-2 and IL-6 in order to generate a proliferative response, however IL-4 was not required for the action of IL-2 plus IL-6 in the generation of a cytotoxic response. DISCUSSION The process by which antigen and lymphokine signals direct a precursor T cell to become an active cytotoxic cell is referred to as cytotoxic differentiation. Our present understanding of the molecular basis of CTL recognition and lysis is incomplete, requiring us to rely upon a functional assayas an indication of CTL differentiation. It is possible that differentiation may simply involve the production of proteins required for the T lymphocyte to carry out the lytic function, such asthe generation of performs, proteases, or other molecules involved in target cell lysis (8). It is also possible that differentiation occurs through changesin the ability of the TCR to trigger a functional response, such as ability of the receptor to generate second messengers.Whereas multiple lymphokines are involved in differentiation, it is not known if each lymphokine causesdifferentiation along the same pathway or if one cytokine causesthe generation of a certain component (i.e., lytic protein) required for an active cytotoxic cell. It is now evident that a single lymphokine can have a functional effect upon a range of different cell types. IL-6 has been found to have the following actions (30): promotion of bone marrow progenitor cell growth (31, 32); promotion of hybridoma and plasmacytoma growth (33); induction of Ig secretion by B lymphocytes (34); mediation of acute phase protein release(35); inhibition of fibroblast growth (36); and induction

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IL-6

TABLE 4 Anti-IL-4

Experiment A.

Added lymphokines (-) IL-2 IL-6 IL-2 IL-2 IL-4 IL-4 (-) (-) IL-2 IL-6 IL-2 IL-2 IL-2 IL-4 IL-4 IL-4 (-) (-1

+ IL-6 + IL-6

+ IL-6 + IL-6 + IL-6

Does Not Block IL-6 Activity Added antibodies

(-) (-) t-1 (-) IlBll (-) IlBll IlBll (-) (-) (-) C-1 1 lBl1 187.1 C-1 1 IBl I 187.1 1 IBl I 187.1

% Specific killing on PHA-P8 15 -0.3 3.2 I .3 28.5 29.8 33.2 3.0 -0.5 -0.1 6.6 I .3 35.3 36.0 32.4 20.1 4.0 20.7 -0.1 0.1

+ k f + f f f + f f f + + f f f f * f

0.2 0.5 0.6 2.1 0.8 1.9 0.2 0.1 0.4 0.9 0.0 0.6 1.0 2.1 2.8 I.0 1.2 0.2 0.5

Note. Thymocytes from C57BL/6 mice (8 X 105) were cultured in 96-well round-bottomed microtiter plates with 3 &ml Con A and the indicated lymphokines in the presence of or in the absence of monoclonal antibodies. Lymphokine levels were: human IL-2 30 U/ml; murine IL-4 1000 U/ml; human IL-6 100 U/ ml. Antibodies, anti-IL-4 (1 lBl1) or anti-x chain (187.1) were added as 12.5% culture supernatant at the onset of culture. Cytotoxicity was determined after 2 days of culture by removing the supernatant and adding targets containing PHA to the remaining cells. Values are expressed as % specific killing f SEM where n = 3.

and release of IL-2 by lymph node responders (8). IL-6 is produced by a variety of cells including T lymphocytes, fibroblasts, macrophages, mast cells, and smooth muscle cells in response to antigen, double-stranded RNA, IL-l, or TNF (30, 35-37). IL-4 is also multifunctional, known to influence the differentiation of B lymphocytes, T lymphocytes, and mast cells, and to act as a colony stimulating factor (38-42). In the murine system, in vitro production of IL-4 is believed to be restricted to the type II helper T cells and this may require the presence of IL- 1 (15, 43). These two lymphokines, IL-6 and IL-4, therefore, have the capacity to regulate diverse components of the immune system and their production may be regulated by other lymphokines. Using the lectin triggered thymocyte assay, we previously established that generation of a CTL response in the presence of IL-2 also required the addition of IL-6 (7, 8). Prolonged culture in the presence of IL-2 resulted in the generation of a CTL response; however, this corresponded to the production of IL-6 by cells contained in the thymocyte culture (11). Thus, finding that a lymphokine will support the generation of a CTL response is not definitive evidence that the added lymphokine is directly mediating the observed action. Since IL-4 has been identified as a lymphokine responsible for CTL generation, we examined the effect of IL-4 in combination with other lymphokines. To determine whether our experience with IL-2 in requiring other lympho-

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AND

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kines to be functional is typical, we looked for the presence of enhanced activity for combinations of lymphokines and used monoclonal antibodies able to block the action of certain lymphokines. Our results indicated that IL-2 and IL-6 play a role in the CTL responsegenerated in the presence of IL-4, as IL-2 enhanced the activity of a low level of IL-4; anti-IL-2 significantly blocked the activity of IL-4; and IL-4 induced the production of IL-2 and increased the expression of IL-2 receptor in thymocyte cultures. IL-6 enhanced the proliferative responsefound for IL-4, and antibody against IL-6 significantly inhibited both the IL-Csupported generation of CTL and the IL-4-mediated proliferation. The addition of the anti-IL-4 mAb to thymocyte cultures inhibited the CTL response to exogenous IL-4 but not to exogenous IL-2 plus IL-6. Thus, IL-4 did not appear to be required for the IL-2 + IL-6 supported generation,of CTL. However, IL-4 may be required for thymocyte proliferation in response to certain lymphokines (Bertagnolli and Herrmann, manuscript submitted). The inability of anti-IL-6 to completely block the response to IL-4 may indicate that two activation pathways exist, one requiring IL-6 and the other IL-6 independent. Becausewe used amounts of antibody that were more than enough to block the levels of endogenous IL-6 as determined by the B9 assay,we do not believe that these data reflect failure of the antibody to completely block endogenous IL-6. These arguments may also be extended to the failure of anti-IL-4 to block the CTL response for the combination of IL-2 and IL-6. Finally, the ability of human IL-2 and IL-6 to overcome the inhibitory effects of the mAbs suggestedthat the inhibition found is specific. Our data do not agree with some previous data concerning the effect of IL-4 on T lymphocytes. In a study by Pfeifer et al. the proliferative response and the cytotoxic response in the presence of IL-4 was not greatly inhibited by anti-IL-2R mAb 7D4. There are two basic differences between this work and our study. The assay in the Pfeifer study involved a 4- to 5-day incubation, compared to this study of about 2 days duration. In addition, the responder cells used in the Pfeifer study were spleen cells, compared to the thymocytes used for our assay. Immature T cells may have a greater requirement for lymphokine signaling and therefore require triggering through IL-2 in order to respond to IL-4. In summary, these data suggestthat the generation of CTL from thymocytes by the addition of IL-4 is dependent upon the presenceof functional IL-2 and IL-6. In culture of thymocytes of IL-4, the enhanced lysis with added IL-2 and the enhanced proliferation in the presence of IL-6 indicate that for CTL activation, IL-4 functions as an amplifying cosignal along with IL-2 and IL-6. We and others have shown that IL-6 is involved in the generation of a CTL response,asthe generation of CTL in the presence of IL-2 requires the addition of IL-6 to the thymocyte culture. In contrast, IL-4 generates a CTL response in the absenceof added IL-6. An IL-6 requirement for this response, however, can be shown by blocking endogenous production of IL-6 with anti-IL-6 mAb. Thus, IL-4 is able to generate a CTL response from thymocytes by using endogenous IL-6 and causing the production of IL-2. REFERENCES I. Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L., and Wiley, D. C., Nature (London) 329, 5 12, 1987. 2. Moller, Goran, Ed. “Immunological Reviews,” pp. l-222. Munksgaard International Booksellers and Publishers, Copenhagen K, Denmark, 1988.

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35. 36. 37. 38. 39. 40. 4 I. 42. 43.

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