- Email: [email protected]
Identification of a novel thymocyte growth-promoting factor derived from B cell lymphomas
CELLULAR
IMMUNOLOGY
Identification
129,228-240
(1990)
of a Novel Thymocyte Growth-Promoting Derived from B Cell Lymphomas
Factor
TAKASHI SUDA, ANNE O’GARRA, IAN MACNEIL, MELISSA FISCHER, MARTHA W. BOND,AND ALBERT~LOTNIK' Department ofImmunology, DNAX Research Institute,’ 901 California Avenue, Palo Alto, California 94304 Received February 26, 1990; accepted April 8, 1990 We found a unique thymocyte growth-promoting activity in supematants (SN) from subclones of the B cell lymphoma CH 12.LX. We have tentatively named this activity B-TCGF (for B cell-derived T cell growth factor) and characterized the activity produced by the CH12.LX.4866 subclone. This SN did not induce thymocyte proliferation alone, however, it enhanced both adult and fetal (Day IS of gestation) murine thymocyte proliferation in the presence of IL-2, IL-4, or K-7. Other known cytokines were screened for a B-TCGF-like activity using both adult and fetal thymocytes. IL-6 was found to be active only on adult thymocytes, while TNFol and GM-CSF were found to be active only on fetal thymocytes. However, neutralizing antibodies against these cytokines did not block the B-TCGF activity present in CH 12.LX.4866 using either adult or fetal thymocytes. These observations suggest that the BTCGF activity is mediated by a novel factor(s). The apparent molecular weight of this novel molecule(s) was 27-50 kDa determined by sizing HPLC. o 1990 Academic hffs IW.
INTRODUCTION The process of T cell development in the thymus has been the subject of intense study. However, the signals that mediate growth and differentiation of thymocytes are still poorly understood. Several cytokines have been shown to induce thymocyte proliferation. These include IL1 (l-3), IL2 (l-3), IL-4 (4, 5), IL-6 (3,6,7), IL-7 (7, 8), and TNFar (9). Some of these cytokines show specificity for different thymocyte subsets (10). We recently observed that various subsets of adult thymocytes including CD4-CD8-, as well as fetal thymocytes at Day 15 of gestation (Day 15 FI) were able to respond to anti-CD3 antibodies in the presence of IL-2 and IL-4 (T. Suda and A. Zlotnik, unpublished observation). We reasoned that this system could be used to explore the specificity of the T cell receptor expressed by these cells, so we performed these experiments using either syngeneic or allogeneic class II MHC-bearing B cell lymphomas as stimulator cells in the presence of IL-2 and K-4. Some of these combinations resulted in strong proliferative responses by either adult or fetal thymocytes. However, control experiments indicated that these responses were not MHC-restricted and were instead mediated by soluble products present in the supematant ’ To whom correspondence should be addressed. * DNAX Research Institute is supported by Schering-Plough Corporation. 228 0008-8749190 $3.00 Copyright 0 1990 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
NOVEL
THYMOCYTE
GROWTH
229
FACTOR
TABLE 1 List of Cytokines Used in This Study Cytokine r-hub IL- I (Y r-mu IL-2 r-mu IL-3 r-mu IL-4 r-mu IL-5 r-mu IL-6 r-mu IL-7 r-mu TNFol r-mu GM-CSF n-po TGFB
Specific activity (host) lo8 units/mg (E. coli) 3 X 10’ units/mg (E. coli) 3 X lo8 units/mg (silkworm) lo8 units/mg (COS 7) 10’ units/mg (COS 7) 4 X 10’ units/mg (COS 7) (crude SN’) (COS 7) 3 X 10’ units/mg (COS 7) 10’ units/mg (COS 7) 7 X 10’ units/mg (platelet)
Response used to determine a unit a Thymocyte proliferation with PHA HT-2 proliferation
Source Genzyme Corp.
HT-2 proliferation
T. Mosmann (DNAX) A. Miyajima (DNAX) N. Harada (DNAX)
BCLl proliferation
R. Coffman (DNAX)
KD 83 proliferation (7)
F. Lee (DNAX)
clone K proliferation (7)
F. Lee (DNAX)
WEHI- 164.13 killing ( 10) NFS-60 proliferation (15)
M. Palladino (Genentech) Schering-Plough Res.
Inhibition of Mv 1Lu proliferation
R&D Systems, Inc.
MC9 proliferation (14)
a A unit of IL-2 was determined using an international standard. The other units were defined as the amount in 1 ml which induced half-maximum responses. ‘Abbreviations: r, recombinant; n, natural; hu, human; mu, murine; po, porcine. ‘Approximately 2 X lo4 units/ml.
(SN) of the B cell lymphomas used as stimulator cells. The best activity was found in subclones of CH 12.LX and CH44 B cell lymphomas, some of which have been reported to induce expansion of non-neoplastic T cells when injected into mice (11, 12). In fact, a previous report has described a T cell growth factor activity produced by the parent cell lines of these subclones ( 12), although this activity was not characterized. In this report, we describe a thymocyte growth-promoting activity present in B lymphoma SN. The results strongly suggest the existence of a novel cytokine which may play a role in T cell development as well as in the non-neoplastic T cell expansion associated with B cell lymphomas. MATERIALS
AND METHODS
Preparation of adult andfetal thymocytes.Three- to seven-week-old female BALB/ c mice (Simonsen Laboratories, Gilroy, CA) were used as the source of adult thymocytes. Day 15 FT were obtained from timed pregnant Balb/c mice (Simonsen Labs). The day of gestation was calculated by plug date and verified by fetal morphology. Fetal thymic lobes were obtained using microdissection forceps under a dissecting microscope. Single-cell suspensions were prepared by gentle teasing between two glass slides. B cell lymphoma cell lines. Various subclones derived from CH12.LX and CH44 B cell lymphomas were generous gifts of Dr. G. Haughton (University of North Caro-
230
SUDA ET AL. TABLE 2 List of Antibodies Used in This Study
Specificity hybridoma mo IL3
19B3
moIL-5
TRFKl
mo IL-6
MP520F3
Species and class Rat IgG2b
Rat IgG I
mo TNFol (antiserum) mo GM-CSF 22E9
Rabbit
mo TGF@ (antiserum)
Rabbit IgG
Neutralizing activity 0.5 &ml neutralized 10 units IL-3 2.5 &ml neutralized 1:25 dil. D 10 sup. 0.5 &ml neutralized 40 units/ml IL6 IO6 neutralizing units/ml 2.5 &ml neutralized 10 rig/ml GM-CSF 30 @g/ml neutralized 1 rig/ml TGF(3
Source J. Abrams ( 16) (DNAX) A. O’Garra ( 17) (DNAX) M. Pearce (7) (DNAX) Genzyme Corp. J. Abrams (DNAX) R&D Systems, Inc.
lina). The A20- 1.11 B cell lymphoma was kindly provided by Dr. P. Marrack (Howard Hughes Medical Institute, Denver, CO). Cells were cultured in RPM1 1640 medium supplemented with 50 N2-mercaptoethanol, 2 mglutamine, 10 mMHepes (pH 7.4), 1 m&f sodium pyruvate and 100 unit/ml penicillin and 100 &ml streptomycin containing 5% FCS (JR Scientific, Woodland, CA). Cytokines and antibodies against cytokines. The cytokines and the neutralizing anti-cytokine antibodies used in this study are listed in Tables 1 and 2, respectively. Separation of thymocyte subsets. Adult thymocyte subsets were sorted on a Becton-Dickinson FACS IV as described (10). Briefly, thymocytes were incubated with either monoclonal anti-CD4 antibodies (RL172) and/or monoclonal anti-CD8 (Lyt2.2) antibodies (AD4, purchased from Cedarlane Laboratories Limited, Ontario, Canada) as needed for 45 min on ice, followed by treatment with low toxicity rabbit complement (Cedarlane) for 30 min at 37°C. Cells were then stained with phycoerythrin-conjugated monoclonal anti-CD4 antibodies (GK1.5) and fluorescein-conjugated anti-CD8 monoclonal antibodies (53-6.7, Becton-Dickinson Mountain View, CA). CD4-8- double-negative (DN) and CD4+ single-positive (SP) cells were routinely greater than 99% pure, while the CD8SP were greater than 98% and CD4+8+ double-positives (DP) were greater than 97% pure. Coating of anti-mouse CD3 monoclonal antibodies on pat-bottom 96-well plates. The anti-CD3 monoclonal antibody producing hybridoma, 145-2Cll (13) was kindly provided by Dr. Jelfrey Bluestone (University of Chicago). Fifty microliters of 145-2Cll serum-free culture SN was added to the wells and was incubated at 37°C for 1 hr. The SN was then removed, and the wells were washed extensively with medium containing 10% FCS. Thymocyte proliferation assay. Cells ( lo*) were cultured with SN from B lymphomas or various cytokines in the presence of IL-2 (500 units/ml) and IL-4 (250 units/ ml) in 300 ~1 of RPM1 medium supplemented by L-glutamine (200 m&Q, MEMamino acids, MEM-vitamins, sodium bicarbonate, penicillin/streptomycin (Sigma), 5 X 10e5 M 2-mercaptoethanol and 10% fetal calf serum (JR Scientific) in a flatbottom 96-well culture plate for 4 days. The wells were then pulsed with 1 &i/well [3H]thymidine and harvested 18 hr later.
NOVEL
THYMOCYTE
GROWTH
113
lit3
231
FACTOR
1:766
1:12
1:192
Dllution
of Supernatant
1~3072
FIG. 1. Dose-dependent growth-promoting effect of culture SN from B cell lymphomas on thymocytes in the presence of IL-2 plus IL-4. Adult thymocytes ( 105) were cultured with serial dilutions of culture SN from various B-lymphomas: A20-1.11 (A); CH 12.LX.4550 (0); CH12.LX.4866 (0); CH 12.LX.4922 (B); CH44.4925 (A) in the presence of IL-2 (500 units/ml) plus IL-4 (250 units/ml) for 4 days. The broken line shows [‘Hlthymidine incorporation induced by IL-2 plus IL-4 without culture SN.
GelJiltration chromatography. CH 12.LX.4866 SN was concentrated lo-fold using an Amicon (Danvers, MA) ultrafiltration system with a YM5 membrane. A 250-~1 aliquot was applied to a 0.75 X 60 cm TSK G3000 SW column (LKB Instruments, Gaithersburg, MD) equilibrated in 0.25 MNaCl, 0.05 MHepes (pH 7.2), and eluted at a flow rate of 0.2 ml/min. One-minute fractions were collected and stored at -70°C until assayed for B-TCGF activity. RESULTS B lymphoma culture SN enhances the proliferation of adult thymocytes in thepresence of IL-2 plus IL-4. We first observed that anti-CD3 antibodies induce thymocyte proliferation in the presence of IL-2 and IG4. Subsequently, we sought to investigate TABLE 3 B-TCGF Enhances Adult Thymocyte Proliferation Induced by IG2, IL-4, or IL-7 [3H]TdR incorporation Cytokine”
Medium
None IL-2 IL-4 IL-4 + IL-2 Mock IL-7
0.1 + o.oc 2.4 + 0.8 0.7 f 0.2 8.5 + 1.6 0.1 f 0.0 0.9 k 0.2
(X lo-’ cpm) CH12.LX.4866
SNb
0.220.1 12.3 f 1.1 22.1 2 1.3 72.7 f 2.6 0.1 +0.1 9.4 + 0.5
’ Final concentrations of cytokines were: IL-2,500 units/ml; IL4,250 units/ml; mock (mock transfected COS7 cell SN as a control for IL7); 1:100 dilution, IL7; 1:100 dilution (approximately 200 units/ml). b Final 1:6 dilution. ’ Standard deviation.
232
SUDA ET AL.
1
2
3 Culture
4
5
6
Days
FIG. 2. Kinetics of thymocyte proliferative response to CH12.LX.4866 SN. Adult thymocytes were cultured with (0) or without (0) CH12.LX.4866 culture SN (final 1:6 dilution) or in wells where anti-CD3 antibodies were precoated (A), in the presence of IL-2 (500 units/ml) plus IL-4 (250 units/ml) for various periods.
the specificity of the CD3+ thymocytes responding under these conditions, by substituting anti-CD3 stimulation with allogeneic MHC antigen stimulation. BALB/c thymocytes were cultured with either syngeneic or allogeneic class II MHC expressing B cell lymphomas in the presence of IL-2 and IL-4. Some B cell lymphomas derived from B10.H-2”H-4bp/Wts (2”4b) mice induced potent thymocyte proliferation under these conditions. However, anti-MHC antibodies specific for this haplotype did not inhibit the responses (data not shown). Therefore, we tested whether these B cell lymphomas produced soluble factor(s) which enhanced thymocyte proliferation. As shown in Fig. 1, culture SN from some of these B cell lymphomas enhanced thymocyte proliferation induced by IL-2 plus IL-4 in a dose-dependent manner. One of the best sources of this activity was the B cell lymphoma CH12.LX.4866. SN from this CH 12 subclone enhanced thymocyte responses to IL-2, IL-4, or IL-7, while the SN alone did not induce significant proliferation (Table 3). We tentatively named this activity B-TCGF (for B-cell derived T cell growth factor). As shown in Fig. 2, the maximum response was observed at 4 days of culture for B-TCGF stimulation, while anti-CD3 induced a quicker response. For this reason, a culture period of 4 days was used in the following experiments. No single known cytokine can account for the B-TCGF activity. We knew that CH 12.LX.4866 cells produce IL-3, IL-4, IL-6, TNFa, GM-CSF, and TGF& but not IL-l, IL-2, IL-5, IFN~, nor IL-73. To investigate whether a single known cytokine could account for the B-TCGF activity observed in this SN, various recombinant cytokines were tested for potential B-TCGF activity in combination with IL-2 and IL-4. As shown in Fig. 3, only IL-6 enhanced the IL-2 plus IL-Cinduced response as much as CH12.LX.4866 SN (when using adult unfractionated thymocytes as responding cells).
3 G’Garra, A., Stapleton, G., Dhar, V., Pearce, M., Schumacher, J., Rugo, H., Barbis, D., Stall, A., Cupp, J., Moore, K., Vieira, P., Mosmann, T., Whitmore, A., Arnold, L., Hat&ton, G., and Howard, M. Znternational Zmmunology, in press.
NOVEL
THYMOCYTE
GROWTH
FACTOR
233
FIG. 3. Only IL-6 can mimic B-TCGF activity for adult thymocytes. Adult thymocytes were cultured with serial dilutions of CH 12.LX.4866 SN or various recombinant cytokines indicated, in the presence (0) or absence (0) of IL-2 (500 units/ml) plus IL-4 (250 units/ml). Broken lines show t3H]thymidine incorporation induced by IL-2 plus IL-4 without the SN or cytokines.
Recently, we obtained a neutralizing monoclonal antibody against murine IL-64 (20F3). Using this antibody we investigated whether the B-TCGF activity present in CH12.LX.4866 SN was due to IL-6. As shown in Fig. 4, 20F3 inhibited partially (20-30%) the response to CH 12.LX.4866 SN, while the IL-6-induced response was abrogated completely. This partial inhibition indicates a minor contribution of IL-6 in the proliferation observed to CH 12.LX.4866 SN. Neutralizing antibodies specific for other known cytokines were also tested, but none of them significantly inhibited B-TCGF activity present in CH 12.LX.4866 SN (Fig. 5). B-TCGF enhances responses of CD4+8-, CD4-8’ and CD4-8- subsets of adult thymocytes. Adult thymocytes were separated on the basis of their expression of CD4 and/or CD8 cell surface markers. CH 12.LX.4866 SN was tested on each thymocyte subset for growth-promoting activity. As shown in Table 4, the SN enhanced both CD4+ SP and DN proliferation in the presence of IG2 plus IL-4. The combination of IL-2 and IL-4 induced potent proliferation of CD8+ SP without CH12.LX.4866 4 Starnes, H. F., Pearce, M. K., Tewari, A., Yim, J. H., Zou, J. C., Sullivan, L. M., and Abrams, J. S. submitted for publication.
234
SUDA ET AL.
A
B 60
60 -
Q--v+ T
YJ 0 40 I % 0 20
01
___.__ . L-___
-A-
0 1:12 I:46 CH12.LX.4666 106
1~192 I:766 SN Dilution
26 646 IL-6 (units/ml)
0
104 Anti-IL-6
10-1 100 10’ Antlbody @g/ml)
1.66’
FIG. 4. Anti-IL6 monoclonal antibodies abrogate IG6-induced but not B-TCGF-induced enhancement of thymocyte response to IL-2 plus IL-4. (A) Adult thymocytes were cultured with serial dilutions of CH12.LX.4866 SN (0,0) or IL6 (&A), and IL2 plus IL-4 in the presence (0,A) or absence (@,A) of I &ml anti-IL-6 monoclonal antibody (MP520F3). (B) Adult thymocytes were cultured with a I:6 dilution of CH12.LX.4866 SN (0) or 200 units/ml of IL6 (0), and IL-2 plus IL4 in the presence or absence of various doses of anti-IL-6 antibody. Vertical lines show standard deviations. Broken lines show [‘Hlthymidine incorporation induced by IL-2 plus IL-4 alone.
SN but some enhancement was reproducibly observed by addition of the SN. The enhancement of the CD8+ SP response was more evident when CH 12.LX.4866 SN was used with either IL-2 or IL4 alone (data not shown). In contrast, the CH12.LX.4866 SN failed to induce DP proliferation in the presence or absence of IL72 plus IL-4.
4~6
SN
antibody against:
-
none
+
non*
+
IL-2
+
IL-6
+
TNFa
+
IFNT
+
CW-CSF
+
TGFp
CPMXlOJ 0
20
40
60
60
100
FIG. 5. Neutralizing antibodies against various cytokines do not interfere with CH 12.LX.4866-induced adult thymocyte proliferation. Adult thymocytes were cultured with a 1:6 dilution of CH 12.LX.4866 SN and IL-2 plus IL-4 in the presence or absence of neutralizing antibodies against various cytokines. Antibody concentrations were; anti-B-3,2 &ml; anti-IG5,2.5 pg/ml; anti-TNFa, 1:300 dilution (3,3 neutralizing units); anti-IFNy, 2 &ml; anti-GM-CSF, 2.5 &ml; anti-TGF@, 10 rcg/ml. Horizontal lines show standard deviations.
NOVEL
THYMOCYTE
GROWTH
235
FACTOR
TABLE 4 Proliferative Response of Various Subsets from Adult Thymocytes to B-TCGF in the Presence of IL2 Plus IL4” [3H]TdR incorporation Expt I 11 III
Thymocyte subset b
Medium
Unseparated CD4-CD8CD4+CD8Unseparated CD4-CD8CD4-CD8+ Unseparated CD4+CD8+
0.2 + O.Od 0.5 + 0.4 0.2kO.l 3.7 + 1.7 4.6 + 1.2 146.1 of:8.9 1.6 2 0.3 0.1 kO.0
1Om3cpm)
(X
CH12.LX.4866
SN’
5.7 * 0.2 23.0 e 0.9 48.1+ 2.7 45.5 + 2.9 101.4*0.3 231.4k4.1 46.2 + 3.5 0.5 * 0.3
’ IL-2,500 units/ml; IL-4,250 units/ml. b Adult thymocytes were separated into four subsets based on their expression of CD4 and CD8 antigens by using complement dependent cell killing and subsequent cell sorting (See Materials and Methods). c Final 1:6 dilution. [‘H]TdR incorporation induced by CH12.LX.4866 SN without IL2 and IL4 were always less than 1000 cpm. d Standard deviation.
B-TCGF enhancesDay 15fetal thymocyteproliferation with IL-2, but not with IL4 or IL- 7. The majority of Day 15 FT are CD3-4-8- except for a small number of CD3+ cells expressing the $-TCR. This makes Day 15 FT a good source of immature T cells. To investigate whether Day 15 FT also respond to B-TCGF, they were cultured with CH12.LX.4866 SN in the presence of IL-2, E-4, IL-2 + IL-4, or IL-7. As shown in Table 5, CH12.LX.4866 SN failed to induce substantial proliferation of Day 15 FT alone or with IL-4, while it enhanced the proliferation induced by IL-2. As reported elsewhere (7, 8), IL-7 induced substantial proliferation of Day 15 FT by itself. However, CH 12.LX.4866 SN did not enhance this response significantly. Thus, B-TCGF can promote proliferation of various maturational stages of thymocytes in the presence of IL-2. TABLE 5 Differential Effects of B-TCGF, TNFa, and GM-CSF on Day I5 Fetal Thymocyte Proliferation Induced by IL-2, B-4, or IL-7 [‘H]TdR incorporation (X lo-’ cpm) in the presence of Cytokine”
Medium
None IL-2 IL-4 IL-4 + IL-2 IL-7
0.1 * 0.0’ 5.2 + 0.2 0.1 + 0.0 1.3 + 0.2 13.6 + 3.8
n See footnote a of Table 3. b Final concentration: CH12.LX.4866 ’ Standard deviation.
CH12.LX.4866 0.2 + 0.0 18.4 + 1.1 1.2 f 0.6 17.0 Ik 0.7 18.3 + 2.3
SNb
TNFa b
GM-CSFb
0.6 L- 0.5 80.8 +- 1.6 0.6 + 0.2 7.1 iT 0.0 16.5 * 4.3
1.8 zk0.5 17.5 f 0.9 1.2f0.3 5.0 zk 0.4 51.4k3.4
SN, 1:6 dilution; TNFol, 100 units/ml; GM-CSF, 10 rig/ml.
236
SUDA ET AL.
madlum 4666 *up. IL-1 0: IL-3 IL-5 IL-6
FIG. 6. Only TNFa and GM-CSF substitute for B-TCGF as growth promoters for fetal thymocytes. Day 15 FT ( 105) were cultured with or without CH 12.LX.4866 SN or various recombinant cytokines in the presence of IL-2 (500 units/ml) plus IL-4 (250 units/ml). Final concentrations of cytokines were: CHi2.LX.4866 SN, 1:6 dilution; IL-I a, 40 units/ml; IL-3, 100 units/ml; IL-5 I .25 ndml; IL-6,20 units/ ml; TNFol, 100 units/ml; IFN-r, 100 Units/ml; GM-CSF, 10 rig/ml; TGF& 10 @ml. Horizontal lines show standard deviations.
No single known cytokine can account for Day 15 fetal thymocyte responses to BTCGF. Various known cytokines were tested for their growth-promoting effects on Day 15 FT. As shown in a representative experiment in Fig. 6, TNFa and GM-CSF induced significant enhancement of the Day 15 FT response in the presence of IL-2 plus IL-4. While this experiment shows single concentrations (this was the only way to test many cytokines in a single experiment due to cell number limitations), these concentrations were selected as optimal from titration experiments (data not shown). There were significant differences between the response patterns of Day 15 FT to TNFa, GM-CSF and CH12.LX.4866 SN (Table 5). Namely, while all three (TNFa, GM-CSF, and CH12.LX.4866 SN) enhanced the response induced by IL-2, TNFa induced significantly greater enhancement than the others. We have previously shown that IL-4 inhibits the TNFcv plus IL-2-induced response ( 18). IL-4 also inhibits the GM-CSF + IL-Zinduced response, but it did not affect the CH12.LX.4866 SN plus IL-2-induced response. GM-CSF but not CH 12.LX.4866 SN enhanced the IL7-induced response. Finally, neutralizing antibodies against TNFa or GM-CSF did not inhibit the proliferation of Day 15 FT in response to CH 12.LX.4866 SN (Fig. 7). cpmxl0” 4666 SN
+
nqne non*
O-
IL-3
+ +
IL-6 TNFa IFNy GM-CSF TGFb
FIG. 7. Neutralizing antibodies against various cytokines do not interfere with CH 12.LX.4866 induced fetal thymocyte proliferation. The conditions used are the same as in Fig. 5 legend except that Day 15 ET were used as responding cells.
NOVEL
THYMOCYTE
GROWTH
FACTOR
237
B-TCGF activity is mediated by a 27-50 kilodalton (kDa) molecule(s). The BTCGF activity present in CH12.LX.4866 SN was destroyed by heating at 60°C for 30 min (data not shown). The approximately lo-fold concentrated (by Amicon ultrafiltration apparatus) CH12.LX.4866 SN was applied to a gel filtration column (TSK G3000 SW), and fractions were tested in B-TCGF assays using either fetal or adult thymocytes. The activity for adult thymocytes eluted as a single peak (or possibly a doublet) with an apparent molecular weight of 27-50 kDa (Fig. 8). The activity for Day 15 FT eluted as a single peak at the same place as activity for adult thymocytes (or the earlier peak of the doublet). DISCUSSION In this study, we have identified a novel cytokine activity produced by a B cell lymphoma, CH12.LX.4866. This novel cytokine, in combination with IL-2 and IL4, induced strong proliferative responses in several adult thymocyte subsets as well as in Day 15 PT. We initially detected this activity during attempts to study the antigen specificity of T cells in immature thymocyte subsets, by coculturing thymocytes with Ia-positive B cell lymphomas. We detected significant thymocyte proliferation but it was apparent that this response was mediated by a soluble factor produced by the B cell lymphomas. We characterized this activity (B-TCGF) in either adult or fetal thymocyte proliferation assays. In summary: (i) B-TCGF does not induce adult or fetal thymocyte proliferation alone; (ii) B-TCGF enhances adult (unseparated) thymocyte proliferation induced by IL-2, IL-4, or IL-7; (iii) B-TCGF enhances proliferation of CD4+SP, CD8+SP, or DN thymocytes induced by IL-2 plus IL-4; (iv) B-TCGF enhances Day 15 fetal thymocyte proliferation induced by IL-2, but not proliferation induced by IL-4 or IL-7. The ability of B-TCGF to promote proliferation of thymocytes at various stages of differentiation suggests a role for B-TCGF in T cell development. The characteristics of B-TCGF seemed to be unique to this molecule, and suggested that the activity is due to a novel cytokine. In an attempt to establish whether the activity could be due to known cytokines, we tested several recombinant cytokines, (including those known to be present in the SN of CH 12.LX.4866: IL-3, IL-6, TNFa, GM-CSF, and TGFp3). The following cytokines were tested for B-TCGF activity using both adult and fetal thymocytes: IL-l, IL-3, IL-S, IL-6, TNFa, GM-CSF, and TGFP (Figs. 3,6). Table 6 summarizes why B-TCGF is not likely to be mediated by known cytokines. In the adult thymocyte assay, only IL-6 had activity that could compare with the CH12.LX.4866 SN (Fig. 3). However, we ruled out that it could be responsible for the B-TCGF activity observed, since anti-IL-6 antibodies did not inhibit B-TCGF in the CH 12.4866 SN (Fig. 4). In contrast, only TNFcv and GM-CSF induced substantial responses of Day 15 FT (Fig. 6). Thus, none of these cytokines induced both adult and fetal thymocyte responses. Furthermore, antibodies against these cytokines did not neutralize the B-TCGF activity present in CH 12.4866 SN, in either the adult or the fetal thymocyte assay, also making it unlikely that the B-TCGF activity observed could be due to combinations of these cytokines. Thus, we conclude that the B-TCGF activity is not mediated by one of these cytokines. We have previously studied the growth-promoting effects of various cytokines on subsets of adult ( 10) and Day 15 PI (18). When we compared B-TCGF activity to these known thymocyte growth factors, it became apparent that the pattern ofthymocyte responses induced by B-TCGF is unique. Our previous findings indicate that:
238
SUDA ET AL. TABLE 6 Evidence for Nonidentity of B-TCGF with Known Cytokines
Cytokines
Present in CH 12.LX.4866 SN
Absence of primary growth factor activity”
IL- 1 IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 TNFcv IFN--f GM-CSF TGF/3 P40
No No Yes Yesc No Yes No Yes No Yes Yes No
Yes No Yes No Yes Yes No Yes Yes Yes Yes Yes”
Has B-TCGF activity in Adult assay
n See text (Discussion). b n.a., not applicable. ’ Only detectable by PCR (see text). d Anti-IL-6 Ab partially (20-30s) inhibits CH 12.LX.4866 thymocyte assay (see Fig. 4). ’ nd., not done.
No b Fit Et
Yes n.d.’ No No No No No“
Fetal assay
Ab blocks B-TCGF activity
No n.a. No n.a. No No n.d. Yes No Yes No Yes”
nd. n.a. No n.a. No Nod n.d. No No No No n.d.
SN activity in the adult (but not the fetal)
(i) Cytokines can be divided into two groups based on their effects on thymocyte proliferation; primary growth factors that induce some proliferation by themselves (IG2, IL-4, or IL7) and secondary growth factors which do not induce thymocyteproliferation alone, but enhance the responses induced by primary growth factors. The latter include cytokines like IL- 1, IL-6, and TNFa! ( 10). According to these definitions, B-TCGF is a secondary growth factor since it does not induce proliferation by itself, but enhances responses of some primary growth factors. (ii) IL- 1, IL-6, and TNFa enhance proliferation of unseparated adult thymocytes (at 5 X lo5 cells/well) induced by IL-2 or IL-7, while only IL-6 enhances that induced by IL-4. It should be emphasized that neither IL-la nor TNFar induced adult thymocyte responses at a lower cell concentration ( lo5 cells/well) which was used in the B-TCGF assay (Fig. 3). Thus, B-TCGF is the second example (besides IL6) of a secondary growth factor which enhances IL-4-induced responses. We have also observed that IL 1(Yand IL6 show specificity for particular thymocyte subsets: e.g., IL- 1(Yfor CD4-8-3+ thymocytes, and IL-6 for CD4+ or CD8+ SP (lo), whereas B-TCGF affects all these subsets (Table 4). (iii) In Day 15 FT, TNFa and P40 (19) enhanced the response induced by IL-2, but not the response induced by IL-4 or IL-7 (18). However, CH12.LX 4866 does not produce detectable P403. In the present study, we found that GM-CSF enhanced the proliferation of Day 15-FT induced by IL-2 or IL7 (Table 5), while adult thymocytes responded poorly to it (Fig. 3). To our knowledge, this is the first report of GM-CSF effects on Day 15 FT. We previously reported that IL-4 inhibits the TNFa- but not the P40-induced response of Day 15 FT (18). Here we show that IL4 inhibits the response to GM-CSF but not to B-TCGF (Table 5). Thus, B-TCGF activity in FT proliferation is similar to P40 rather than to TNFa or GM-CSF.
NOVEL
2 .-
% '; E gi,.
THYMOCYTE
GROWTH
29OK14OK67K vvvv
-
32K
239
FACTOR
12.4K 7
lo-
50
-4o-
ia2. 3 2 I
5!+ .E
.z. p
lo-
%
z
r2$
O30
40
50
60
70
80
90
100
110
120
130
140
min
FIG. 8. B-TCGF activity elutes with an apparent molecular weight of 27-50 kDa. Tenfold concentrated CH12.LX.4866 SN (250 ~1) was fractionated in a TSK G3000 SW. Fifty or twenty-five microliters of each fraction were added to adult (0) or F’T (a), respectively, together with IL-2 (500 units/ml) and IL-4 (250 units/ml) in a total volume of 300 rl/culture. The solid line shows the absorbance at 280 nm. The arrows show elution positions of proteins of known molecular weight.
We previously reported that IL-7 induces proliferation of adult SP and DN as well as Day 15 FlY without other costimulants (7). This makes IL-7 a primary growth factor also different from B-TCGF. Furthermore, CH12.LX.4866 cells do not produce IL-73 and is therefore unlikely to mediate B-TCGF activity in CH12.LX. 4866 SN. These observations indicate that B-TCGF induces a unique pattern of mature and immature thymocyte responses, and strongly suggest that B-TCGF activity is due to a novel cytokine(s) that may play a role in T cell development. Physicochemical characterization demonstrated that B-TCGF is a heat-labile molecule with an apparent molecular weight of 27-50 kDa (Fig. 8). The activity eluted in the same area for the Day 15 FT assay, suggesting that the same molecular species mediates both responses. The slight variation between the adult and the fetal assays may represent molecular heterogenity ofthe molecule(s) responsible, or different sensitivity of the target cells. Recently, Willoughby et al. reported that SN from CH44 and CH12.LX B cell lymphomas (parent cell lines of subclones in which we found B-TCGF activity) induce proliferation of thymocyte or spleen cells without another costimulant (12). Although CH12.LX.4866 cells produce a limited amount of IL-4 (only detectable by Polymerase Chain Reaction (PCR) amplification of reverse-transcribed mRNA3), some other subclones produce substantial amounts of IL-4 (A. O’Garra, unpublished observations). Thus, it is possible that the reported direct growth induction was due to a combination of B-TCGF plus IL-4 which may have existed in the same SN. A recent paper by Sperling and Wortis (20) has described some T cells (CD4-8-3+ bearing the -ya T cell receptor) that are directly stimulated by a CH 12.LX B cell lymphoma. While the authors considered that this stimulation is mediated by direct cellcell interaction between the lymphoma and the responding T cells, it is also possible that the lymphoma may be producing B-TCGF and may play a role in the effects
240
SUDA ET AL.
observed in that system. As shown in Table 4, B-TCGF is a powerful growth stimulus for CD4-8- thymocytes in the presence of IL-2 plus IL-4. Recently, Fiorentino et al. (21) have described a novel activity produced by a T cell clone D lo-G4.1 that inhibits cytokine production from activated spleen cells. Specifically, these investigators have measured IFNr production and called their activity cytokine synthesis inhibitory factor (CSIF). We have preliminary evidence that CSIF and B-TCGF may be related cytokines, since CH12.LX.4866 produces CSIF3 and semipurified CSIF has activity in the B-TCGF assay. Further experiments will aim to establish the relationship between these two activities. ACKNOWLEDGMENTS The authors thank the sources listed in Tables 1 and 2 for their gifts of recombinant cytokines and antibodies against cytokines. We thank Dr. Geoffrey Haughton for the CH I2 B cell lymphomas and Dr. Allan Waitz for perusing the manuscript. We also thank Dr. James Cupp and Josephine Polakoff for their help with the FACS studies. Finally, we thank Dan Finn for excellent assistance in preparing the manuscript. Note added in proof: We have now confirmed that the B-TCGF activity described in this study is mediated by a novel cytokine that we have designated Interleukin 10 (&IO). This novel cytokine also mediates the activity described in reference 2 1, called “Cytokine Synthesis Inhibitory Factor” (CSIF). A cDNA encoding IL- 10 has been isolated and will be described soon (Moore, K. et aZ.,Science, in press).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Conlon, P., Henney, C., and Gillis, S., J. Immunol. 128,797, 1982. Mannel, D., Mizel, S., Diamanstein, T., and Falk, W., J. Immunol. 134,3108, 1985. Suda, T., Hodgkin, P., Lee, F., and Zlotnik, A., J. Immunol. Methods 120, 173, 1989. Zlotnik, A., Ransom, J., Frank, G., Fischer, M., and Howard, M., Proc. Natl. Acad. Sci. USA 84,3856, 1987. Palacios, R., Sideras, P., Boehmer, H. V., EMBO J. 6,91, 1987. Hodgkin, P. D., Bond, M. W., D’Garra, A., Frank, G., Lee, F., Colfman, R. L., Zlotnik, A., and Howard, M., J. Immunol. 141, 151, 1988. Murray, R., Suda, T., Wrighton, N., Lee, F., and Zlotnik, A., Int. Immunol. 1,526,1989. Watson, J., Morrisey, P., Namen, A., Conlon, P., and Widmer, M., J. Immunol. 143, 1215, 1989. Ranges, G. E., Zlotnik, A., Espevik, T., Dinarello, C., Cerami, A., and Palladino, M. A., J. Exp. Med. 167,1472, 1988. Suda, T., Murray, R., Guidos, C., and Zlotnik, A., J. Immunol., 144,3039. Willoughby, P., and Haughton, G., J. Natl. Cancer Inst. 80,35 1, 1988. Willoughby, P., Jennette, J., and Hat&ton, G., J. Pathol. 133,507, 1988. Leo, D., Foo, M., Sachs, D. H., Samelson, L. E., and Bluestone, J. A., Proc. Natl. Acad. Sci. USA 84, 1374,1987. Miyajima, A., Schreurs, J., Gtsu, K., Kondo, A., Arai, K., and Mae&, S., Gene 58,273, 1987. Chiu, C. P., Moulds, C., Coffman, R. L., Rennick, D., and Lee, F., Proc. Natl. Acad. Sci. USA 85, 7099, 1988. Abrams, J. S., and Pearce, M. K., J. Immunol., 140,131,1988. Schumacher, J. H., O’Garra, A., Shrader, B., vanKimmenade, A., Bond, M. W., Mosmann, T. R., and Coffman, R. L., J. Immunol. 141,1576.1988. Suda, T., Murray, R., Fisher, M., Yokota, T., and Zlotnik, A., J. Immunol., 144, 1789. Van Snick, J., Goethals, A., Renauld, J., Van Boost, E., Uyttenhove, C., Rubria, M., Moritz, R., Simpson, R., J. Exp. Med. 169,363, 1989. Sperling, A. I., and Wortis, H. H., Int. Immunoi. 1,434,1989. Fiorentino, D. F., Bond, M. W., and Mosmann, T. R., J. Exp. Med. 170,2081, 1989.
IMMUNOLOGY
Identification
129,228-240
(1990)
of a Novel Thymocyte Growth-Promoting Derived from B Cell Lymphomas
Factor
TAKASHI SUDA, ANNE O’GARRA, IAN MACNEIL, MELISSA FISCHER, MARTHA W. BOND,AND ALBERT~LOTNIK' Department ofImmunology, DNAX Research Institute,’ 901 California Avenue, Palo Alto, California 94304 Received February 26, 1990; accepted April 8, 1990 We found a unique thymocyte growth-promoting activity in supematants (SN) from subclones of the B cell lymphoma CH 12.LX. We have tentatively named this activity B-TCGF (for B cell-derived T cell growth factor) and characterized the activity produced by the CH12.LX.4866 subclone. This SN did not induce thymocyte proliferation alone, however, it enhanced both adult and fetal (Day IS of gestation) murine thymocyte proliferation in the presence of IL-2, IL-4, or K-7. Other known cytokines were screened for a B-TCGF-like activity using both adult and fetal thymocytes. IL-6 was found to be active only on adult thymocytes, while TNFol and GM-CSF were found to be active only on fetal thymocytes. However, neutralizing antibodies against these cytokines did not block the B-TCGF activity present in CH 12.LX.4866 using either adult or fetal thymocytes. These observations suggest that the BTCGF activity is mediated by a novel factor(s). The apparent molecular weight of this novel molecule(s) was 27-50 kDa determined by sizing HPLC. o 1990 Academic hffs IW.
INTRODUCTION The process of T cell development in the thymus has been the subject of intense study. However, the signals that mediate growth and differentiation of thymocytes are still poorly understood. Several cytokines have been shown to induce thymocyte proliferation. These include IL1 (l-3), IL2 (l-3), IL-4 (4, 5), IL-6 (3,6,7), IL-7 (7, 8), and TNFar (9). Some of these cytokines show specificity for different thymocyte subsets (10). We recently observed that various subsets of adult thymocytes including CD4-CD8-, as well as fetal thymocytes at Day 15 of gestation (Day 15 FI) were able to respond to anti-CD3 antibodies in the presence of IL-2 and IL-4 (T. Suda and A. Zlotnik, unpublished observation). We reasoned that this system could be used to explore the specificity of the T cell receptor expressed by these cells, so we performed these experiments using either syngeneic or allogeneic class II MHC-bearing B cell lymphomas as stimulator cells in the presence of IL-2 and K-4. Some of these combinations resulted in strong proliferative responses by either adult or fetal thymocytes. However, control experiments indicated that these responses were not MHC-restricted and were instead mediated by soluble products present in the supematant ’ To whom correspondence should be addressed. * DNAX Research Institute is supported by Schering-Plough Corporation. 228 0008-8749190 $3.00 Copyright 0 1990 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.
NOVEL
THYMOCYTE
GROWTH
229
FACTOR
TABLE 1 List of Cytokines Used in This Study Cytokine r-hub IL- I (Y r-mu IL-2 r-mu IL-3 r-mu IL-4 r-mu IL-5 r-mu IL-6 r-mu IL-7 r-mu TNFol r-mu GM-CSF n-po TGFB
Specific activity (host) lo8 units/mg (E. coli) 3 X 10’ units/mg (E. coli) 3 X lo8 units/mg (silkworm) lo8 units/mg (COS 7) 10’ units/mg (COS 7) 4 X 10’ units/mg (COS 7) (crude SN’) (COS 7) 3 X 10’ units/mg (COS 7) 10’ units/mg (COS 7) 7 X 10’ units/mg (platelet)
Response used to determine a unit a Thymocyte proliferation with PHA HT-2 proliferation
Source Genzyme Corp.
HT-2 proliferation
T. Mosmann (DNAX) A. Miyajima (DNAX) N. Harada (DNAX)
BCLl proliferation
R. Coffman (DNAX)
KD 83 proliferation (7)
F. Lee (DNAX)
clone K proliferation (7)
F. Lee (DNAX)
WEHI- 164.13 killing ( 10) NFS-60 proliferation (15)
M. Palladino (Genentech) Schering-Plough Res.
Inhibition of Mv 1Lu proliferation
R&D Systems, Inc.
MC9 proliferation (14)
a A unit of IL-2 was determined using an international standard. The other units were defined as the amount in 1 ml which induced half-maximum responses. ‘Abbreviations: r, recombinant; n, natural; hu, human; mu, murine; po, porcine. ‘Approximately 2 X lo4 units/ml.
(SN) of the B cell lymphomas used as stimulator cells. The best activity was found in subclones of CH 12.LX and CH44 B cell lymphomas, some of which have been reported to induce expansion of non-neoplastic T cells when injected into mice (11, 12). In fact, a previous report has described a T cell growth factor activity produced by the parent cell lines of these subclones ( 12), although this activity was not characterized. In this report, we describe a thymocyte growth-promoting activity present in B lymphoma SN. The results strongly suggest the existence of a novel cytokine which may play a role in T cell development as well as in the non-neoplastic T cell expansion associated with B cell lymphomas. MATERIALS
AND METHODS
Preparation of adult andfetal thymocytes.Three- to seven-week-old female BALB/ c mice (Simonsen Laboratories, Gilroy, CA) were used as the source of adult thymocytes. Day 15 FT were obtained from timed pregnant Balb/c mice (Simonsen Labs). The day of gestation was calculated by plug date and verified by fetal morphology. Fetal thymic lobes were obtained using microdissection forceps under a dissecting microscope. Single-cell suspensions were prepared by gentle teasing between two glass slides. B cell lymphoma cell lines. Various subclones derived from CH12.LX and CH44 B cell lymphomas were generous gifts of Dr. G. Haughton (University of North Caro-
230
SUDA ET AL. TABLE 2 List of Antibodies Used in This Study
Specificity hybridoma mo IL3
19B3
moIL-5
TRFKl
mo IL-6
MP520F3
Species and class Rat IgG2b
Rat IgG I
mo TNFol (antiserum) mo GM-CSF 22E9
Rabbit
mo TGF@ (antiserum)
Rabbit IgG
Neutralizing activity 0.5 &ml neutralized 10 units IL-3 2.5 &ml neutralized 1:25 dil. D 10 sup. 0.5 &ml neutralized 40 units/ml IL6 IO6 neutralizing units/ml 2.5 &ml neutralized 10 rig/ml GM-CSF 30 @g/ml neutralized 1 rig/ml TGF(3
Source J. Abrams ( 16) (DNAX) A. O’Garra ( 17) (DNAX) M. Pearce (7) (DNAX) Genzyme Corp. J. Abrams (DNAX) R&D Systems, Inc.
lina). The A20- 1.11 B cell lymphoma was kindly provided by Dr. P. Marrack (Howard Hughes Medical Institute, Denver, CO). Cells were cultured in RPM1 1640 medium supplemented with 50 N2-mercaptoethanol, 2 mglutamine, 10 mMHepes (pH 7.4), 1 m&f sodium pyruvate and 100 unit/ml penicillin and 100 &ml streptomycin containing 5% FCS (JR Scientific, Woodland, CA). Cytokines and antibodies against cytokines. The cytokines and the neutralizing anti-cytokine antibodies used in this study are listed in Tables 1 and 2, respectively. Separation of thymocyte subsets. Adult thymocyte subsets were sorted on a Becton-Dickinson FACS IV as described (10). Briefly, thymocytes were incubated with either monoclonal anti-CD4 antibodies (RL172) and/or monoclonal anti-CD8 (Lyt2.2) antibodies (AD4, purchased from Cedarlane Laboratories Limited, Ontario, Canada) as needed for 45 min on ice, followed by treatment with low toxicity rabbit complement (Cedarlane) for 30 min at 37°C. Cells were then stained with phycoerythrin-conjugated monoclonal anti-CD4 antibodies (GK1.5) and fluorescein-conjugated anti-CD8 monoclonal antibodies (53-6.7, Becton-Dickinson Mountain View, CA). CD4-8- double-negative (DN) and CD4+ single-positive (SP) cells were routinely greater than 99% pure, while the CD8SP were greater than 98% and CD4+8+ double-positives (DP) were greater than 97% pure. Coating of anti-mouse CD3 monoclonal antibodies on pat-bottom 96-well plates. The anti-CD3 monoclonal antibody producing hybridoma, 145-2Cll (13) was kindly provided by Dr. Jelfrey Bluestone (University of Chicago). Fifty microliters of 145-2Cll serum-free culture SN was added to the wells and was incubated at 37°C for 1 hr. The SN was then removed, and the wells were washed extensively with medium containing 10% FCS. Thymocyte proliferation assay. Cells ( lo*) were cultured with SN from B lymphomas or various cytokines in the presence of IL-2 (500 units/ml) and IL-4 (250 units/ ml) in 300 ~1 of RPM1 medium supplemented by L-glutamine (200 m&Q, MEMamino acids, MEM-vitamins, sodium bicarbonate, penicillin/streptomycin (Sigma), 5 X 10e5 M 2-mercaptoethanol and 10% fetal calf serum (JR Scientific) in a flatbottom 96-well culture plate for 4 days. The wells were then pulsed with 1 &i/well [3H]thymidine and harvested 18 hr later.
NOVEL
THYMOCYTE
GROWTH
113
lit3
231
FACTOR
1:766
1:12
1:192
Dllution
of Supernatant
1~3072
FIG. 1. Dose-dependent growth-promoting effect of culture SN from B cell lymphomas on thymocytes in the presence of IL-2 plus IL-4. Adult thymocytes ( 105) were cultured with serial dilutions of culture SN from various B-lymphomas: A20-1.11 (A); CH 12.LX.4550 (0); CH12.LX.4866 (0); CH 12.LX.4922 (B); CH44.4925 (A) in the presence of IL-2 (500 units/ml) plus IL-4 (250 units/ml) for 4 days. The broken line shows [‘Hlthymidine incorporation induced by IL-2 plus IL-4 without culture SN.
GelJiltration chromatography. CH 12.LX.4866 SN was concentrated lo-fold using an Amicon (Danvers, MA) ultrafiltration system with a YM5 membrane. A 250-~1 aliquot was applied to a 0.75 X 60 cm TSK G3000 SW column (LKB Instruments, Gaithersburg, MD) equilibrated in 0.25 MNaCl, 0.05 MHepes (pH 7.2), and eluted at a flow rate of 0.2 ml/min. One-minute fractions were collected and stored at -70°C until assayed for B-TCGF activity. RESULTS B lymphoma culture SN enhances the proliferation of adult thymocytes in thepresence of IL-2 plus IL-4. We first observed that anti-CD3 antibodies induce thymocyte proliferation in the presence of IL-2 and IG4. Subsequently, we sought to investigate TABLE 3 B-TCGF Enhances Adult Thymocyte Proliferation Induced by IG2, IL-4, or IL-7 [3H]TdR incorporation Cytokine”
Medium
None IL-2 IL-4 IL-4 + IL-2 Mock IL-7
0.1 + o.oc 2.4 + 0.8 0.7 f 0.2 8.5 + 1.6 0.1 f 0.0 0.9 k 0.2
(X lo-’ cpm) CH12.LX.4866
SNb
0.220.1 12.3 f 1.1 22.1 2 1.3 72.7 f 2.6 0.1 +0.1 9.4 + 0.5
’ Final concentrations of cytokines were: IL-2,500 units/ml; IL4,250 units/ml; mock (mock transfected COS7 cell SN as a control for IL7); 1:100 dilution, IL7; 1:100 dilution (approximately 200 units/ml). b Final 1:6 dilution. ’ Standard deviation.
232
SUDA ET AL.
1
2
3 Culture
4
5
6
Days
FIG. 2. Kinetics of thymocyte proliferative response to CH12.LX.4866 SN. Adult thymocytes were cultured with (0) or without (0) CH12.LX.4866 culture SN (final 1:6 dilution) or in wells where anti-CD3 antibodies were precoated (A), in the presence of IL-2 (500 units/ml) plus IL-4 (250 units/ml) for various periods.
the specificity of the CD3+ thymocytes responding under these conditions, by substituting anti-CD3 stimulation with allogeneic MHC antigen stimulation. BALB/c thymocytes were cultured with either syngeneic or allogeneic class II MHC expressing B cell lymphomas in the presence of IL-2 and IL-4. Some B cell lymphomas derived from B10.H-2”H-4bp/Wts (2”4b) mice induced potent thymocyte proliferation under these conditions. However, anti-MHC antibodies specific for this haplotype did not inhibit the responses (data not shown). Therefore, we tested whether these B cell lymphomas produced soluble factor(s) which enhanced thymocyte proliferation. As shown in Fig. 1, culture SN from some of these B cell lymphomas enhanced thymocyte proliferation induced by IL-2 plus IL-4 in a dose-dependent manner. One of the best sources of this activity was the B cell lymphoma CH12.LX.4866. SN from this CH 12 subclone enhanced thymocyte responses to IL-2, IL-4, or IL-7, while the SN alone did not induce significant proliferation (Table 3). We tentatively named this activity B-TCGF (for B-cell derived T cell growth factor). As shown in Fig. 2, the maximum response was observed at 4 days of culture for B-TCGF stimulation, while anti-CD3 induced a quicker response. For this reason, a culture period of 4 days was used in the following experiments. No single known cytokine can account for the B-TCGF activity. We knew that CH 12.LX.4866 cells produce IL-3, IL-4, IL-6, TNFa, GM-CSF, and TGF& but not IL-l, IL-2, IL-5, IFN~, nor IL-73. To investigate whether a single known cytokine could account for the B-TCGF activity observed in this SN, various recombinant cytokines were tested for potential B-TCGF activity in combination with IL-2 and IL-4. As shown in Fig. 3, only IL-6 enhanced the IL-2 plus IL-Cinduced response as much as CH12.LX.4866 SN (when using adult unfractionated thymocytes as responding cells).
3 G’Garra, A., Stapleton, G., Dhar, V., Pearce, M., Schumacher, J., Rugo, H., Barbis, D., Stall, A., Cupp, J., Moore, K., Vieira, P., Mosmann, T., Whitmore, A., Arnold, L., Hat&ton, G., and Howard, M. Znternational Zmmunology, in press.
NOVEL
THYMOCYTE
GROWTH
FACTOR
233
FIG. 3. Only IL-6 can mimic B-TCGF activity for adult thymocytes. Adult thymocytes were cultured with serial dilutions of CH 12.LX.4866 SN or various recombinant cytokines indicated, in the presence (0) or absence (0) of IL-2 (500 units/ml) plus IL-4 (250 units/ml). Broken lines show t3H]thymidine incorporation induced by IL-2 plus IL-4 without the SN or cytokines.
Recently, we obtained a neutralizing monoclonal antibody against murine IL-64 (20F3). Using this antibody we investigated whether the B-TCGF activity present in CH12.LX.4866 SN was due to IL-6. As shown in Fig. 4, 20F3 inhibited partially (20-30%) the response to CH 12.LX.4866 SN, while the IL-6-induced response was abrogated completely. This partial inhibition indicates a minor contribution of IL-6 in the proliferation observed to CH 12.LX.4866 SN. Neutralizing antibodies specific for other known cytokines were also tested, but none of them significantly inhibited B-TCGF activity present in CH 12.LX.4866 SN (Fig. 5). B-TCGF enhances responses of CD4+8-, CD4-8’ and CD4-8- subsets of adult thymocytes. Adult thymocytes were separated on the basis of their expression of CD4 and/or CD8 cell surface markers. CH 12.LX.4866 SN was tested on each thymocyte subset for growth-promoting activity. As shown in Table 4, the SN enhanced both CD4+ SP and DN proliferation in the presence of IG2 plus IL-4. The combination of IL-2 and IL-4 induced potent proliferation of CD8+ SP without CH12.LX.4866 4 Starnes, H. F., Pearce, M. K., Tewari, A., Yim, J. H., Zou, J. C., Sullivan, L. M., and Abrams, J. S. submitted for publication.
234
SUDA ET AL.
A
B 60
60 -
Q--v+ T
YJ 0 40 I % 0 20
01
___.__ . L-___
-A-
0 1:12 I:46 CH12.LX.4666 106
1~192 I:766 SN Dilution
26 646 IL-6 (units/ml)
0
104 Anti-IL-6
10-1 100 10’ Antlbody @g/ml)
1.66’
FIG. 4. Anti-IL6 monoclonal antibodies abrogate IG6-induced but not B-TCGF-induced enhancement of thymocyte response to IL-2 plus IL-4. (A) Adult thymocytes were cultured with serial dilutions of CH12.LX.4866 SN (0,0) or IL6 (&A), and IL2 plus IL-4 in the presence (0,A) or absence (@,A) of I &ml anti-IL-6 monoclonal antibody (MP520F3). (B) Adult thymocytes were cultured with a I:6 dilution of CH12.LX.4866 SN (0) or 200 units/ml of IL6 (0), and IL-2 plus IL4 in the presence or absence of various doses of anti-IL-6 antibody. Vertical lines show standard deviations. Broken lines show [‘Hlthymidine incorporation induced by IL-2 plus IL-4 alone.
SN but some enhancement was reproducibly observed by addition of the SN. The enhancement of the CD8+ SP response was more evident when CH 12.LX.4866 SN was used with either IL-2 or IL4 alone (data not shown). In contrast, the CH12.LX.4866 SN failed to induce DP proliferation in the presence or absence of IL72 plus IL-4.
4~6
SN
antibody against:
-
none
+
non*
+
IL-2
+
IL-6
+
TNFa
+
IFNT
+
CW-CSF
+
TGFp
CPMXlOJ 0
20
40
60
60
100
FIG. 5. Neutralizing antibodies against various cytokines do not interfere with CH 12.LX.4866-induced adult thymocyte proliferation. Adult thymocytes were cultured with a 1:6 dilution of CH 12.LX.4866 SN and IL-2 plus IL-4 in the presence or absence of neutralizing antibodies against various cytokines. Antibody concentrations were; anti-B-3,2 &ml; anti-IG5,2.5 pg/ml; anti-TNFa, 1:300 dilution (3,3 neutralizing units); anti-IFNy, 2 &ml; anti-GM-CSF, 2.5 &ml; anti-TGF@, 10 rcg/ml. Horizontal lines show standard deviations.
NOVEL
THYMOCYTE
GROWTH
235
FACTOR
TABLE 4 Proliferative Response of Various Subsets from Adult Thymocytes to B-TCGF in the Presence of IL2 Plus IL4” [3H]TdR incorporation Expt I 11 III
Thymocyte subset b
Medium
Unseparated CD4-CD8CD4+CD8Unseparated CD4-CD8CD4-CD8+ Unseparated CD4+CD8+
0.2 + O.Od 0.5 + 0.4 0.2kO.l 3.7 + 1.7 4.6 + 1.2 146.1 of:8.9 1.6 2 0.3 0.1 kO.0
1Om3cpm)
(X
CH12.LX.4866
SN’
5.7 * 0.2 23.0 e 0.9 48.1+ 2.7 45.5 + 2.9 101.4*0.3 231.4k4.1 46.2 + 3.5 0.5 * 0.3
’ IL-2,500 units/ml; IL-4,250 units/ml. b Adult thymocytes were separated into four subsets based on their expression of CD4 and CD8 antigens by using complement dependent cell killing and subsequent cell sorting (See Materials and Methods). c Final 1:6 dilution. [‘H]TdR incorporation induced by CH12.LX.4866 SN without IL2 and IL4 were always less than 1000 cpm. d Standard deviation.
B-TCGF enhancesDay 15fetal thymocyteproliferation with IL-2, but not with IL4 or IL- 7. The majority of Day 15 FT are CD3-4-8- except for a small number of CD3+ cells expressing the $-TCR. This makes Day 15 FT a good source of immature T cells. To investigate whether Day 15 FT also respond to B-TCGF, they were cultured with CH12.LX.4866 SN in the presence of IL-2, E-4, IL-2 + IL-4, or IL-7. As shown in Table 5, CH12.LX.4866 SN failed to induce substantial proliferation of Day 15 FT alone or with IL-4, while it enhanced the proliferation induced by IL-2. As reported elsewhere (7, 8), IL-7 induced substantial proliferation of Day 15 FT by itself. However, CH 12.LX.4866 SN did not enhance this response significantly. Thus, B-TCGF can promote proliferation of various maturational stages of thymocytes in the presence of IL-2. TABLE 5 Differential Effects of B-TCGF, TNFa, and GM-CSF on Day I5 Fetal Thymocyte Proliferation Induced by IL-2, B-4, or IL-7 [‘H]TdR incorporation (X lo-’ cpm) in the presence of Cytokine”
Medium
None IL-2 IL-4 IL-4 + IL-2 IL-7
0.1 * 0.0’ 5.2 + 0.2 0.1 + 0.0 1.3 + 0.2 13.6 + 3.8
n See footnote a of Table 3. b Final concentration: CH12.LX.4866 ’ Standard deviation.
CH12.LX.4866 0.2 + 0.0 18.4 + 1.1 1.2 f 0.6 17.0 Ik 0.7 18.3 + 2.3
SNb
TNFa b
GM-CSFb
0.6 L- 0.5 80.8 +- 1.6 0.6 + 0.2 7.1 iT 0.0 16.5 * 4.3
1.8 zk0.5 17.5 f 0.9 1.2f0.3 5.0 zk 0.4 51.4k3.4
SN, 1:6 dilution; TNFol, 100 units/ml; GM-CSF, 10 rig/ml.
236
SUDA ET AL.
madlum 4666 *up. IL-1 0: IL-3 IL-5 IL-6
FIG. 6. Only TNFa and GM-CSF substitute for B-TCGF as growth promoters for fetal thymocytes. Day 15 FT ( 105) were cultured with or without CH 12.LX.4866 SN or various recombinant cytokines in the presence of IL-2 (500 units/ml) plus IL-4 (250 units/ml). Final concentrations of cytokines were: CHi2.LX.4866 SN, 1:6 dilution; IL-I a, 40 units/ml; IL-3, 100 units/ml; IL-5 I .25 ndml; IL-6,20 units/ ml; TNFol, 100 units/ml; IFN-r, 100 Units/ml; GM-CSF, 10 rig/ml; TGF& 10 @ml. Horizontal lines show standard deviations.
No single known cytokine can account for Day 15 fetal thymocyte responses to BTCGF. Various known cytokines were tested for their growth-promoting effects on Day 15 FT. As shown in a representative experiment in Fig. 6, TNFa and GM-CSF induced significant enhancement of the Day 15 FT response in the presence of IL-2 plus IL-4. While this experiment shows single concentrations (this was the only way to test many cytokines in a single experiment due to cell number limitations), these concentrations were selected as optimal from titration experiments (data not shown). There were significant differences between the response patterns of Day 15 FT to TNFa, GM-CSF and CH12.LX.4866 SN (Table 5). Namely, while all three (TNFa, GM-CSF, and CH12.LX.4866 SN) enhanced the response induced by IL-2, TNFa induced significantly greater enhancement than the others. We have previously shown that IL-4 inhibits the TNFcv plus IL-2-induced response ( 18). IL-4 also inhibits the GM-CSF + IL-Zinduced response, but it did not affect the CH12.LX.4866 SN plus IL-2-induced response. GM-CSF but not CH 12.LX.4866 SN enhanced the IL7-induced response. Finally, neutralizing antibodies against TNFa or GM-CSF did not inhibit the proliferation of Day 15 FT in response to CH 12.LX.4866 SN (Fig. 7). cpmxl0” 4666 SN
+
nqne non*
O-
IL-3
+ +
IL-6 TNFa IFNy GM-CSF TGFb
FIG. 7. Neutralizing antibodies against various cytokines do not interfere with CH 12.LX.4866 induced fetal thymocyte proliferation. The conditions used are the same as in Fig. 5 legend except that Day 15 ET were used as responding cells.
NOVEL
THYMOCYTE
GROWTH
FACTOR
237
B-TCGF activity is mediated by a 27-50 kilodalton (kDa) molecule(s). The BTCGF activity present in CH12.LX.4866 SN was destroyed by heating at 60°C for 30 min (data not shown). The approximately lo-fold concentrated (by Amicon ultrafiltration apparatus) CH12.LX.4866 SN was applied to a gel filtration column (TSK G3000 SW), and fractions were tested in B-TCGF assays using either fetal or adult thymocytes. The activity for adult thymocytes eluted as a single peak (or possibly a doublet) with an apparent molecular weight of 27-50 kDa (Fig. 8). The activity for Day 15 FT eluted as a single peak at the same place as activity for adult thymocytes (or the earlier peak of the doublet). DISCUSSION In this study, we have identified a novel cytokine activity produced by a B cell lymphoma, CH12.LX.4866. This novel cytokine, in combination with IL-2 and IL4, induced strong proliferative responses in several adult thymocyte subsets as well as in Day 15 PT. We initially detected this activity during attempts to study the antigen specificity of T cells in immature thymocyte subsets, by coculturing thymocytes with Ia-positive B cell lymphomas. We detected significant thymocyte proliferation but it was apparent that this response was mediated by a soluble factor produced by the B cell lymphomas. We characterized this activity (B-TCGF) in either adult or fetal thymocyte proliferation assays. In summary: (i) B-TCGF does not induce adult or fetal thymocyte proliferation alone; (ii) B-TCGF enhances adult (unseparated) thymocyte proliferation induced by IL-2, IL-4, or IL-7; (iii) B-TCGF enhances proliferation of CD4+SP, CD8+SP, or DN thymocytes induced by IL-2 plus IL-4; (iv) B-TCGF enhances Day 15 fetal thymocyte proliferation induced by IL-2, but not proliferation induced by IL-4 or IL-7. The ability of B-TCGF to promote proliferation of thymocytes at various stages of differentiation suggests a role for B-TCGF in T cell development. The characteristics of B-TCGF seemed to be unique to this molecule, and suggested that the activity is due to a novel cytokine. In an attempt to establish whether the activity could be due to known cytokines, we tested several recombinant cytokines, (including those known to be present in the SN of CH 12.LX.4866: IL-3, IL-6, TNFa, GM-CSF, and TGFp3). The following cytokines were tested for B-TCGF activity using both adult and fetal thymocytes: IL-l, IL-3, IL-S, IL-6, TNFa, GM-CSF, and TGFP (Figs. 3,6). Table 6 summarizes why B-TCGF is not likely to be mediated by known cytokines. In the adult thymocyte assay, only IL-6 had activity that could compare with the CH12.LX.4866 SN (Fig. 3). However, we ruled out that it could be responsible for the B-TCGF activity observed, since anti-IL-6 antibodies did not inhibit B-TCGF in the CH 12.4866 SN (Fig. 4). In contrast, only TNFcv and GM-CSF induced substantial responses of Day 15 FT (Fig. 6). Thus, none of these cytokines induced both adult and fetal thymocyte responses. Furthermore, antibodies against these cytokines did not neutralize the B-TCGF activity present in CH 12.4866 SN, in either the adult or the fetal thymocyte assay, also making it unlikely that the B-TCGF activity observed could be due to combinations of these cytokines. Thus, we conclude that the B-TCGF activity is not mediated by one of these cytokines. We have previously studied the growth-promoting effects of various cytokines on subsets of adult ( 10) and Day 15 PI (18). When we compared B-TCGF activity to these known thymocyte growth factors, it became apparent that the pattern ofthymocyte responses induced by B-TCGF is unique. Our previous findings indicate that:
238
SUDA ET AL. TABLE 6 Evidence for Nonidentity of B-TCGF with Known Cytokines
Cytokines
Present in CH 12.LX.4866 SN
Absence of primary growth factor activity”
IL- 1 IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 TNFcv IFN--f GM-CSF TGF/3 P40
No No Yes Yesc No Yes No Yes No Yes Yes No
Yes No Yes No Yes Yes No Yes Yes Yes Yes Yes”
Has B-TCGF activity in Adult assay
n See text (Discussion). b n.a., not applicable. ’ Only detectable by PCR (see text). d Anti-IL-6 Ab partially (20-30s) inhibits CH 12.LX.4866 thymocyte assay (see Fig. 4). ’ nd., not done.
No b Fit Et
Yes n.d.’ No No No No No“
Fetal assay
Ab blocks B-TCGF activity
No n.a. No n.a. No No n.d. Yes No Yes No Yes”
nd. n.a. No n.a. No Nod n.d. No No No No n.d.
SN activity in the adult (but not the fetal)
(i) Cytokines can be divided into two groups based on their effects on thymocyte proliferation; primary growth factors that induce some proliferation by themselves (IG2, IL-4, or IL7) and secondary growth factors which do not induce thymocyteproliferation alone, but enhance the responses induced by primary growth factors. The latter include cytokines like IL- 1, IL-6, and TNFa! ( 10). According to these definitions, B-TCGF is a secondary growth factor since it does not induce proliferation by itself, but enhances responses of some primary growth factors. (ii) IL- 1, IL-6, and TNFa enhance proliferation of unseparated adult thymocytes (at 5 X lo5 cells/well) induced by IL-2 or IL-7, while only IL-6 enhances that induced by IL-4. It should be emphasized that neither IL-la nor TNFar induced adult thymocyte responses at a lower cell concentration ( lo5 cells/well) which was used in the B-TCGF assay (Fig. 3). Thus, B-TCGF is the second example (besides IL6) of a secondary growth factor which enhances IL-4-induced responses. We have also observed that IL 1(Yand IL6 show specificity for particular thymocyte subsets: e.g., IL- 1(Yfor CD4-8-3+ thymocytes, and IL-6 for CD4+ or CD8+ SP (lo), whereas B-TCGF affects all these subsets (Table 4). (iii) In Day 15 FT, TNFa and P40 (19) enhanced the response induced by IL-2, but not the response induced by IL-4 or IL-7 (18). However, CH12.LX 4866 does not produce detectable P403. In the present study, we found that GM-CSF enhanced the proliferation of Day 15-FT induced by IL-2 or IL7 (Table 5), while adult thymocytes responded poorly to it (Fig. 3). To our knowledge, this is the first report of GM-CSF effects on Day 15 FT. We previously reported that IL-4 inhibits the TNFa- but not the P40-induced response of Day 15 FT (18). Here we show that IL4 inhibits the response to GM-CSF but not to B-TCGF (Table 5). Thus, B-TCGF activity in FT proliferation is similar to P40 rather than to TNFa or GM-CSF.
NOVEL
2 .-
% '; E gi,.
THYMOCYTE
GROWTH
29OK14OK67K vvvv
-
32K
239
FACTOR
12.4K 7
lo-
50
-4o-
ia2. 3 2 I
5!+ .E
.z. p
lo-
%
z
r2$
O30
40
50
60
70
80
90
100
110
120
130
140
min
FIG. 8. B-TCGF activity elutes with an apparent molecular weight of 27-50 kDa. Tenfold concentrated CH12.LX.4866 SN (250 ~1) was fractionated in a TSK G3000 SW. Fifty or twenty-five microliters of each fraction were added to adult (0) or F’T (a), respectively, together with IL-2 (500 units/ml) and IL-4 (250 units/ml) in a total volume of 300 rl/culture. The solid line shows the absorbance at 280 nm. The arrows show elution positions of proteins of known molecular weight.
We previously reported that IL-7 induces proliferation of adult SP and DN as well as Day 15 FlY without other costimulants (7). This makes IL-7 a primary growth factor also different from B-TCGF. Furthermore, CH12.LX.4866 cells do not produce IL-73 and is therefore unlikely to mediate B-TCGF activity in CH12.LX. 4866 SN. These observations indicate that B-TCGF induces a unique pattern of mature and immature thymocyte responses, and strongly suggest that B-TCGF activity is due to a novel cytokine(s) that may play a role in T cell development. Physicochemical characterization demonstrated that B-TCGF is a heat-labile molecule with an apparent molecular weight of 27-50 kDa (Fig. 8). The activity eluted in the same area for the Day 15 FT assay, suggesting that the same molecular species mediates both responses. The slight variation between the adult and the fetal assays may represent molecular heterogenity ofthe molecule(s) responsible, or different sensitivity of the target cells. Recently, Willoughby et al. reported that SN from CH44 and CH12.LX B cell lymphomas (parent cell lines of subclones in which we found B-TCGF activity) induce proliferation of thymocyte or spleen cells without another costimulant (12). Although CH12.LX.4866 cells produce a limited amount of IL-4 (only detectable by Polymerase Chain Reaction (PCR) amplification of reverse-transcribed mRNA3), some other subclones produce substantial amounts of IL-4 (A. O’Garra, unpublished observations). Thus, it is possible that the reported direct growth induction was due to a combination of B-TCGF plus IL-4 which may have existed in the same SN. A recent paper by Sperling and Wortis (20) has described some T cells (CD4-8-3+ bearing the -ya T cell receptor) that are directly stimulated by a CH 12.LX B cell lymphoma. While the authors considered that this stimulation is mediated by direct cellcell interaction between the lymphoma and the responding T cells, it is also possible that the lymphoma may be producing B-TCGF and may play a role in the effects
240
SUDA ET AL.
observed in that system. As shown in Table 4, B-TCGF is a powerful growth stimulus for CD4-8- thymocytes in the presence of IL-2 plus IL-4. Recently, Fiorentino et al. (21) have described a novel activity produced by a T cell clone D lo-G4.1 that inhibits cytokine production from activated spleen cells. Specifically, these investigators have measured IFNr production and called their activity cytokine synthesis inhibitory factor (CSIF). We have preliminary evidence that CSIF and B-TCGF may be related cytokines, since CH12.LX.4866 produces CSIF3 and semipurified CSIF has activity in the B-TCGF assay. Further experiments will aim to establish the relationship between these two activities. ACKNOWLEDGMENTS The authors thank the sources listed in Tables 1 and 2 for their gifts of recombinant cytokines and antibodies against cytokines. We thank Dr. Geoffrey Haughton for the CH I2 B cell lymphomas and Dr. Allan Waitz for perusing the manuscript. We also thank Dr. James Cupp and Josephine Polakoff for their help with the FACS studies. Finally, we thank Dan Finn for excellent assistance in preparing the manuscript. Note added in proof: We have now confirmed that the B-TCGF activity described in this study is mediated by a novel cytokine that we have designated Interleukin 10 (&IO). This novel cytokine also mediates the activity described in reference 2 1, called “Cytokine Synthesis Inhibitory Factor” (CSIF). A cDNA encoding IL- 10 has been isolated and will be described soon (Moore, K. et aZ.,Science, in press).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
Conlon, P., Henney, C., and Gillis, S., J. Immunol. 128,797, 1982. Mannel, D., Mizel, S., Diamanstein, T., and Falk, W., J. Immunol. 134,3108, 1985. Suda, T., Hodgkin, P., Lee, F., and Zlotnik, A., J. Immunol. Methods 120, 173, 1989. Zlotnik, A., Ransom, J., Frank, G., Fischer, M., and Howard, M., Proc. Natl. Acad. Sci. USA 84,3856, 1987. Palacios, R., Sideras, P., Boehmer, H. V., EMBO J. 6,91, 1987. Hodgkin, P. D., Bond, M. W., D’Garra, A., Frank, G., Lee, F., Colfman, R. L., Zlotnik, A., and Howard, M., J. Immunol. 141, 151, 1988. Murray, R., Suda, T., Wrighton, N., Lee, F., and Zlotnik, A., Int. Immunol. 1,526,1989. Watson, J., Morrisey, P., Namen, A., Conlon, P., and Widmer, M., J. Immunol. 143, 1215, 1989. Ranges, G. E., Zlotnik, A., Espevik, T., Dinarello, C., Cerami, A., and Palladino, M. A., J. Exp. Med. 167,1472, 1988. Suda, T., Murray, R., Guidos, C., and Zlotnik, A., J. Immunol., 144,3039. Willoughby, P., and Haughton, G., J. Natl. Cancer Inst. 80,35 1, 1988. Willoughby, P., Jennette, J., and Hat&ton, G., J. Pathol. 133,507, 1988. Leo, D., Foo, M., Sachs, D. H., Samelson, L. E., and Bluestone, J. A., Proc. Natl. Acad. Sci. USA 84, 1374,1987. Miyajima, A., Schreurs, J., Gtsu, K., Kondo, A., Arai, K., and Mae&, S., Gene 58,273, 1987. Chiu, C. P., Moulds, C., Coffman, R. L., Rennick, D., and Lee, F., Proc. Natl. Acad. Sci. USA 85, 7099, 1988. Abrams, J. S., and Pearce, M. K., J. Immunol., 140,131,1988. Schumacher, J. H., O’Garra, A., Shrader, B., vanKimmenade, A., Bond, M. W., Mosmann, T. R., and Coffman, R. L., J. Immunol. 141,1576.1988. Suda, T., Murray, R., Fisher, M., Yokota, T., and Zlotnik, A., J. Immunol., 144, 1789. Van Snick, J., Goethals, A., Renauld, J., Van Boost, E., Uyttenhove, C., Rubria, M., Moritz, R., Simpson, R., J. Exp. Med. 169,363, 1989. Sperling, A. I., and Wortis, H. H., Int. Immunoi. 1,434,1989. Fiorentino, D. F., Bond, M. W., and Mosmann, T. R., J. Exp. Med. 170,2081, 1989.