Production of IL-2 and IFN by TH2 clones

Immunology Letters, 21 (1989) 119-126

Elsevier IML 01224

Production of IL-2 and IFN by TH2 clones A k i h i t o Yokoyama, Brian Evavold, Daniel E. D u n n a n d Jose Q u i n t a n s Department of Pathology, Committee on Immunology, University of Chicago, Chicago, IL 60637, U.S.A.

(Received 3 February 1989; accepted 27 February 1989)

1. Summary

We have studied the production of IL-2, IL-4 and IFN- 7 by a panel of CD4 ÷ clones produced in our laboratory. The clones were classified as TH1 and Tiq2 because of their ability to secrete IL-2 or IL-4, respectively, following stimulation with APC + Ag and by their characteristic proliferative responses to exogenous IL-2 or IL-4. Some of the TH2 clones, all of which happened to be autoreactive, produced IL-2 and one of these, as well as one antigen-reactive TH2 clone, also secreted IFN- 7 following stimulation with immobilized anti-CD3 mAb. IL-2 production by Tn2 cells required higher concentrations of anti-CD3 mAb than IL-4 production. Thus, the TH2 clones seem to be heterogeneous. We designate the IL-2/IL-4 secretors as TH2B and those making IL-4 as TH2A clones. 2. Introduction

Recent studies have demonstrated that murine helper T cell clones can be separated into at least two distinct subsets on the basis of lymphokine production and utilization [1-9]. One subset, designated TH1, produces IL-2 and IFN- 7, while the other, TH2, secretes IL-4 and IL-5. TH1 clones proliferate Key words: Helper T cells; Interleukins; Immune regulation Abbreviations." APC, antigen presenting cells; Ag, antigen; SN,

supernatant(s); PFA, paraformaldehyde;KLH, keyholelimpet hemocyanin;FGG,fowlgamma globulin;mAb,monoclonalantibody; rlL, recombinant interleukin; TH, T helper cell. Correspondence to." JoseQuintans, Committeeon Immunology, Department of Pathology, Box 414, 5841 S. Maryland Ave., University of Chicago, Chicago, IL 60637, U.S.A.

to exogenous IL-2, whereas TH2 clones require IL-1 as an accessory molecule for the maximal response to IL-2 or IL-4 [5]. Functionally, TH1 clones are involved in cell-mediated immunity like delayed-type hypersensitivity (DTH), and the TH2 subset participates in humoral immunity, providing cognate B cell help. In addition, we have recently observed that TH2 but not TH1 cells have co-stimulatory properties that allow them to mediate T - T interactions [I0, 11]. Some groups have also designated TH1 inflammatory cells, Tinf, and TH2 as the T helper cells, Thelp [5]. Exceptions to these general characterizations have been reported, with some THI clones failing to induce DTH [12] and others providing B cell help [3-6]. In the present study, we have examined lymphokine production by a panel of 10 CD4 ÷ clones generated and maintained in our laboratory. Following stimulation with APC 3 + Ag we detected two THI (IL-2 secretors) and eight TH2 (IL-4 secretors). Four TH2 cells could be induced to secrete IL-2 if stimulated by high concentrations of immobilized anti-CD3 mAb. One of the double IL-2/IL-4 secretors and one of the prototypic TH2 cells also produced low levels of IFN-7. These findings suggest that the heterogeneity of CD4 ÷ cells extends beyond the TH1/TH2 dichotomy. 3. Materials and Methods 3.1. A n i m a l s

Female CBA/Ca mice 6 - 1 2 weeks of age were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in laminar flow racks at the University of Chicago animal facility.

0165-2478 / 89 / $ 3.50 © 1989 ElsevierSciencePublishers B.V.(BiomedicalDivision)

119

3.2. T cell clones' D10.G4.1 [14] was a gift of Dr. Charles Janeway (Yale University). The remaining T cell clones used in this study were generated and maintained in our laboratory [10, 11, 13]. [.7.4 and LF2 are subclones of Lbd [13] derived from primed lymph nodes of a C B A / N mouse. Lbe, FGG-3, AK-4, PC-l, PC-15, and 5A were derived from CBA/CaJ mice. FGG-8 and FGG-I1 are FGG-reactive clones derived from CNKF1 (CBA/Ca x BALB.K) F1 mice [10]. The autoreactive clones were stimulated weekly with irradiated, syngeneic splenocytes. The antigen-reactive clones were maintained on a schedule of 7-days stimulation with antigen (FGG 100/~g/1.5 ml or KLH 20 t~g/1.5 ml) and irradiated (2000 rads) syngeneic spleen, followed by 7 days of rest with irradiated (2000 rads) syngeneic spleen and no antigen. All clones were maintained in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% FCS (Hyclone Laboratories Inc., Logan, UT), 2 mM Lglutamine, penicillin, streptomyocin and 5 x 10 5 M 2ME. 3.3. Reagents Murine recombinant rlL-4 and human rlL-ll3 were purchased from Genzyme (Boston, MA). Human rIL-2 from Hoffman-LaRoche was a gift of Dr. J. Richards (University of Chicago). Concentrations are expressed in units as defined by the suppliers. Anti-CD3 (145-2Cll) hybridoma [15] was a gift of Dr. J. Bluestone (University of Chicago). Anti-IL-2 ($4B6) was a gift of Dr. T. Mossman (DNAX, Palo Alto, CA). Anti-IL-4 (llBll) was obtained from American Type Culture Collection (ATCC, Rockville, MD). MAbs were purified from culture supernatant using 50% ammonium sulfate precipitation. 3.4. Generation of lymphokine containing supernatants 5 - 1 0 × 105 viable T H cells were stimulated by syngeneic spleen cells and appropriate antigen (100/~g/ml) or immobilized anti-CD3 mAb in 24well plates (final volume 2 ml). Immobilization of anti-CD3 mAb was achieved by incubation of purified antibody, unless otherwise stated at 1 #g/ml, for 4 - 1 5 hours in a CO2-free incubator at 37°C. 120

SN were collected after 2 4 - 4 8 h. IL-4 containing SN (IL-4SN) from the antigen-reactive clone FGG-3 stimulated with immobilized anti-CD3 was used as an IL-4 standard. Such SN typically contained 10-14×103 units of IL-4/ml as compared to the rlL-4. 3.5. Assays The proliferative responses of the T H clones (2xl04/well) were determined from triplicate cultures containing irradiated spleen cells and appropriate antigen in 96-well flat bottomed plates. Cultures were incubated for 3 days at 37 °C in 5°70 CO2, pulsed for the final 6 - 1 5 h with 1 t~Ci[3H]thymidine (Amersham Corp., Arlington Heights, IL). The cells were harvested in a P H D 200A cell harvester (Cambridge Technology, Cambridge, MA) and assayed in a Packard 1500 scintillation counter (United Technologies/Packard, Downers Grove, IL). IL-2/IL-4 was determined using HT-2 (1 X 104) indicator cells as described [1]. HT-2 cells were cultured for 24 h (0.2 ml), the last 6 h containing tritiated thymidine in the presence or absence of anti-IL-2 ($4B6) and/or anti-IL-4 (llB11) mAb. The clone's proliferative responses to exogenous IL-1, IL2 and IL-4 were tested by co-culturing 2x104 cells for 3 days with human rIL-2 or murine IL-4 SN in the presence or absence of human rIbl/3. For the IL2 assay, a typical TH1 clone (2x 104), PC-l, was used and cultured for 3 days with supernatant of various clones. This clone displayed no sensitivity to r I ~ 4 (see Fig. 1). IFN-2/was assayed by an ELISA assay as previously described [16]. 4. Results

4.1. Initial characterization of Ttt clones The MHC Class II restricted T H clones used in this study are listed in Table 1. By FACS analysis, all clones expressed Thy-1, CD3, and CD4 determinants (data not shown). Of the eleven clones tested, five were operationally defined as autoreactive because of their ability to grow in the presence of syngeneic filler cells. Their growth was specifically inhibited by the appropriate anti-class II antibodies (not shown). With the reported exception of Lbd [13] no attempt was made to exclude reactivity of the autoreactive

TABLE 1 Specificities of T cell clones and determination of lymphokine production following stimulation with APC + Ag. Clones

TH2 L7.4 LF2 Lbe.l 5A FGG-3 FGG-8 FGG-II PC-15 D10.G4.1 TH1 PC-1 AK-4

Specificitya

HT-2 cell proliferation Clonal SNb

+ anti-IL-2 mAbc

+ anti-lL-4 mAbc

+ both mAb

IEr IEr IEr IEK FGG + lEt FGG + IEr FGG + IE~ PC + IEK Conaldb + IAK or IAb

37950 38744 18144 15953 17588 14978 22458 21076 16738

37909 37 189 16819 13860 16655 12392 20538 18519 14848

1711 2 101 460 374 303 1259 441 727 731

1028 437 393 335 371 671 329 319 486

IAK KLH + IEK

25 345 2414

694 549

23 123 2167

567 403

a The MHC restriction of T H cells is based on the inhibition of proliferation by mAbs specific for IA or IE determinants, b Serial dilutions of the supernatants were tested. The results shown correspond to the following dilutions: 1:2 for AK-4, 1:4 for L7.4 and LF2, and 1:8 for the other clones, c Anti-IL-2 mAb ($4B6 hybridoma culture SN) and/or anti-IL-4 mAb (11BI 1 hybridoma culture SN) were used at a 1:8 dilution.

cells with F C S c o m p o n e n t s . C h a r a c t e r i z a t i o n o f the cells as TH1 or TH2 was b a s e d on the detection o f I ~ 2 or IL-4, respectively, in culture s u p e r n a t a n t s (SN) following s t i m u l a t i o n with A P C + Ag. We detected I L - 2 / I L - 4 activity as s u p p o r t o f growth o f an I L - 2 / I L - 4 reactive cell line (HT-2) in the presence o r absence o f specific neutralizing m A b s . Serial dilutions o f the clonal SN allowed us to classify nine o f the clones as TH2 a n d two as THI clones (Table 1). To c o n f i r m the classifications b a s e d o n the results presented in Table 1, the proliferative response o f the helper clones to IL-2 or IL-4 was measured in the presence or absence o f rIL-1. C o n s i s t e n t with previous reports [2, 5, 7 - 9 ] , TH1 clones (for example: PC-1 in Fig. 1) proliferated to rIL-2 b u t not IL-4, a n d displayed no sensitivity to rIL-1. O n the o t h e r h a n d , TH2 clones (Lbd7.4 a n d F G G - 3 in Fig. l) r e s p o n d e d to b o t h rIL-2 a n d IL-4SN, a n d the a d d i t i o n o f rIL-1 caused an increase in their prolifera t i o n [5]. F r o m the results d e s c r i b e d in Table 1 a n d Fig. l, we c a t e g o r i z e d the clones as TH1 o r TH2.

4.2. Detection of 1L-2 secretion by TH2 clones

stimulated with immobilized anti-CD3 mAb A l l the helper clones can be activited to p r o d u c e l y m p h o k i n e s following s t i m u l a t i o n with i m m o b i lized a n t i - C D 3 mAb. We a t t e m p t e d to c o n f i r m o u r classifications f r o m Table 1 by collecting SN from clones s t i m u l a t e d with i m m o b i l i z e d a n t i - C D 3 m A b (1 # g / m l ) . S N from Lbd7.4 o r L F 2 s u p p o r t e d HT-2 cell growth which was inhibited by b o t h anti-IL-2 a n d anti-IL-4 m A b s , with c o m p l e t e b l o c k a g e observed when b o t h m A b s were used (Fig. 2). This suggested that IL-2 a n d IL-4 were c o n t a i n e d in the SN. Since TH1 clones proliferate in response to exo g e n o u s IL-2 a n d not IL-4, IL-2 p r o d u c t i o n by these clones was c o n f i r m e d by using a TH1 clone ( P C - l ) as a r e a d o u t (Fig. 3). A g a i n SN from Lbd7.4 or L F 2 caused I L - 2 - d e p e n d e n t proliferation, a n d their ability to p r o d u c e b o t h l y m p h o k i n e s was c o n f i r m e d after recloning at 0.6 cells/well ( d a t a n o t shown). O t h e r TH2 clones s t i m u l a t e d with i m m o b i l i z e d a n t i - C D 3 m A b did not secrete detectable levels o f IL-2 even after s t i m u l a t i o n with high doses (2 # g / m l ) o f the antiCD3 m A b ( d a t a n o t shown). Interestingly, the f o u r 121

rlL-2

IL-4

SN

0000fl 0001I HT-2 PROLIFERATION

CLONES

cpm

c.p.m. 30,000 f 20,000 A

c p m 20,000 15,000 !

.'" ~""

B

e ." "e

10,000

20,000

L7.4

5,000

10,000

cpm

B

10,000

20,000]---10,00

0 I

I

I

I

80,000

50

60,000

~'~'" "" ~

I

40,000

50

0 L7.4

20,000 15,000 I D

20,000 ~ =

25

0

125

~

20,000

10,000

C

~

ano n t i -ml LA b2 mAb

10,000

0

0 ~ I I [ I 0 50 125 150,000

anti IL-4 0

25

50 0

20,000 F

both rnAb FGG-3

15,000 t PC-t

10,000 5,000

Fig. 2. HT-2 assay of supernatants from T helper clones stimulated with immobilized anti-CD3 mAb. SN were used diluted 1:200 for L7.4 (panel A) and LF2 (panel B), and 1:40 for FGG-3 (panel C).

0

0 0

50

125 (uniUml)

0

20

50 (%)

Fig. 1. Proliferative responses of T H cells to rlL-2 and IL-4SN. T helper clones L7.4 (panels A, B), FGG-3 (panels C, D), and PC1 (panels E, F) were cultured with increasing concentrations of rlL-2 (15 to 125 U/ml, panels A, C, E) and IL-4SN (6 to 50% vol/vol, panels B, D, F) in the presence ( o ) or absence ( • ) or riLl (1 U/ml).

TH2 clones (Lbd7.4, LF2.2, Lbe.1, 5A.1) that produced IL-2 were also autoreactive. For convenience, we have designated the IL-2/IL-4 producing clones as TH2B and the clones secreting IL-4 as TH2A.

4.3. IL-2 production by the TH2B clones correlated with anti-CD3 mAb dose Since activation with APC + Ag versus anti-CD3 mAb [17, 18] presumably differs only in the intensity of stimulation, we titrated the dose of anti-CD3 mAb required for IL-2 secretion. As shown in Fig. 4, Lbd7.4 failed to produce detectable amounts of lymphokine(s) at 0.002 t~g/ml. IL-4 secretion was detected at 0.02 #g/ml, but I ~ 2 activity became detectable at 0.2 gg/ml. Thus, IL-2 secretion required a stronger stimulatory signal than IL-4 production. 122

t

FGG-3

5,000

-"

LF2

This is likely to account for the failure to detect IL-2 in the experiments reported in Table 1 (stimulation with APC + Ag). 4.4.

Some TH2 clones produce IFN-'y following anti-CD3 mAb stimulation

As demonstrated above, there are several clones (TH2B) that secrete both IL-2 and IL-4 if appropriately stimulated. We next tested the clones for IFN-~ production following stimulation with APC + Ag or immobilized anti-CD3 mAb. The results are shown in Table 2. Among TH2B clones, IFN-7 was detected in the SN of LF2.2 but not Lbd7.4 or 5A.1. One TH2A (FGG-3), produced IFN-7 following stimulation with immobilized anti-CD3 mAb. The amount of IFN-~ detected from the TH2 clones was much less than typically observed from TH 1 clones. These results suggested that IFN-~ production by these TH2 clones was also dependent on the intensity of stimulation. 5. Discussion Helper T cells constitute heterogeneous populations of lymphocytes with different immunoregula-

HT-2 PC-1 PROLIFERATION cpm 50,000

100,000

150,000

I

TH2B

20,000

40,000

60,000

I

I

l

r

L7.4 LF2.2 Lbe. 1 5A.1 TH2B FGG-3 FGG-8

0.2

FGG-11 PC-15 D10 TH 1 PC-1 KA-4

0.02 rlL-4 2,000 rlL-2 125 (U/ml)

62 31 15 7.5 3.7 1.8 0

I

3

0.002 no mAb anti-lL-2 mAb

no Ab anti-lL2 a nti-I L4

Fig. 3. Proliferative responses of PC-1 (T Hi) cells induced by SN from cultures of clones stimulated with immobilized anti-CD3 mAb for 24 h. PC-1 cells (2× 104/microwell), were cultured for 3 days with the indicated T H SN (1:4 dilution), rlL-2 (0 to 125 U/ml), or rlL-4 (2000 U/ml).

tory properties, phenotypic markers, and the capability to secrete a variety of lymphokines following activation [1 - 9, 19]. It has been reported that in mufine systems discrete subsets of helper T cell clones carry out different helper functions and produce some but not all lymphokines. Basically, TH1 clones secrete IL-2 and IFN-7 and TH2 cells IL-4 and IL-5. Although much interest has been generated by the T H1/TI~2 paradigm, a number of observations indi-

Fig. 4. HT2 assay of SN from Lbd7.4 cells stimulated with the indicated concentrations of immobilized anti-CD3 ab (#g/ml).

cate that it must be interpreted with some caution. A source of concern is the extensive selection required to generate T cell clones in vitro. It is very likely that the complex mixtures of growth factors used for cloning introduce a selective bias. For instance, in our laboratory we most frequently generate TH2 clones which are hardy cells capable of surviving extensive rest periods (2 weeks) and of supporting antigen-induced growth in the absence of added growth factors. In contrast, our Tnl cells are more fragile and rather susceptible to the absence of exogenous IL-2. We have also noticed that the antigenprimed (KLH or FGG) populations of lymph node

123

TABLE 2 Determination of IFN-3' production by clones stimulated with A P C + Ag or immobilized anti-CD3 a. A P C + Ag Stimulation b

Anti-CD3 mAb Stimulation c

<5 <5 <5

<5 52 <5

<5 <5 <5

<5 92 <5

501

3690

Tn2B L7.4 LF2.2 5A.1 Tn2A FGG-8 FGG-3 D10 TH1 PC-1

a The results shown here were obtained by ELISA assay and expressed as units/ml, b Same supernatants were used in Table 1. c Same supernatants were used in Fig. 2.

cells used to generate our clones initially secrete IL-2 but after 2 weeks in culture become IL-4 secretors, suggesting that some kind of differentiation and/or selection occurred in vitro. The lymphokine switch can be induced in vivo because stimulation of unprimed T cells with anti-CD3 in vitro causes IL-2 but not IL-4 secretion. In contrast, the same stimulation protocol yields both IL-2 and IL-4 if the T cells originate from mice injected with anti-CD3 7 days previously [20]. These observations are related to those published by Swain et al. [21] and in agreement with the reports of low frequencies of IL-4 secreting cells as detected by in situ mRNA hybridization [22] and limiting dilution analysis [23]. They clearly indicate that IL-2 is the predominant lymphokine secreted in the course of primary T cell responses. In this paper we report the results of screening CD4 ÷ clones generated and maintained in our laboratory. We undertook this project not only because of an interest in the placement of our clones in the TH1/TH2 categories but more importantly, because we had noted that they differed in their immunoregulatory properties, particularly with regard to their ability to mediate T - T interactions [10, 11]. For instance, we had reported that the lEd-reactive Lbd clone could mediate amplification, suppression, and contrasuppression of T cell-mediated immune responses [13]. Melchers et al. have recently 124

described similar immunoregulatory properties for EL4 cells [24] which secrete both IL-2 and IL-4, as do Lbd cells (this paper). Also Evavold et al. have discovered that IL-2-secreting T H ceils lack a costimulatory function characteristic of activated IL-4 secreting cells (manuscript submitted). Although we found prototypic T n l (PC1, AK4) and TH2 (FGG8) cells, we observed that one of our TH2 clones (FGG-3) secreted low but detectable levels of IFN--y and that four autoreactive CD4 + clones secreted both IL-2 and IL-4 (Lbd 7.4, LF2, Lbe.1 and 5A). LF2 cells, an early subclone of Lbd [13], secreted IFN-3, as well as I b 2 / I L - 4 . The double producers represent stable, long-term cultured cells (9 months to 4 years) which resemble Tn2 cells in their responsiveness to rIL-I and share their ability to induce proliferation of Tn2 but not T n l cells (Yokoyama, unpublished data). We can thus subdivide Tn2 cells into prototypic IL-4 secretors (Tn2A) and IL-2/IL4 double producers (Tn2B). Others have reported that some human T cells produce IL-2, IL-4, and IFN-3, [25] while some murine T cells produce IL-2 and IL-4 [26] or IL-4 and IFN-3, [27]. Glasebrook et al. have cloned helper cells called Tn0 because they secrete most lymphokines [26]. These results indicate that the heterogeneity of CD4 + cells extends beyond the T n 1 and TH2 dichotomy. Although the findings presented herein may only require a modest revision of the paradigm, it is our impression that the heterogeneity of our clones is restricted by our own cloning procedures and that additional heterogeneity of T n cells will be detected when more diverse cloning protocols are utilized.

Acknowledgements This work was supported by NIH grants PO1 CA19266 and T32 AI-7090.

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