Analysis of T-T lymphocytes and T-accessory cell interactions using cloned T cells: MHC I restricted cloned T cells activate MHC II restricted T helper cells

CELLULAR

IMMUNOLOGY

99, 182-l 95 ( 1986)

Analysis of T-T Lymphocytes and T-Accessory Cell Interactions Using Cloned T Cells: MHC I Restricted Cloned T Cells Activate MHC II Restricted T Helper Cells’ THOMAS BRUCKNERAND RAIMUND DI PAUL? Department

of Immunology, Hygiene Institut, University Miinster, Federal Republic of Germany

Received

September

6, 1985; accepted

December

ofhfiinster,

3, 1985

We evaluated the role of molecules of the major histocompatibility complex (MHC) involved in the cellular interactions of two T-cell clones by testing the effect of monoclonal antibodies on the responses of the clones in vitro. The two T-cell clones used in the study are specific for minor histocompatibility antigens and restricted to the H-2K’. In the absence of exogenous IL2 the clones require the presence of Ia+, Thy-l- accessory cells and of Thy-l+, Lyt-1+2- cells in the irradiated spleen cell suspension used as stimulator. It is also necessary that both the accessory cells and the T cells in the stimulator cell populations are recognized specifically by the clones. Monoclonal antibodies specific for the H-2K product inhibited the lytic effector function of the cytolytic clone. These antibodies when added to cultures of stimulator cells and clones inhibited also the proliferation of this clone and of a nonlytic clone. When antigen recognition was measured by the increase in sensitivity of the clones to IL-2 while confronted with uv-irradiated stimulator cells, both clones were blocked efficiently by anti-H-2K antibodies. Thus, these results suggest that the interaction of monoclonal antibodies with the restricting H-2K molecule is sufficient to block the recognition signal, a prerequisite for proliferation. In contrast, monoclonal antibodies specific for A.& and/or E,E, had no effect on cytolysis or on restricted recognition. However, they inhibited the proliferative responses as efficiently as the H-2K specific antibodies. Inhibition by class II-specific antibodies was not abolished when stimulator cell populations were depleted of Lyt-2+ cells. The blocking effect, however, was reversed by the addition of IL-2. No inhibition was obtained with antibody specific for E,E, when B 1O.A(4R) spleen cells, which do not express E,E,, or when BlO.A(4R) accessory cells, which were reconstituted with (BALB/c X BlO.A(4R)) Fl T cells, were used as stimulators. Stimulator cells heterozygous for H-2 could be inhibited by antibodies to the parental haplotype not encoded in the clones (H-2Kd). These and previous results suggest that H-ZK-restricted minor histocompatibility antigen-specific recognition transmits an activating signal to the clones and to the stimulator cells. The clones probably are induced to express more IL-2 receptors. The stimulator T cells seem to interact through A,A, and E,EB molecules with syngeneic accessory cells. This interaction results in IL2 production by the stimulator T cells and thus in the proliferation of the clones. o is86 Academic PBS, I~C.

INTRODUCTION

Cell interactions among lymphoid cells are pivotal events in the immune response to foreign antigens. These interactions are mediated by antigens and restricted by the ’ Supported by a grant to R.D.P. from the Deutsche Forschungsgemeinschaft. 2 To whom correspondence should be addressed: Department of Immunology, Hygiene Institut, Westftiische Wilhelms-Universit, Domagkstrasse 3, D-4400 Milnster, FRG. 182 0008-8749186 $3.00 Copytigbt 0 1986 by Academic Press, Inc. All rights of reproduction in any form resaved.

CELLULAR

INTERACTIONS

IN VITRO

183

major histocompatibility complex (MHC).3 Such interactions lead to the activation of specific clones followed by the release of antigen-unspecific growth and/or differentiation factors. Class I MHC products, which are encoded in the H-2K and H-2D loci, are usually recognized by cytolytic effector cells (CTL), whereas the class II determinants, which are encoded in the I-A and I-E loci, are recognized mainly by T helper lymphocytes (Tn). A CTL response to antigen, manifested in the activation and proliferation of CTL precursors, can be amplified by T helper cells ( l-7). Recently the concept has evolved that CTL are activated by the recognition of antigen as well as class I products on accessory cells (ACC). Through this activation the CTL become sensitive to IL-2, which is produced by Tn activated in a similar way as are the CTL (8). In addition other factors produced by ACC may be required by all participating cells. In the above model T-cell help is mediated by a growth factor and thus a direct cellular T-T interaction is not required. On the other hand, using an in vitro model with T-cell clones we have shown that such interactions can occur (9, 10). They are restricted by the H-2K and specific for a minor histocompatibility antigen (miHA). In our experimental system the T-cell clones were activated by antigen on macrophages, yet they did not proliferate in the absence of Tn cells syngeneic with the stimulating macrophages. These Tn would only support proliferation if they carried the miHA and the H-2K specifically recognized by the clones. From this result we concluded that the T clones, regardless of whether they were CTL or only of proliferative phenotype (PTL), interacted through their receptor(s) with the restricting H-2K molecule and an unknown miHA on the surface of the Tn. Thus the two interactions of the clone, one with the ACC and the other with the Tn cells, were both class I (H-2K)restricted and could have been sufficient to induce all factors necessary for the eventual proliferation of the clones. On the other hand, Tn usually interact with the ACC in a class II-restricted fashion and it has been shown that class I-restricted CTL can require class II-restricted Tn ( 1 I- 14) even if this is not the rule ( 15- 17). This led us to study which MHC molecules were involved in these cellular interactions. To do so we carried out blocking experiments with monoclonal antibodies (mAb) specific for class I and class II products of the MHC. The results show that mAb specific for H-2K inhibit the effector function (cytolysis), the induction of IL-2 sensitivity and the proliferation of the clones. Anti-Ia-specific mAb inhibit only the proliferative response of the clones. Whereas the anti-H-2K mAb block the interaction of the clones with target or stimulator cells including Tn and ACC, the anti-Ia probably block a syngeneic interaction between stimulator Tn and stimulator ACC. MATERIALS

AND

METHODS

Animals. BALB.K, BlO.BR, BlO.A(4R), and AKR/J of both sexes were obtained from Olac, Bicester, Great Britain. Fl hybrids were bred in our own animal facilities. T-cell clones. The isolation of the T-cell clones used in this study and some of their ’ Abbreviations used: ACC, accessory cells; C, complement; Con A, Concanavalin A; CTL, cytolytic T lymphocytes; IL-2, interleukin 2; [‘H]dThd, tritiated thymidine; mAb, monoclonal antibodies; MHC, major histocompatibility complex; miHA, minor histocompatibihty antigen(s); MIS, M locus; PTL, proliferating T lymphocytes; SAC, splenic adherent cells; SMLR, syngeneic mixed lymphocyte reaction; Tn, T helper cells; Ts, T suppressor cells; uv, ultraviolet light.

184

BRikKNER

AND

DI

PAUL1

characteristics and growth requirements have been described in detail previously (9, 10). AKR. lOk-23.21 (AKR-2 1) originated from a mixed lymphocyte culture (MLC) of AKR spleen cells stimulated with BlO.BR cells. CBA. lOk-4.6 (CBA-6) was obtained similarly from a CBA/J spleen. Both clones showed better and more reproducible growth on irradiated BALB.K stimulator spleen cells on which they can grow without the addition of exogenous IL-2. They have been maintained continuously on these stimulator cells for over 4 years. Both clone are specific for an undefined miHA which is shared by B 10 and BALB and restricted by the H-2Kk. AKR-2 1 shows cytotoxicity, whereas no definite function could be assigned to CBA-6. The AKR clone is Thy-l+, Lyt- l-2+, Ia-, and the CBA clone is Thy-l+, Lyt- l-2+, Ia- (unpublished data). Monoclonal antibodies. The monoclonal antibodies were obtained from Cedarlane Laboratories (Homby, Ontario, Canada). They had the following determinant specificity, main haplotype specificity and hydridoma designation (18, 19): H-2.m2, H-2Dbyd, B 22-249.Rl; H-2.ml1, H-2KkPq, H lOO-5.R28; H-2.m25, H-2Kk*‘, H lOO27.R55; Ia.m2, I-Ak, B15-124.Rl; Ia.ml9, I-Ak,‘, H 116-32.R5; Ia.m30, I-Ab,d,q*v,B17263.Rl; Ia.m7, I-Ek,d*p,r*“,v, l3/4.R5; Ia.7, I-Ek,d,p,r, 14-4-48; Lyt- 1.2, CG 16; Lyt 2.1, 49-11,l; Lyt-2.2, AD4( 15). The monoclonal anti-Thy-l.2 (20) was kindly provided by Dr. H. G. Opitz (Bayer AG, Wuppertal, FRG). Cell separation and antibody plus complement treatement. These methods have been described in detail previously (10). In short, splenic adherent cells (SAC) were obtained by adherence to plastic tissue culture dishes and treatment with monoclonal anti-Thy- 1.2 antibodies plus complement. T cells were obtained by filtration through nylon wool columns (2 1). Assayfor cell proliferation. To evaluate the proliferative response upon stimulation, the incorporation of tritiated thymidine ([3H]dThd) was measured. T-cell clones to be tested were harvested from flasks at least seven days after stimulation with irradiated spleen cells. At this stage they show practically no spontaneous [3H]dThd incorporation. A given number of clone cells, usually 2 X 104, was placed together with stimulator cells or any compound to be tested in flatbottom microtiter wells (Falcon, 3040F, Oxnard, Calif.) to give a final volume of 200 ~1. All experiments were done in triplicate. After incubation at 37°C usually for 48-72 hr, 0.4 PCi [3H]dThd in 50 ~1 of DME was added to each well. Six hours later, the cells were harvested onto glass fiber filters with a Titertek cell harvester (Skatron, Flow Laboratories, Bonn, FRG). The [3H]dThd incorporated was measured by standard liquid scintillation counting. Since the standard error of the means of triplicate cultures did not exceed 20% except when values were below 100 cpm, the results are expressed as mean cpm. Ultraviolet light irradiation. Spleen cells in tissue culture dishes (10 cm diam) in a volume of 5 ml were exposed to ultraviolet light (uv). The source of uv was a Sterisol lamp (Heraeus, Hanau, FRG) with a maximum energy at 254 nm. The lamp was located 30 cm from the culture dishes and the time of exposure was 10 min. T-cell growth factor (IL-2). IL-2 was a crude supematant from rat spleen cells cultured for 24 hr in serum-free Dulbecco’s modified Eagle’s containing 5 &ml of concanavalin A (Con A) (Sigma, Munich, FRG). After centrifugation and subsequent filtration through Nalgene filters with a pore size of 0.1 pm (Nalge Company, Rochester, N.Y.), the IL-2 was stored in aliquots at -20°C. Assay for cell-mediated lysis and generation of CTL. CTL specific for allogeneic cells were induced by culturing 2 X lo7 responder spleen cells with an equal number

CELLULAR

INTERACTIONS

185

IN VITRO

of irradiated (1500 rad) stimulator spleen cells. Culture conditions and preparation of Con A-stimulated target cells were standard and have been described (9). The release of “Cr into the supernatant was calculated according to the formula: %lysis = ((cpm experimental release - cpm spontaneous rel.)/(cpm maximal rel. - cpm spont. rel.)) x 100. RESULTS Inhibition of the cytolytic response of CTL clone Am-21 with mAb specific for H-2 (K, A, and E). In a proliferation assay using spleens from mice of different congeneic strains as a source of stimulator cells we showed that the two clones employed in the present study are specific for miHA and restricted by the K end of the H-2 complex (10). In addition, the specificity of clone AKR-2 1 was demonstrated by its killing specificity. It is known that antibodies (including mAb) specific for H-2 products can inhibit the cytolytic action of CTL recognizing H-2K or H-2D encoded al10 determinants, as well as CTL recognizing K or D as restricting elements (22-24). These experiments established also that the antibodies, even if they react with the effector cells, are only effective if they are specific for the H-2 molecule on the target cell. Since only AKR-21 is cytolytic, we could test blocking of cytolysis only in this line. Two different mAb were used (Table 1). One was specific for an H-2K determinant. The other was a mixture of two mAb, one of which was specific for an I-A-encoded determinant, and the second of which was directed against an I-E molecule. Only the H-ZK-specific antibody was able to inhibit the lytic activity of AKR-21. Addition of Con A restored the lytic activity in the presence of mAb to K. Thus a direct toxic or blocking effect on the T-cell clone can be excluded. We assume that clone AKR-21 interacts with the target cell through the H-2K encoded molecule and the miHA to deliver the killing signal. This effect could be blocked by antibodies specific for the TABLE 1 Inhibition of Cytolysis by T-Cell Clone AKR-2 1 with Anti-H-2K Anti-I-A-Specific Monoclonal Antibodies”

but Not

Ratio effecter/target Antibody specificity

Con A

0.7

2.1 BALB.K 39.7 28.5

6.3

6.3

38.3 33.1

AKR/J -6.8 -

+

21.2b 16.8

+

13.8

jJ 19.3

I-A + I-E (Ia.rn19 + Ia.7)

+

23.5 21.7

38.2 32.8

38.3 38.8

-

Lyt-2.1

-

24.7

43.7

42.0

-

-

u

H-2K (H-2.mll)

-5.1 1.5

DGiven numbers of cloned CTL were incubated with 2 X lo4 BALB.K Con A blasts, labeled with “Cr. The release of 5’Cr was measured after 4 hr incubation. The antibody-containing ascites was diluted l/500 and added at the beginning of the test. b % lysis of Con A blasts.

186

BRijCKNER

AND DI PAUL1

restricting H-2K molecule, but not by anti-A or anti-E. Thus I-A- and I-E-regionencoded molecules are not involved in this interaction. In addition, we also tested a monoclonal anti-Lyt-2 antibody in the cytolysis assay. No inhibition of lysis was observed with our clone. Heterogeneity of T-cell clones in their susceptibility to inhibition by anti-Lyt-2 antibodies has been observed by others (25). The clone AKR-2 1 probably belongs to the CTL class which cannot be inhibited by anti-Lyt-2. Inhibition of T-cell proliferation with mAb specljic for H-2 (K, A, and E). We had demonstrated that the antigenic signal, as mediated by miHA in the context of self H-2 on irradiated stimulator spleen cells, is required for continuous growth by both clones in spite of their IL-2 sensitivity. The novelty of the system is that in the absence of exogenously added IL-2 the clones require not only Ia+ ACC but also Tu in the stimulating cell population. To provide the conditions leading to proliferation, these two cell populations have to be specifically recognized by the clones. It is, however, not clear whether the interaction of the clones with ACC and Tu, resulting in the production of factors is sufficient to induce proliferation or if other cellular interactions, like those mediated by Ia molecules on ACC, may also take place. To dissect this problem we undertook inhibition experiments in the proliferation assay with mAb specific for H-2K, I-A, and I-E determinants. If no reaction other than the H-2K-restricted, miHA-specific interaction between the clones and the stimulator cells is involved, only anti-K should inhibit the proliferation of the clones. The results in Fig. 1 show that this was not the case. K-specific antibodies almost completely inhibited the proliferation of both clones. This result was in agreement with the findings of others that K,D-specific antibodies can inhibit generation as well as proliferation of CTL (24, 26, 27). However, in addition, A- and E-specific mAb also inhibited the proliferative responses by about 50%. When anti-A and anti-E were used together their activity was additive. Since both the A and the E molecules are present on the same ACC (28-30), these results suggest that both molecules were bound by mAb in order for the inhibition to be nearly complete. Thus, the effect on proliferation is probably not due to the blocking of ACC as such by the mAb, but to the blocking of an interaction between the ACC and the T-cell clones. The addition of IL2 abolished the inhibitory effect of all of these antibodies regardless of whether irradiated stimulator spleen cells were present or not. Considering the fact that the clones do not express Ia molecules, this constitutes additional evidence that the effect of the antibodies is to block the interaction between ACC and the T-cell clones and not the T-cell clones themselves. To show that inhibition of proliferation was not due to an activation of Lyt-2+ suppressor cells by the anti-Ia-antibodies (3 I), we pretreated the stimulator spleen cells with Lyt-2 specific mAb (Table 2) plus C. The Lyt-2+ cells were eliminated when exposed to this antibody plus C because no allogeneic CTL response occurred after treatment. As we had observed before (lo), the elimination of Lyt-2+ cells from the stimulator spleen cells had no effect on the capacity of these cells to induce proliferation. Furthermore there was evidently no effect on the blocking of anti-Ia antibodies. This suggests that suppression by Lyt-2+ cells does not play a role in the mechanism of inhibition. H-2K-specific mAb block the activation of T-cell clones to become IL-2-sensitive. T cells require at least two signals to proliferate. One signal is delivered by recognition of antigen in the context of an H-2 molecule. The second signal is mediated by a growth factor (IL-2). This has been shown for many T-cell clones, and by using

CELLULAR v b

v anti A onti

INTERACTIONS I-A I-E

q

l

0

l

IN VITRO

187

anti II-A+I-El anti H-2K

a6-

AKRlOk-23

0

6.4

Reciprocal

3.2 antibody

21

1.6 dilution

0.8

0.L

0.2

x 10m3

FIG. 1. Inhibition of proliferative responses of T-cell clones stimulated with irradiated BALB.K spleen cells or with IL-2 by monoclonal antibodies specific for products of H-2K, I-A, I-E, or a mixture of anti-IA and anti-I-E. The T-cell clones were cultured in the presence of mAb (ascites)at the indicated concentrations for 48 hr and proliferation measured by [‘H]dThd incorporation. The monoclonal antibodies added to the cultures were anti-Ia.ml9 specific for I-A, anit-Ia. specific for I-E, anti-H-2.mll specific for H-2K, and a mixture of anti-1a.m 19 and anti-Ia.7. Open symbols represent the T-cell clones in the presence of IL-2; closed symbols, T-cell clones in the presence of irradiated BALB.K spleen cells.

uv-irradiated spleen cells as stimulators in the presence of limiting amounts of IL-2, has been also been substantiated for the two anti-miHA clones used here (10). Such uv-treated cells do not induce proliferation of the clones, probably because they are unable to produce interleukins. Yet their antigen-presenting capacity is still intact; in the presence of limiting amounts of IL-2 these uv-irradiated stimulator cells carrying the appropriate miHA and H-2 are capable of inducing proliferation. Since in the experiments described above in which metabolically active spleen cells were used as stimulators, both anti-K and anti-Ia mAb inhibited the proliferation, it was important to assesswhether the recognition of Ia on accessory cells played a role in the delivery of signal 1 to the clones. Therefore, we performed inhibition experiments by using uv-irradiated spleen cells as the antigen (signal 1) and limiting amounts of IL-2 (signal 2). The experiments in Fig. 2 confirm the previous findings that neither uv-irradiated spleen cells nor limiting (!) concentrations IL-2 alone or in the presence of syngeneic uv-irradiated spleen cells stimulate the proliferative response of T-cell clones. Only uv-irradiated spleen cells possessing the appropriate miHA and H-2 can induce proliferation in the presence of IL-2. This proliferation was blocked completely by adding

188

BRUCKNER

AND DI PAUL1 TABLE 2

Elimination of Lyt-2+ (T,) in the Stimulator Population Has No Influence on Blocking by Anti-Ia mAb Treatment of BALB.K stimulator cells” Specificity of mAb added

Anti-Lyt-2. I + C (control)

A Anti-A’ Anti-Ek Anti-A + Anti-E B Treatment of BALB.K cells

Anti-Lyt-2.2 + C

Proliferation of clone AKR-2 1 23,724 3,724 4,095 255

(0)b (84) (83) (99)

17,950 2,678 4,544 27

(0) (85) (75) (100)

Ratio effecter/target I

10

Cytolytic activity of BALB.K cells stimulated with irradiated DBA/2 cells Anti-Lyt-2.1 + C Anti-Lyt-2.2 + C

16.3’ 0.8

36.5 $J

a BALB.K spleen cells (Lyt-2.2 genotype) were treated with anti-Lyt 2.1 plus C as a negative control, or with anti-Lyt-2.2 plus C to eliminate Lyt-2+ cells. The treated cells were either irradiated and used as stimulator cells in the proliferative assay with clone AKR-21 or left unirradiated and stimulated with DBA/Z spleen cells in a primary allogeneic MLC. After 5 days, the cells from the MLC were tested for lytic activity on S’Cr-labeled DBA/2 Con A blasts. b cpm (% inhibition). ’ %s’Cr release.

anti-K, but not by a mixture of anti-A plus anti-E antibodies (Fig. 2). The lack of restoration of the proliferative response of the clones cannot be ascribed to a possible lack of Con A in limiting concentrations of IL-2. We had shown previously ((9) and unpublished results) that Con A inhibits rather than stimulates the proliferative responses of the clones. The results thus suggest that the H-Zrestricted interaction between the clones and the spleen cells, leading to sensitivity for IL-2, is K end specific, and no A- nor E-mediated recognition is involved in this step. Inhibition of T-cell proliferation on heterozygous stimulator cells. All results so far obtained on the specificity of the T-cell clones strongly indicate that the H-2Kencoded molecule is the restricting element recognized by the clones. It seems justified to assume that the inhibition observed with anti-A and/or anti-E is due not to blocking of the interaction between clones and stimulator cells, but rather to blocking of an interaction between the TH and the ACC. This assumption is supported by the fact that both cell types are required in the stimulator population for an effective proliferation to occur. The inhibiton could be explained by the presence of a second clone specific for A and E only if this clone were completely silent under all testing conditions such as (a) lack of proliferation on BlO.AQR (KqAkEk), (b) no role in cytolysis (Table I), and (c) no role in activation by uv-irradiated cells (Fig. 2). This is unlikely. Finally it is aho quite improbable that the clones possess receptors with different functions and with at least three specificities.

i rredisted

CBA

CBA

CBA

No

No

+

+ + +

I.

antibodies

3.

0

3,

IL-2

+ + +

I

1 2

I 0

I 6

13HI-Thymldlne

1

AKR.lOk-23.21

I

I

incorporstlon

10

I

1 0

I

cells

lcpm

x

10m3i

CBA.

1 1

IOk-4.6

I 2

FIG. 2. Inhibition of the synergistic interaction between m-irradiated spleen cells (antigen) and the proliferative signal IL-2 by antibodies specific for H-2K (anti-H-2.m25) but not by a mixture of antibodies specific for I-A and I-E (anti-Ia.ml9 + anti-Ia.7). 2 X lo4 cloned T cells were cultured in microtiter wells as indicated, either with uvirradiated syngeneic (either AKR/J or CBA/J) or BALB.K stimulator spleen cells at 8 X lo5 per well. The monoclonal antibodies were added at a final concentration of l/200 at the start of the cultures. IL-2 in form of a crude Con A-stimulated rat spleen cell supernatant was used at a limiting concentration (6%). [3H]dThd incorporation was measured after 48 hr.

AKR

or

tolls

BAL6.K

No

AKR

or

cell8

BALB.K

No

H-2K H2.m25

I-E

+ la.7

IL-2

I llmltlng

4.

or

..

antlbodies

antibodies

AKR

la.mlQ

I-A

No

of

Sp83cificity

+

cells

CBA

cells

0ALB.K

No

AKR

or

calls

BALB.K

No

8ple.n

Uv

T

G

190

BRUCKNER

AND DI PAUL1

An additional piece of evidence that the blocking occurs between the syngeneic stimulator cells would be the inhibition by antibodies specific for haplotypes not encoded in the genetic background of the clones, as in (H-2d X H-2k)F1 cells. If the clones activate TH indiscriminately from their Ia haplotype specificity, not only antiAk and anti-Ek but also anti-Ad should inhibit the proliferation. In Table 3 we show four experiments performed with Fl stimulator cells. In no case was the inhibition with anti-Ad comparable to that obtained with anit-Ak. Even if only one-fourth of the T cells in a heterozygous population is restricted to the I-A molecules of one parent, the inhibition obtained with the anti-Ad is much lower than expected. It is not yet clear whether this is due to a property of the antibodies or to an intrinsic property of the Tn from these Fl mice. Nonetheless the inhibition was siginificant. When anti-E, which is reactive with both haplotypes, was added, a cumulative effect could be observed in two of the experiments. Neither the capacity to inhibit with an anti-Ad nor the variability in inhibition in Fl should have occurred if the T clones were endowed with receptors which do not discriminate between different Ia molecules and different haplotypes. E,EB moleculeshave to be expressedby the stimulator ACC. We had shown that Ia+, Thy-l+, adherent ACC in the stimulator spleen were necessary for proliferation to be induced (10). Taken together with the results from the Fl cells (Table 3) and the fact that no class II molecules could be demonstrated on the surface of the clones, one can expect that the target of anti-A inhibition is the accessory cell in the stimulator cell population. To test this supposition we used B 1O.A(4R) spleen cells as stimulators, because BlO.A(4R) do not express I-E-encoded molecules (32). The experiments in Table 4 show that the target of anti-E blocking is the stimulator cell. When BlO.BR TABLE 3 Inhibition of the Proliferative Responses of T-Cell Clones to H-2 Heterozygous Fl Spleen Cells by mAb Specific for Either Parental Class 2 MHC Determinantsa AKR-2 I Expt 1

Specificity of mAbb Locus (hadotwd

Determinant designation

No antibodies

CBA-6 Expt 2

Expt 1

90

% cpm Inhibition

cpm

7812

0

12,499

Inhibition

Expt 2

%

%

cpm Inhibition

cpm

Inhibition

0

5067

0

2310

0

I-Ad 1-A’

Ia.m30 Ia.ml9

3724 2145

52 73

9,920 5,705

21 54

3838 2695

24 47

2186 1175

5 49

I-Ekd

Ia.

2476

68

4,168

67

2263

55

1236

46

1361 1056

83 86

3,520 842

72 93

2129 1395

58 72

993 619

57 73

886

89

214

98

427

92

203

94

I-Ad + I-Ekd I-A’ + I-Ek,d H-2K’

H-2.m 11

LI2 X lo4 cloned T cells and 1 X lo6 irradiated stimulator cells were incubated in microtiter wells together with the monoclonal antibodies at a final dilution of l/400 for 48 hr. Thereafter they were pulsed with [‘H]dThd for 6 hr. In Experiment 1 (BALB/c X BlO.A(4R))Fl and in Experiment 2 (BALB/c X BALB.K)Fl were used. b Only specificities expressed by the stimulator cells are listed.

CELLULAR

INTERACTIONS

IN

191

VITRO

TABLE 4 Monoclonal Antibodies Specific for I-E-Encoded Molecules Do Not Inhibit the Proliferative Response of AKR-2 I to Stimulator Cells Not Expressing E Molecules (BlO.A(4R))” Stimulator spleen cells Specificity of mAb* Locus (haplow

Determinant designation

BlO.BR

cm

BlO.A(4R)

% Inhibition

cm

% Inhibition

No antibodies

Expt 1 Expt 2 Expt 3

19,054 9,510 18,225

0 0 0

3126 5252 4913

0 0 0

I-A’

1a.m 19

5,181 5,569 5,491

73 41 70

1375 2623 2330

56 50 52

1-E’

Ia.

7,016 5,630 7,019

63 41 61

5265

I-A’ + I-Ek

Ia.ml9 + Ia.

845 2,920 1,402

96 69 92

1535 2296 2905

50 57 41

H-2Kk

H-2.m 11

107 n.d. n.d.

99

109 n.d. n.d.

96

’ See Table 3. * Only specificities expressed by the stimulator cells are listed.

spleen cells were used as stimulators, the clone was inhibited with anti-E. Inhibition was also observed with a mixture of anti-E and anti-A the effect was additive. When BlO.A(4R) cells were used as stimulators we observed no inhibiton with anti-E but rather a slight enhancement of proliferation. It is interesting to note that anti-A did not inhibit the stimulating capacity of B lO.A(4R) completely, or at least not as strongly as when anti-A plus anti-E were used with BlO.BR. One would expect an inhibition similar to that obtained with a mixture of anti-A and anti-E in the case of BlO.BR or BALBK, if the only class II element expressed in BlO.A(4R) is A,AB. To test whether adherent, Thy-l- cells in the stimulator spleen are blocked by antiE, we isolated BlO.A(4R) adherent, Thy-l- cells which do not express I-E molecules and do not stimulate the clones to proliferate (10). These ACC were reconstituted with nylon wool-isolated T cells from (BALB/c X B 1O.A(4R))Fl mice which are genetically endowed to express E molecules. The reconstitution (Table 5) was successful and proliferation by clone AKR-21 could be inhibited by anti-A, but not by anti-E antibodies. This confirms that adherent Thy- I- ACC from the stimulator population were the target of the anti-E blocking. DISCUSSION In the experiments described above, we have analyzed the MHC molecules involved in the interactions occurring between cloned T cells and antigen-carrying stimulator

192

BRUCKNER

AND DI PAUL1 TABLE 5

I-E-Encoded Molecules Have to Be Expressed on Adherent Cells for Monoclonal Antibodies to Inhibit the Proliferation of the AKR-2 1 Clone’ Stimulator cells BlO.A(4R)-SAC (I-E-) + Fl T cells

(BALB/c X BlO.A(4R))Fl Spleen cells (I-E ‘)

Specificity of mAb Locus

Determinant

No antibodies

cpm

90 Inhibition

90 cpm

Inhibition

8,785

0

9,795

0

I-AL

Ia.ml9

3,645

59

4,145

52

I-Ekd

Ia.

9,304

-6

4,136

52

H-2Kk

H-2.mll

384

96

158

98

’ Splenic adherent cells (SAC) were obtained from BlO.A(4R) by adherence to plastic dishes and treatment with monoclonal anti-Thy- 1.2 antibodies and complement. SAC alone, unless reconstituted with NW-nonadherent spleen cells (T cells) from Fl mice, induced the clone to incorporate 2 105 cpm of thymidine only. Similarly, the Fl T cells alone stimulated the clone to incorporate 2234 cpm of [3H]dThd. In the above experiments 8 X lo5 of each cell type were used for reconstitution. In the same experiment BALB/c SAC induced 3100 cpm when reconstituted with Fl T cells. See also Table 3.

cells. In addition to being dependent on the specific recognition of miHA and H-2K on ACC. The T clones studied require TH in the stimulating cell population. These Tn cells have to carry the appropriate miHA and H-2K on their surface, as do the ACC (9, 10). We found that monoclonal antibodies specific for the H-2K-encoded determinant blocked not only the cytolytic effector function of the CTL clone, but also the proliferative response of this and of a PTL clone. Thus at the molecular level the K molecule is the restricting element in these anti-miHA T-cell clones. This has been determined already by mapping on stimulator cells from congenic mice. We observed that proliferation could also be inhibited by mAb specific for A,AB and E,E, . These mAb specific for class II MHC products, however, had no effect on the cytolytic function of the CTL clone or on the induction of increased IL-2 sensitivity of the CTL and the PTL clone by uv-irradiated stimulator cells. Blocking by anti-E,EB was possible only if E,EB molecules were expressed on the ACC cell in the stimulator spleen and not on the T cells. In addition, when we used H-2 heterozygous stimulator cells it was possible to inhibit proliferation with antibodies to determinants .of the parental I-A haplotype not encoded in the clones. Taken together, these results suggest that recognition of class I MHC molecules + miHA activates the clones. Class II molecules on the ACC in the stimulating population have to be recognized by syn&eneic TH in order for the clones to proliferate. In Fig. 3 we have summarized schematically how we think the activation of the T-cell clones and the cellular interactions in our in vitro model occur. The cloned T cells, whether CTL or PTL, recognize miHA and H-2K on the ACC. This is a signal for the T clone to express more IL-2 receptors. The cloned T cells also bind to the irradiated Tu, evidently with the same miHA/H-2K specificity. This seems to be

CELLULAR

INTERACTIONS

IN VITRO

-

193

PROLIFERATION

FIG. 3. Schematic representation of cellular interactions occurring in the activation of T-cell clones. The T clone (CTL or PTL) requires an H-2-restricted recognition of antigen (miHA) on the ACC leading to increased expression of IL-2 receptors. A Tu in the stimulator spleen has to be recognized by the clone. This leads to an interaction with the syngeneic ACC, mediated by AJA, and E,E,, and resulting in IL-2 production to be utilized for proliferation by the activated clone.

required to activate the Tn to interact with the ACC through the Ia molecules. This results in the production of IL-2 which eventually leads the clones to proliferate. There are several points at present which have to be further clarified. The possibility that some surface molecules are shed from the clones and presented to the Tn by the ACC as an antigen ( 14) has not been ruled out completely. This would, however, require that those Tn clones recognizing the antigen give an exceptionally rapid and quantitiatively strong response, because the optimum of proliferation by the clones is obtained after 48 hr. In this case sufficient IL-2 has to be produced several hours earlier. Since the several combinations between T clones, Tn and ACC which we have tested did not differ at the H-2, the shedded antigen could be a miHA. For the reasons cited above we consider it very improbable. Alternatively, the antigen could be an Mls structure well known to induce a relatively strong primary proliferative response (33) involving IL-2 production (34). Another explanation is that T clones recognize a majority, if not all, of the Tn cells and activate them to interact with the ACC. This interaction bears all the characteristics of a syngeneic mixed lymphocyte reaction (SMLR) (35) because it shows specificity for A,AB and E,EB (36) and occurs between syngeneic cells with concurrent IL-2 production (37). It differs, however, from the standard SMLR which does not need a demonstrable activation of T cells; the clones do not proliferate on syngeneic, semisyngeneic or allogeneic spleen cells if any one of the recognition elements (Kk or miHA) is missing from the T cells. Thus recognition would be a prerequisite for the kind of SMLR to occur. The direct binding of the clones to the ACC and Tn is of paramount importance. Furthermore, it is probable that the clones, the ACC and the Tn produce factors we have not yet detected. Nonetheless, such undetected factors may be important for the outcome of clonal activation. It is possible that the ACC not only present antigen to the clones, but that ACC are themselves activated by this process to produce factors like IL-l (38). This idea is supported by the results obtained with metabolically in-

194

BRUCKNER

AND DI PAUL1

activated ACC, which do not support growth in the presence of X-irradiated Tu ( 10). The activation signal for the ACC could also come from the Tu. Generally it is accepted that T-help is a prerequisite in some CTL responses (l-6). T-help is also implicated in the response to minor antigens (7, 17). In some cases it has been shown that class I-restricted response requires or is supported by class II-restricted help ( 1 1- 14). With regard to anti-miHA T cell clones, some authors suggest that all anti-miHA CTL clones can grow in vitro without help (39). Others assign the K,D-restricted anti-miHA T cell clones into at least two classes (40, 41). One class produces its own IL-2 and thus does not require help. The second is thought to be dependent on IL-2 from another cell, possibly a Tn cell type. Our T clones belong to the second type because they cannot produce IL-2, at least not in amounts sufficient for their own proliferation and thus require help. There is certainly heterogeneity at the clonal level with regard to the requirements for help. The way clones are selected for growth might not only influence the outcome but might give some rare clones the chance to be established. Our T-cell clones were selected under strict antigen-dependent conditions and without exogeneous IL-2. With these clones we have shown that not only T-ACC-specific but also T-T-specific cellular interactions resulting in help are possible in vitro. It is, however, important to stress that the activation of Tu in our system is class I restricted and extends from the T clone to the TH. The outcome then, the Tu-ACC interaction, is class II restricted. The other experimental system where such an interaction between T cells has been shown is the so called “veto cell” in the anti-miHA response (42,43). These T cells differ from ours in that they do not recognize the responding target cell and that they suppress the immune response to miHA. Our clones recognize the target cells and activate them. This activation results in the expression of help. Nevertheless, killing of all of the stimulator cells occurs after a certain period of time, at least when the CTL clone is used. We do not know if the cellular interactions taking place in our model reflect an actual in vivo pathway. The availability, however, of T-cell clones which interact with different cell populations might facilitate the study of cells and molecules in the exchange of information among cells of the immune system. ACKNOWLEDGMENTS We thank Heike Weber for excellent assistance and Dr. R. M. Zinkemagel and Dr. D. Troyer for critical reading of the manuscript.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Cantor, H., and Boyse, E. A., J. Exp. Med. 141, 1390, 1975. Altman, A., Bechtold, T. E., and Katz, D., J. Immunol. 122, 2484, 1979. Ashman, R. B., and Mtlllbacher, A., J. Exp. Med. 150, 1277, 1979. Cooley, M. A., and Schmitt-Verhulst, A.-M., J. Immunol. 123, 2328, 1979. Finberg, R., Greene, M. I., Benacerraf, B., and Burakoff, S. J., J. Zmmunol. 123, 1205, 1979. Melief, C. J., van der Meulen, M. Y., Christiaans, B. J., and de Greeve, P., Eur. J. Immunol. 9,7, 1979. Wettstein, P. J., and Frelinger, J. A., J. Immunol. 127,43, 1981. Wagner, H., and Rollinghoff, M., J. Exp. Med. 148, 1523, 1978. Di Pauli, R., and Opalka, B., Eur. J. Immunol. 12, 365, 1982. Di Pauli, R., and Bruckner, T., Eur. J. Immunol. 14, 915, 1984. Zinkernagel, RM., Callahan, G. N., Althage, A., Cooper, S., Streilein, J. W., and Klein, J., J. Exp. Med. 147,897,

1978.

12. Von Biihmer, H., and Haas, W., J. Exp. Med. 150, 1134, 1979.

CELLULAR 13. 14. 15. 16. 17. 18.

INTERACTIONS

IN

VITRO

195

19. 20. 21. 22.

Komgold, R., and Sprent, J., J. Exp. Med. 151, 314, 1980. Kruisbeek, A. M., Andrysiak, P. M., and Singer, A., J. Zmmunol. 131, 1650, 1983. Bennick, J. R., and Doherty, P. C., Nature (London) 276, 829, 1978. Fink, P. J., and Bevan, M. J., Proc. Natl. Acad. Sci. USA 78, 6401, 1981. Raulet, D. H., and Bevan, M. J., J. Exp. Med. 155, 1766, 1982. Klein, J., Huang, H.-J. S., Lemke, H., Hammerling, G. J., and Hlmmerling, U., Immunogenetics 8, 419, 1979. Ozato, K., Mayer, N., and Sachs, D. H., J. Zmmunol. 124, 533, 1980. Opitz, H. G., Opitz, U., Hewlett, G., and Schlumberger, H. D., Zmmunobiology 160,438, 1982. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Zmmunol. 3,645, 1973. Nabholz, M., Vives, J., Young, H. M., Meo, T., Miggiano, V., Rijnbeek, A., and Shreffler, D. C., Eur.

23. 24. 25. 26.

Schmitt-Verhulst, A.-M., Sachs, D. H., and Shearer, G. M., J. Exp. Med. 143, 211, 1976. Fischer Lindahl, K., and Lemke, H., Eur. J. Zmmunol. 9,526, 1979. Glasebrook, A. L., Kelso, A., and MacDonald, H. R., J. Zmmunol. 130, 1545, 1983. Lemonnier, F., Burakoff, S. J., Mescher, M., Dorf, M. E., and Benacerraf, B., J. Zmmunol. 120, 17 17,

27.

Albert, F., Boyer, C., Bufeme, M., and Schmitt-Verhulst, A.-M., Immunogenetics 19,279, 1984. Cowing, C., Pincus, S. H., Sachs, D. H., and Dickler, H. B., J. Zmmunol. 121, 1680, 1978. Ahmann, G. B., Nadler, P. I., Bimkrant, A., and Hodes, R. J., J. Zmmunol. 123,903, 1979. Pettinelli, C. B., Ahmann, G. B., and Shearer, G. M., J. Zmmunol. 124, 1911, 1980. Perry, L. L., and Greene, M. E., J. Exp. Med. 156,480, 1982. Murphy, D. B., Jones, P. P., Loken, M. R., and McDevitt, H. O., Proc. Natl. Acad. Sci. USA 77, 5404, 1980. Festenstein, H., Transplant. Rev. 15,62, 1973. Ely, J. M., Prystowsky, M. B., Eisenberg, L., Quintans, J., Goldwasser, E., Glasebrook, A. L., and Fitch, F. W., J. Zmmunol. 127, 2345, 1981. Howe, M. L., J. Zmmunol. 110, 1090, 1972. Glimcber, L. H., Longo, D. L., Green, I., and Schwartz, R. H., J. Exp. Med. 154, 1652, 1981. Lattime, E. C., Gillis, S., Pecoraro, G., and Stutman, O., J. Zmmunol. 128,480, 1982. Smith, K. A., Lachman, L. B., Oppenheim, J. J., and Favata, M. F., J. Exp. Med. 151, 1551, 1980. Von Bohmer, H., and Turton, K., Eur. J. Zmmunol. 13, 176, 1983. Widmer, M. B., and Bach, F. H., Nature (London) 294,750, 1981. Roopenian, D. C., Widmer, M. B., Orosz, C. G., and Bach, F., J. Zmmunol. 131,2135, 1983. Rammensee, H.-G., Nagy, Z. A., and Klein, J., Eur. J. Zmmunol. 12, 930, 1982. Fink, P. J., Rammensee, H.-G., and Bevan, M. J., J. Zmmunol. 133, 1775, 1984.

J. Zmmunol.

4,318,

1974.

1978.

28. 29. 30. 31. 32. 33. 34. 35.

36. 37. 38. 39. 40.

41. 42. 43.