Journal of Immunogenetics (1988) 15, 161-168.

A N E X T E R N A L S T I M U L U S T H A T MIMICS Mls LOCUS RESPONSES

c. A . JANEWAY, J . CHALUPNY, P. J . CONRAD A N D s. BUXSER*

Department of Pathology and Section of Immunobiology, Howard Hughes Institute at the Yale University School of Medicine, New Haven, CT 06510 and *The Upjohn Company,

Department of Cell Biology, Kalamazoo, MI 49001, U.S.A.

S U M M A R Y

The response to a novel set of T cell mitogens has been analysed and compared to the response to Mls locus differences. These polyclonal T cell activators, staphylococcal enterotoxins A and B, stimulate T cells in a way that requires an antigen-presenting cell bearing class I1 MHC products and involves the CD4:T cell receptor complex. However, the specificity of MHC recognition by the T cell receptor is lost in this response. Thus, these mitogens produce a response with characteristics similar to that induced by MIS locus differences. These mitogens can be used to analyse the immunobiology of this response, and may help in understanding and identifying the MIS locus product as well.

INTRODUCTION

The intense T cell proliferative response that develops when T cells from Mlsb strains are stimulated by spleen cells from MISO strains has long fascinated immunobiologists. This response has engendered a large number of different explanations, none of which has yet been experimentally verified. A major difficulty has been the lack of a chemical definition of the MIS locus product.

The characteristics of the primary and cloned T cell responses to MIS that make it so interesting to immunobiologists are listed in Table 1. The most striking aspects of this response are the very high precursor frequency, the involvement of the T cell receptor :CD4 complex in recognizing class I1 major histocompatibility complex (MHC) in this response, and the loss of MHC specificity, both in terms of MHC rstriction and in terms of distinguishing I-A from I-E. Such a profound alteration in the specificity of the T cell receptor is certainly worthy of an explanation.

Based on these findings, we (Katz & Janeway, 1985) had proposed that MIS encoded differences in expression of an antigen-presenting cell (APC) molecule which promoted interactions between class I1 MHC molecules and the T cell receptor. To test this idea, we

Correspondence: Charles A. Janeway, Department of Pathology and Section of Immunobiology, Howard Hughes Institute at the Yale University School of Medicine, New Haven, CT 06510, U.S.A.

161

162 C. A . Janeway et al. TABLE 1. Characteristics of the T cell response to Mls locusdisparate stimulator cells

The response is unidirectional (Festenstein, 1973; Janeway & Katz, 1985). The precursor frequency is 3-5-fold higher than that to MHC differences (Janeway er ol., 1980; Lutz er ol., 1981; Miller & Stutman, 1982). Anti-class I1 antibodies block the response to Mls (Janeway ef a/., 1980; DeBreuil er ol., 1982). Cloned T cell lines of many specificities respond to Mls locus differences (Janeway er ol., 1983; Janeway & Katz, 1985). Cloned T cell lines are not MHC restricted in their responses to MIS locus differences, but are influenced by MHC(Webbera1.. 1981; Janewayefol., 1980; Jones& Janeway, 1982; Lynchefal., 1985; Janeway&Katz, 1985) Antibodies to CD4 and the T cell receptor inhibit responses to Mls locus disparities (Katz & Janeway, 1985). I-E molecules are more involved in responses to Mls locus differences than are I-A molecules (Peck er ol., 1977; DeBreuil er a/., 1982; Janeway et al., 1983). Antigen presentation to a variety of cloned T cell lines is influenced by Mls locus differences (Janeway er a/., 1983).

attempted to substitute exogenous substances which should increase interactions between T cells and APCs. These substances, including a non-mitogenic plant lectin which could agglutinate cells, polyethylene glycol, and anti-class I MHC antibodies, all failed to reproduce the MIS phenomenon. Meanwhile, studies on CD4 have shown clearly that this putative adhesion-strengthening molecule is actually intimately associate'd with the T cell receptor during T cell activation, and the T cell receptor functions approximately 100-fold more effectively when it is associated with CD4 than when it is not (Saizawa et al., 1987). This suggested to us that adhesion-strengthening such as we had proposed is unlikely to lead to T cell activation, unless the molecule actually directly links class I1 MHC to the T cell receptor.

In the present studies, we have taken a different approach to the MIS phenomenon: we have searched for a protein of known structure that mimics the Mls phenomenon when added as an external stimulus. We have asked whether the exogenous protein, when added to whole spleen cells or cloned T cell lines, can mimic the behaviour of differences at the MIS locus. We report here on our studies with two such proteins, staphylococcal enterotoxins A and B (SEA and SEB). Similar studies with a less well-characterized protein found in a filtrate of Mycoplusma arthritidis have been reported by Lynch and colleagues (1986). It is our hope that the analysis of this protein of known structure will lead us to an understanding of the MIS phenomenon, or at least give us an experimental system of comparable characteristics so that we can explore the biology of the MIS response and the means by which a T cell receptor so alters its specificity for MHC.

MATERIALS A N D METHODS Animals

Dona1 B. Murphy under grant CA-29606.

Cloned T cell lines The cloned T cell line D10.G4.1 has been described elsewhere (Kaye et al., 1983). It is

specific for a fragment of the egg white protein conalbumin presented by I-Ak, and cross-

Mice were from the breeding nucleus maintained at Yale University School of Medicine by

An Mls-like T cell mitogen 163 reacts on allogeneic stimulator cells bearing I-Ab."*p.q. A weak reactivity to I-Ad has been reported in a recept study (Horowitz et al., 1987). The line D8 was also described by Kaye and associates (1983); it is specific for I-Ak and the protein ovalbumin. Cloned line AK8 is specific for conalbumin:I-A', carries the F23.1 determinant, and lacks alloreactivity or D10 idiotypic determinants. It has not previously been described.

Antigen-presenting B lymphoma cells To avoid responses on the part of contaminating T cells in spleen cell populations, the B cell lymphoma A20/2J (Kim et al., 1979) was used as an antigen-presenting cell.

Mitogens

The mitogens used were phytohaemagglutinin (PHA) (Sigma, St Louis, MO, U.S.A.) and staphylococcal enterotoxins A and B (SEA and SEB) (see Buxser & Vroegop, 1988, for details). Both were dissolved in phosphate-buffered saline (PBS), diluted in medium and added at various concentrations to the assays.

Monoclonal antibodies Monoclonal antibodies to I-Ad (MKD6, 212), I-E (14.4.43), CD4 (GKlS), the T cell

receptor on D10 cells (3D3, in the form of an Fab fragment, prepared as described by Kaye & Janeway, 1984), and the T cell receptor on AK8 cells (F23.1, Staerz et al., 1985) have all been described previously (Tite & Janeway, 1984).

Assays for response The cloned T cell lines D8, D10 and AK8 produce abundant IL-4 on stimulation (Kaye et

al., 1984). This can be assayed by induction of proliferation in the cell line HT-2, which is responsive to both IL-2 and IL-4. Thus, we have used 24 h supernatants added to lo4 HT-2 cells to assay responses. D10 supernatant is added to a final concentration of lo%, and the incorporation of )H-TdR is measured during a 12 h pulse at the end of a 48 h culture period. Cultures are run in triplicate in flat-bottomed microtitre trays in Click's EHAA medium with 10% foetal calf serum added, as described previously (Kaye et al., 1984).

RESULTS

The staphylococcal enterotoxins A and B are potent mitogens for whole spleen cells The purified staphylococcal enterotoxins A and B were added to spleen cells from many

strains of mice at a wide range of doses, and the proliferative response measured at 72 h by incorporation of )H-TdR. The results of one such experiment are shown in Fig. 1. Strains B10.D2 and BlO.GD, which differ in expression of I-E molecules, are compared. Both respond well to both PHA and SEA, with SEA having a very broad dose-response curve and a lower maximum response than PHA. SEB is considerably less potent than SEA, and stimulates only B 10.D2, not B1O.GD spleen cells. This same difference was observed using BlO.A(2R) (I-E+), which responded to SEB, and BlO.A(4R) (I-E-), which did not respond to SEB. This suggests that SEB responses require, or are greatly augmented by, the presence of I-E molecules, similar to results using Mycoplasma arthritidis mitogen (Lynch et al., 1986).

Cloned T cell lines respond differently to SEA and SEB presented by B lymphoma cells The cloned T cell lines D10 and D8 (Kaye et al., 1983) are both restricted by I-Ak in

responses to different protein antigens. Clone D10 produces T cell growth factors in response

1 64 C. A . Janeway et al. Effect of I-E on response to SEE

Mitogen

FIG. 1. The response of whole spleen cells to different mitogens at varying doses. PHA (IOOO= 100pg/ml)(., u),SEA(1000= 100ng/rnl)(O,O),andSEB(1000=SWng/ml)(*, m) were added to the cultures of 2 x lo5 fresh whole spleen cells from normal mice of strains B10.D2 (Kd, Ad, Ed, W) (D2: 0, H, 13) or B1O.GD (Kd. Ad, E-, Db) (GD: 0, *, 0). Responses of triplicate cultures in cpm thymidine incorporated on day 3 of culture. The role of I-E in the response to SEB is clearly illustrated.

( 0 1

Response of DIO cells in presence of A 2 0

I/dilution

( b l Responses of cloned lines

40,000 r

20,000 30,mu 10.000

0 001 01 I lo 100 lpoo lop00

Mitogen

FIG. 2. The release of IL-4 induced by SEA, SEB and PHA by cloned lines D10 (a), or D8 and AK8 (b) in the presence of A20 cells, measured by thymidine uptake of HT-2 cells given supernatant of 104 T cells cultured with lo5 A20 B lymphoma cells. Means of triplicate original and assay cultures, with supernatants taken at 24 h of culture, added to 104 HT-2 cells for 24 h, followed by a 12 h pulse of thymidine. Starting concentrations of mitogens are: for (a) SEA, 2 = 500 ng/ml; SEB,2 = 500 ng/ml; PHA, 2 = 100 pg/ml; for (b), lo00 ng/ml for both SEA and SEB.

to SEB but not SEA, while clone D8 responds to SEA and not SEB. Clone AK8, also specific for conalbumin :I-Ak responds similarly to clone D10 (Fig. 2). The responses of these cloned lines to SEA and SEB require the presence of an antigen-presenting cell, unlike the response to PHA (Fig. 3). The antigen-presenting cell we used was the cloned B lymphoma line A20/2J in order to avoid the possible production of lymphokine by contaminating T cells in the APC population. That responses are seen with D10 and AK8 with SEB but not SEA, while with D8 the opposite is observed, rules out the possibility that the lymphokine is produced by the feeder cells.

The response of DIO cells to SEB presented by B lymphoma cells is inhibited by anti-I-E but not by anti-I-A antibodies

To determine whether class I1 molecules were involved in the response to D10 and AK8 cells to SEB presented by A20 cells, anti-class I1 antibody blocking studies were carried out

An MIS-like T cell mitogen 165 Response of DIO cells to SEE and PHA

I 10 100 1,000 lop00 l00,ooo VDilution

FIG. 3.ThereleaseofIL-4inducedinDlOcellsbyPHA(2 = lOOpg/ml)(~,O)andSEB(O,+) (2 = 500 ng/ml), measured as in Fig. 2. D10 cells cultured alone (+, 0) or with los A20 cells as APC.

Inhibition of DIO t A20 responses * O SEB + O PHA

0 f

o 60,0000 C 0

2 40.000 e 20,000 c

= o I

SEE PHA SEB PHA S EB PHA

I/Dilution

FIG. 4. The effects of monoclonal antibodies on the response of D10 cells to PHA (2 = 100 pg/ml) and SEB (2 = 1000 ng/ml) in the presence of A20 cells. Monoclonal antibodies added to A20 cells and mitogen prior to adding D10 cells. Responses measured as in Fig. 2.

( 0 ) ( b ) Inhibition of DIO resDonses to SEB Inhibit ion of AK8 rewonses to SEE

v Y I U - 60,000 1 DIO .

+ o nin . W

40,000 :: e N 20.000 E - " I "

I IU

+ O

I00 1,000 SEE

& I

0 A L L d lO,000

AK8 t 14.4.4 0 AK8 + F23.1

I 10 100 '1,000 10,000 SEE

FIG. 5. The effect of monoclonal anti-class I1 and anti-T cell receptor antibodies (3D3 Fab fragment on D10, F23.1, IgG on AK8)on responses of D10 cells (a) or AK8 cells (b) to A20 and SEB. Starting concentration of SEB = 500 ng/ml in both experiments. Responses carried out as described in Fig. 2.

(Figs 4 and 5). Anti-I-E antibodies inhibited this response, while anti-I-A antibodies actually augmented it slightly. The same antibodies had little effect on the response to PHA under identical conditions. Anti-CD4 inhibited both responses markedly, in keeping with our

166 C. A . Junewuy et al. earlier results (Tite et al., 1986). Thus, it cannot be determined from this experiment whether CD4 is directly involved in the response to SEB, or whether negative signalling via CD4 is inhibiting the response to SEE as well as the response to PHA.

The Fab fragment of a monoclonal anti-DI0 receptor antibody can block the response to SEB Our earlier studies of the response of Mlsb T cells to MIS" stimulator cells had implicated the

T cell receptor in this response. To determine whether the T cell receptor was also involved in the response of D10 cells to SEB presented by I-E molecules on A20 cells, we added the Fab fragment of an anti-receptor antibody to the culture. This Fab fragment is known to inhibit responses by D10 cells to all class I1 MHC ligands, but not the response to anti-Thy-1, T cell mitogens or anti-CD3. Thus, inhibition with this antibody implies recognition by the variable portion of the T cell receptor. As seen in Fig. Sa, the Fab fragment of 3D3 is as effective as anti-I-E antibody in inhibiting this response. Comparable blocking of the AK8 response to SEB with F23.1 anti-receptor antibody is shown in Fig. 5b. Thus, the T cell receptor is involved in the response of D10 and AK8 cells to SEB.

DISCUSSION

These results are of interest for two reasons. First, SEA and SEB are unusual mitogens in that responses to them are readily blocked with antibodies to class I1 MHC products (see Buxser & Vroegop, 1988). These mitogens act selectively on different cloned cell lines, using both class I1 MHC and the T cell receptor (probably including CD4) to make this response. In the case of SEB, cloned T cell lines specific for antigen associated with I-Ak are now stimulated by I-Ed. Second, these mitogens appear to mimic the behaviour of Mls-disparate mixed lymphocyte responses.

As our major interest in the MIS phenomenon is in the mechanism by which class I1 MHC can be presented in such a non-specific fashion to so many different T cell receptors, a protein of known structure like SEB which has these same properties is of great value. It allows the interesting immunobiological questions raised by the MIS phenomenon to be pursued at the structural level, using well defined.experimenta1 systems. In addition, it may lead to an understanding of the product of the MIS locus itself, which it mimics biologically.

Given that a protein can reproduce the effects of differences in the Mls locus, what might one deduce about the MIS locus product itself? The most likely possibility, supported by the similar earlier studies of Mycoplusma arthritidis mitogen, are that SEE binds directly to I-E, and somehow mediates binding of I-E to the T cell receptor on D10, AK8 and many other T cells as well, as evidenced by its ability to stimulate many different cloned T cell lines as well as a significant proportion of normal T cells (Fig. 1). By fragmenting SEB, we hope to isolate the relevant peptide and examine its binding to I-E, and the binding of putative I-E :SEB- peptide complexes to the T cell receptor.

If indeed SEE contains a peptide that binds I-E and allows I-E to be recognized by many (but certainly not all: see D8) T cell receptors, could this also be true of autologous peptides, including the product of MIS? And if so, could such peptides play a role in the expansion of the self MHC-specific T cell repertoire, or in self tolerance? These are interesting questions which remain to be answered. One difficulty with such a proposal is that it does not explain the strict MHC restriction of ontogenetic selective events, as the hallmark of MIS locus responses is that they lose strict MHC restriction. However, Mlsmay be an aberrant form of a normal protein whose peptide performs this critical function. For this reason, as well as the sheer puzzlement of it, we continue to find the MIS phenomenon of great interest.

An Mls-like T cell mitogen 167 A C K N O W L E D G M E N T S

The authors wish to thank Michael Katz, Jon Kaye, Pilar Portoles and Don Murphy for valuable reagents used in these studies, and for sharing data and ideas with the authors. This work was supported in part by NIH grant AI-14579 and training grant AI-07019 funds to JC.

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