Intern. Rev. Zmmunol. Vol. 8, 1992, pp. 311-325 Reprints available directly from the publisher Photocopying permitted by license only 01992 Harwood Academic Publishers GmbH Printed in the United States of America
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Retroviral Super-Antigens and T Cells DAVID L. WOODLAND and MARCIA A. BLACKMAN Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA (Received December 16, 1991)
For many years immunologists have been intrigued by a series of potent antigens encoded in the murine genome. These antigens, originally termed minor lymphocyte stimulating (Mls) antigens, are capable of inducing extremely strong T cell proliferative responses when presented in the context of MHC class I1 molecules. Recently, Mls antigens have been shown to stimulate T cells bearing particular T cell receptor Vp elements, leading to the designation of super-antigens. The endogenous expression of these super-antigens in mice results in the clonal elimination of large numbers of T cells in order to maintain self-tolerance. In this review we discuss the recent identification of endogenous super-antigens as retroviral gene products. In addition, we analyze the role of class I1 MHC molecules in the presentation of endogenous super-antigens to T cells. Finally, we discuss the dramatic effect of retroviral super-antigens on the T cell repertoire. KEYWORDS: T cell, T cell receptor, super-antigen, MMTY Mls
INTRODUCTION T cells recognize foreign antigens which have been degraded into small peptides and presented on the cell surface complexed with Major Histocompatibility Complex (MHC) molecules. The T cell antigen receptor (Tcr) is thought to contact both the foreign antigen fragment and the self MHC molecule in this complex. Typically, less than one in 105 T cells bear the appropriate combination of T cell receptor elements required to interact with any given MHC/peptide complex [l, 21. Recently, a separate class of T cell antigens, termed super-antigens, has been described [3, 41. Super-antigens are distinguished from conventional antigens in three fundamental ways. First, they stimulate T cells with high frequency (usually one in ten T cells respond to a super-antigen). Second, T cell recognition of superantigens is mediated primarily through the Vp element of the Tcr and essentially independent of other elements of the Tcr. Since only a few Vp genes are present in the gene pool, each Vp element is represented at a relatively high frequency among T cells. Thus, a superantigen which interacts with several Vp elements is capable of stimulating a large proportion of the T cell repertoire. Third, super-antigens do not exhibit classical MHC restriction. Although they require presentation on class I1 molecules, they are promiscuous in terms of their haplotype specificity. Two classes of super-antigen have been described-bacterial and endogenous. The bacterial super-antigens are toxins secreted by several species of Gram positive bacteria, including Staphylococcus aureus and Streptococcus pyogenes . These toxins are responsible for a variety of diseases in man and animal, including toxic shock and food poisoning [ 5 ] . It 31 1
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D. L. WOODLAND AND M. A. BLACKMAN
has been shown that at least some of the pathogenic effects of these toxins are mediated by the hyper-stimulation of T cells [6]. The endogenous super-antigens are encoded by genes present in the genetic backgrounds of some strains of mice. Since endogenous superantigens are expressed as self antigens during T cell ontogeny, they have a profound effect on the T cell repertoire due to the clonal elimination of large numbers of potentially autoreactive T cells [7]. Endogenous super-antigens have recently been shown to be encoded by retroviruses [8].
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MULTIPLE ENDOGENOUS SUPER-ANTIGENSARE EXPRESSED IN MICE The Minor Lymphocyte Stimulating (Mls) antigens were originally characterized by their ability to induce extremely strong proliferative responses between MHC-matched murine lymphocytes [9, lo]. Analysis of proliferative responses between Mls-expressing and nonexpressing strains of mice revealed that the responding population was comprised predominantly of T cells, and that the major Mls-presenting cells were B cells. The stimulation of T cells was dependent on the expression of MHC class 11molecules on the presenting cells, but was promiscuous in terms of MHC haplotype 111-141. Despite the strong antigenicity of these gene products, it has proven to be surprisingly difficult to generate antibodies specific for them and to biochemically characterize them. Recently, several groups have established that the Mls series of antigens are in fact endogenous super-antigens which stimulate T cells bearing certain Vp elements. For example, the MIS-1 super-antigen, encoded on chromosome 1 of several strains of mice, stimulates the majority of T cells expressing Vp6+, Vp7+, Vp8.l+ and Vp9+ elements [13, 15-18]. In Mls-1-expressing strains of mice this antigen acts as a self molecule and mediates the clonal elimination of these subsets during T cell ontogeny in the thymus [13]. The elimination of these cells renders the animal tolerant to Mls-1, but at the expense of T cell diversity. Several Mls super-antigens have been described. Each of these super-antigens has essentially the same characteristics as Mls-1 described above, except that the Vp specificity varies [4]. The expression of several Mls genes in a given strain of mouse can result in a profound reduction in the T cell repertoire. In addition to the Mls family of super-antigens, studies from several laboratories have reported the existence of a second type of endogenous super-antigen which functions in association with MHC I-E molecules. The I-E associated super-antigens are discussed below.
ENDOGENOUS SUPER-ANTIGENSMEDIATE THE CLONAL ELIMINATION OF I-E REACTIVE T CELLS In 1987, Kappler et al., demonstrated that T cells bearing Vp17 elements of the Tcr tended to recognize I-E molecules, and that Vp17+ T cells were clonally deleted in the thymuses of I-E+ strains of mice [19,20]. This reactivity was apparently independent of other elements of the Tcr and was not self-MHC restricted, in that different alleles I-E were able to stimulate Vp17+ hybridomas. In later studies, several laboratories generated antibodies specific for Vp5+ and Vp11+ T cells and demonstratedthat these T cells were also clonally eliminated in most strains of mice which express functional I-E molecules [21-231. In addition, various other studies have suggested that Vp12+, Vp16+, Vp19a+, and Vp20+ T
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cells are I-E reactive, although there are currently no antibodies available to confirm these observations [24-291. A role for an endogenous super-antigen in the interaction between I-E molecules and I-Ereactive T cells was suggested by several observations. First, Marrack and Kappler demonstrated that Vp17+ hybridomas responded to I-E presented on B cell lines, but not macrophage lines, implicating a role for a B cell-specific gene product [30]. Second, studies by Tomonari’s laboratory suggested that not all I-E+ strains of mice clonally eliminated I-E reactive T cells [21]. For example, the C58 strain of mouse (I-Ek) expresses 10.8% VpII+ T cells, a level comparable to that found in I-E-negative strains of mice. Backcross analyses with this strain revealed a requirement for non-MHC gene products in the deletion of Vp11+ T cells [31,32]. Finally, Bill et al., and Woodland et al., demonstrated that several strains of recombinant inbred mice express high frequencies of V95+ and Vp11’- T cells, despite the fact that they express I-E molecules [22, 331. Taken together, these studies illustrated that I-E molecules were necessary, but not sufficient, to mediate the clonal deletion of these T cells and implicated a role for one or more I-E-associated super-antigens. In order to identify the super-antigens involved in the clonal deletion of I-E reactive T cells, Woodland et al. performed a detailed analysis of Vp5+ and VPll+ expression in C57BL/6 X DBN2 (BXD) recombinant inbred strains of mice [33, 34). Approximately half of the B X D strains had inherited the H-2b MHC locus from the C57BL/6 parent and expressed high frequencies of Vp5+ and VPll+ T cells (approximately 6-8%), typical of the C57BL/6 parent and consistent with the fact that the H-2b locus does not encode a functional I-E molecule. The remaining B X D strains expressed the I-Ed molecule encoded by the H-2d locus inherited from the DBN2 parent. The frequencies of Vp5+ and Vp11+ T cells in these strains varied widely from 1280t
>I280 11280
6 8
6
320 1280 160
2 8 13
80 320 80
11-40
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kMlsl3-11 bbml8- 19 bbml8- 109 3HB113 2HB79 5426
>1280
A20/2J
Etc- 1+ 1B4
lHlO
2C3
> I280
>1280 160
160
>1280 1280 >1280
*AU of the hybridomas, B cell lines and transfectantshave been described elsewhere [24,40,91]. LBB-1 is a B cell lymphoma which expresses MIS-1 and Etc-I. kMlsl3-11, bbm18-19 and bbm18-109 have all been shown to be strongly Mls-1 reactive. tHybridoma responses are presented as unitdml IL-2. Responses which were below detection threshold (< 10 U/ml)are shown as -. Stimulation assays were performed as described previously [33]. Xoncanavalin A was added to the responding hybridomas at 2pg/ml.
(controls)did not stimulate KE8. Table IV shows that the stimulation was specific for Vp5+ and Vp11+ Etc-1-reactive hybridomas. No IL-2 was produced by the hybridomas expressing non-Etc-1-reactive Vp elements, Vp2, 6, 8 and 13. These data confirmed that endogenous super-antigen expression, like that of infectious virus, is also controlled by the ORFs of integrated MMTV viruses. Surprisingly, transfectants generated with Mtv-8 constructs also stimulated the Vp5 + KE8 hybridoma (Table 111). This result was unexpected because there was no genetic evidence linking the deletion of Vp5+ T cells with the presence of the Mtv-8 integrant. It is currently unclear why this is the case. One possible explanation is that Mtv-8 is normally poorly expressed in most strains of mice and the observation that the Mtv-8 locus is heavily methylated in some strains is consistent with this [53]. An alternative explanation is that only a small fraction of VP5+ T cells recognize the Mtv-8 super-antigen, perhaps depending on contributions from the Tcr a chain (see below). Thus the deletion of a small fraction of Vp5+ T cells may not have been noticed, especially since Mtv-8 is present in almost all inbred strains of mice [54]. Studies by several groups have shown that Vp12+, Vp16+ and Vp17+ T cells are clonally eliminated in strains of mice which express functional I-E molecules [19, 20, 25-27]. The availability of MMTV transfectants made it possible to investigate whether this I-E reactivity is mediated by Etc-1. T cell hybridomas expressing Vp12, Vp16 and Vp17 elements were tested for their ability to recognize Etc-1 presented by B cell lines (A20/2J and CH12.LBK) transfected with Mtv-9 ORF DNA. The majority of Vp12+ and Vp17+ hybridomas responded to the transfectants (but not the parental cell lines), demonstrating that Etc-1 interacts with T cells bearing these Vps 140,551. In contrast, none of the Vp16+ hybridomas responded to the Etc-l+ transfectants. However, this result is inconclusive because most of the Vp16+ hybridomas did not express CD4 which is normally required for in vitro responses to Etc-1 (DLW, unpublished observations). In similar studies it has been shown that at least some Vp17+ hybridomas respond to the Mtv-8 encoded super-antigen [40].
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319
More recently, other endogenous Mtv 3’0RF genes have been successfully transfected into appropriate recipient cells. For example, the transfection of B cell lines with Mtv-7 3’LTR genes resulted in the expression of Mls-1 (personal communication, B. Hubex) and the transfection of B cell lines with Mtv-6 3’LTR resulted in the expression of Mls-3 (personal communication, A. Pullen).
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THE STRUCTURE OF RETROVIRAL SUPER-ANTIGENS The identification of endogenous super-antigens as retroviral gene products should allow the structure and function of these molecules to be rapidly determined. An examination of the known MMTV 3’LTR DNA sequences reveals several features [43]. First, there are several potential methionine translation starts which could result in the expression of proteins ranging in size from 37-39 kD. Second, a comparison between the known aminoacid sequences of MMTV ORFs reveals only 10-15% variation and suggests that the C-terminal region may control Vp specificity. Third, a 23 amino-acid hydrophobic region near the amino terminus suggests that the super-antigen is an integral membrane protein. Finally, there are multiple potential glycosylation sites. Recently, Choi et al. and Knight et al. have shown that MMTV super-antigen genes encode integral membrane glycoproteins which have the carboxy terminus exposed on the outside of the cell (type 11) [56, 571. This correlates with the suggestion that the carboxy terminus controls Vp specificity. Functional super-antigen is translated from the first methionine start codon and the protein is glycosylated at 4/5 potential sites.
REGULATED EXPRESSION OF RETROVIRAL SUPER-ANTIGENS The majority of data suggests that retroviral super-antigens are expressed predominantly on the surface of B cells [58-621. However, it is not clear how this expression is regulated during B cell development. Woodland et al. and Gollob and Palmer have shown that a 24 hour pre-treatment of splenic B cells with LPS and IL-4 greatly enhances Etc-1 expression [33, 611. The enhanced presentation of Etc-1 is not due simply to the increased levels of class I1 molecules, since IL-4 alone (which also upregulates surface class II), enhances Etc-1 expression only weakly [61]. If the LPS/IL-4 pretreatment is extended beyond 24 hours, the expression of Etc-1 drops off quickly despite the fact that I-E expression remains elevated for some time (Woodland and Blackman, unpublished observations). Corley and colleagues have measured the level of Mtv-9 transcription and Etc-1 cell-surface expression upon activation of two B cell lines, CH12 and BCL-1, and shown that various combinations of lymphokines regulate Etc-1 expression [62, 631. The expression of Etc-1 under these conditions generally correlated with the density of class I1 molecules on the surface of the presenting cell, suggesting that the regulation is at the level of class I1 expression. However, treatment of CH12 and BCL-1 with LPS resulted in the virtual elimination of super-antigen activity, despite the fact that Mtv-9 transcription was strongly upregulated and surface class U density was only moderately reduced. Since LPS is thought to drive these cells to a more mature phenotype, Lund et al. have suggested a model in which super-antigens can be posttranscriptionallyregulated by (i) class 11expression and (ii) an unknown mechanism which is correlated with B cell development [63]. This model is consistent with the finding that LPS treatment of normal spleen cells induces the transient expression of Etc-1.
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D. L. WOODLAND AND M.A. BLACKMAN
An alternative mechanism for regulating the expression of retroviral super-antigens has been suggested from studies in which multiple super-antigens were expressed in B cell tumor lines [40]. The A20/2Jline contains the proviral integrants Mtv-6, Mtv-8 and Mtv-9, but northern analysis has revealed that only the Mtv-6 locus is transcriptionallyactive. This line also functionally expresses the Mtv-6 linked super-antigen and stimulates Vp3+ hybridomas. Transfection of A20/2J with the Mtv-9 ORF gene (to convert it into an Etc-I+ line) resulted in a substantial reduction in the ability of A20/2J to stimulate Vp3+ T cells. These observationssuggest that endogenous super-antigens may under some circumstances compete with each another for functional expression.
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THE ROLE OF MHC CLASS II MOLECULES? Several models have been proposed to account for the interaction between T cells and retroviral super-antigens. However, current data is consistent with the model originally proposed by Janeway in which the super-antigen acts as a cross-linking molecule by binding directly to the Tcr and class II MHC molecule [64]. This model is based partly on analogy with bacterial super-antigens and partly on a molecular analysis of super-antigen/Tcr interaction. First, experiments with the functionally analogous, bacterial super-antigens have demonstratedthat the toxins bind directly to class II molecules and are fully functional in association with fixed presenting cells, indicating that no antigen processing is required for presentation [4, 65-67]. Second, in vitro mutagenesis of bacterial toxins has identified amino-acid residues which are involved in the interaction with either the Tcr or the class I1 molecule (J. Kappler, personal communication) [68, 691. Third, mutational analyses have revealed sites on both the human and the murine Tcr which seem to be involved in binding both bacterial and retroviral super-antigens, but which are distinct from the MHC/peptide binding site [70-731. For the purposes of this review we will discuss data in the context of the Janeway model; however, it should be borne in mind that this model remains hypothetical. Class II molecules vary significantlyin their ability to present retroviral super-antigens to T cells. For example, several super-antigens (such as Etc-1 encoded by the Mtv-9 OW) appear to be presented exclusively in association with I-E molecules [23]. Similarly, class I1 molecules vary in their ability to present Mls-1 to T cells (H-2k and H-2d are generally the strongest and H-2s the weakest) [13, 741. In terms of the Janeway model, the simplest explanation for these data is that different class II molecules vary in their ability to bind, and thus present, retroviral super-antigens to T cells. Thus, the I-E restriction of Etc-1 would be due to the inability of this super-antigen to bind to I-A. However, recent data suggest that this explanation is invalid. We have analyzed the response of an Etc-1 (Mtv-9 OW) reactive hybridoma, 5412 [33,34], to spleen cells from a number of different strains of mice (Table V). This hybridoma clearly responded to Etc-1 presented in the context of I-E molecules from several different haplotypes. However, this hybridoma also responded to C57BW6 presentor cells which express I-Ab but not I-E molecules and this response could be inhibited by antibodies specific for I-A (D.L. W and E. palmer, unpublished observations). This reactivity was not a generic I-Ab alloreactivity since not all H-2b spleen cells stimulated 5412. For example, the D1.LP strain (H-2b on a DBA/2 genetic background) does not stimulate 5Q12 suggesting a role for a non-MHC gene product. In order to investigate this further, we analyzed the ability of H-2b B x D FU strains to stimulate 5Q12 (Table V). Although only a limited number of strains have been analyzed, there is a perfect correlation between the stimulation of 5Q12 and the presence of the Mtv-9 retroviral
RETROVIRAL SUPER-ANTIGENS
321
TABLE V Response of the Etc-1 Reactive Hybndoma, 5412, to Spleen Cells from Recombinant Inbred Strains of Mice ~~
MHC
MW-9
5412 response* (IL-2 secreted)
B10.D2 DBN2
d d
+
16CI
C51BLl6 D1.LP
b b
+
6CI
-
-
BxD 2 BxD 14 BxD 8
b
+ +
33 87
Strain
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~~~
Concanavalin A
b b
-
-
-
-
160
*Stimulation assays were performed as described by Woodland, et al. [33]. Responses are presented as units/ml IL-2. Responses which were below detection threshold (< 10 Ulml) are shown as -.
integrant. These data suggest that Etc-1 is able to bind I-A molecules and stimulate at least some T cells bearing appropriate Tcr. Thus, the correlation between the clonal deletion of Vp5+ and Vp11+ T cells and I-E expression cannot be due to a failure of Etc-1 to bind to I-A. One possible explanation may be that the recognition of the I-Msuper-antigen complex by Vp5+ and Vp11+ T cells requires contributions from other components of the Tcr (e.g. the Tcr a chain) and that very few cells express the appropriate receptors. Clonal deletion of only a small percent of cells would not be readily noticed. There is additional evidence that T cells distinguish between super-antigens presented on different class I1 molecules. First, we have found that Mls-1-reactive VP7+ T cells were clonally deleted in H-2d, but not H-2b, B x D strains of mice (D.L.W., M. I? Happ and E. Palmer, unpublished observations) [17]. In other words, these T cells were unable to recognize MIS-1 presented in the context of I-Ab molecules. This was not due to the failure of I-Ab molecules to present Mls-1 because Vp6+ T cells were efficiently deleted in the H-2b strains. Second, we have analyzed the response of Vp17+ hybridomas to the Etc-1 superantigen presented on B cell Mtv-9 3’LTR transfectants and shown that the hybridomas were able to distinguish between Etc-1 presented on different class I1 molecules [ 5 5 ] . For example, whereas some hybridomas recognized Etc-1 exclusively in the context of I-Ed, others recognized Etc-1 only in the context of I-Ek. A third group of hybridomas recognized Etc-1 in the context of both I-Ed and I-Ek. A summary of the reactivity of these hybridomas is presented in Table VI. (Interestingly, some of the Vf517+ hybridomas were also able to recognize Etc-1 presented in the context of I-Ak. This is consistent with the observations of Kappler et al. that genetic loci tightly linked to I-E were involved in the deletion of some Vf517+ T cells [75].) Third, several groups have shown that T cells can distinguish between bacterial super-antigens presented by different class I1 molecules [76-781. Taken together, these data do not support a simple bridging model for super-antigen-mediated T cell stimulation and argue for a more intimate role of class I1 in the interaction. The simplest model to explain these observations is that there is a direct interaction between the Tcr and the class IYpeptide complex during super-antigen-mediated T cell activation [ 5 5 , 77, 781. In other words, non-Vp elements of the Tcr might enhance or destabilize the interaction,
D. L. WOODLAND AND M. A. BLACKMAN
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TABLE VI
MHC Restricted Recognition of Etc-1 by a Panel of T Cell Hybridomas
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Restriction* Hybridomat
V$
Ek
Ed
KE8
5.1
11-40
11
5447 4443.5
12 12
+ + + +
2B23-10 2B23-11 2B23-12 2B23-40 2B23-53 2B23-18
17 17 17 17 17 17
+ + + + + + +
-
+
-
-
+ + +
*MHC restriction was determined by measuring the response of hybridomas presenting Etc-1 in the context of I-Ek (CHl2.LBK) or I-Ed (A20/2). The symbols + and - indicate the presence or absence of a response. +Allhybridomas and transfectants have been described elsewhere [33, 551.
depending on the haplotype of the class I1 molecule. There is some evidence to support the view that non-vp elements of the Tcr contribute to super-antigen reactivity [79]. For example, we have analyzed a Vp8.1+ Tcr p transgenic mouse which expresses the Tcr Vp8. 1 p transgene in association with a full repertoire of Tcr a chains [80]. Although this Vp8.l element is associated with reactivity to the super-antigen, Mls-1 (Mtv-7), the data suggested that the Tcr 01 chain had a drastic effect on the ability of T cells bearing the transgenic f3 chain to recognize Mls-I. The precise role of the a chain in the recognition of Mls-1 in this system is not clear. It is possible that this chain directly contacts the Mls molecule or the class IVpeptide complex. Alternatively, the 01 chain may indirectly affect Mls-1 reactivity by inducing a slight conformational twist in the p chain [81].
RETROVIRAL SUPER-ANTIGENS AND THE T CELL REPERTOIRE The primary target of infectious MMTV is the mammary gland. During lactation, enormous quantities of infectious virus are produced and transmitted via the milk to the pups. The virus then finds its way from the gut to the mammary gland where the cycle is repeated. Occasionally, the virus, along with its super-antigen gene, becomes integrated into the murine germline and expressed in permissive tissues [82]. Endogenous expression of retroviral super-antigens in mice has a dramatic effect on the T cell repertoire because they behave as self antigens and mediate the clonal deletion of large numbers of T cells during ontogeny. Analysis of both wild and inbred strains of mice has revealed that any one individual may carry up to ten or more proviral integrants which may result in the elimination of more than 50% of the entire T cell repertoire [54, 831. AchaOrbea and Palmer have suggested that super-antigen-mediated clonal deletion of T cells by endogenous MMTV is an immunological accident, and that the mouse survives this because of the redundancy of the T cell repertoire [28]. On the other hand, it has been
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RETROVIRAL SUPER-ANTIGENS
323
proposed that endogenous super-antigen expression may confer a selective advantage on the mouse. For example, some Vp elements have been associated with autoimmune diseases and it has been suggested that the deletion of T cells bearing these Vp elements may confer a degree of protection from these diseases at the expense of repertoire diversity [84]. This would be especially significant if some autoimmune diseases were initiated by bacterial or viral super-antigens in the first place [85]. Alternatively, integrated MMTV proviruses may serve to protect the animal from the toxic effects of bacterial super-antigens, as has been suggested by Marrack et al. [6]. Indirect support for the idea that endogenous super-antigen genes confer a selective advantage in mice comes from two observations. First, the MMTV 3’0RFgenes, which encode the super-antigens,remain functional even when other genes in the virus are heavily mutated or deleted. The preservation of gene structure suggests that these genes are selectively maintained in the mouse genome. Second, both inbred and wild mice have been shown to maintain structural deletions of up to 50% of Vp genes in the Tcr p locus [83]. It is conceivable that this represents an alternative strategy for eliminating sets of potentially harmful Vp elements from the T cell repertoire. The advantage of retroviral (or bacterial) super-antigens for the microorganism is far from clear. These molecules presumably confer some selective advantage on the virus, although it is not immediately obvious how the non-specific stimulation of large numbers of T cells would benefit the virus. It has been suggested that the massive production of inflammatory lymphokines induced by super-antigen stimulated T cells may temporarily suppress the immune system at a critical stage of infection [4]. An alternative hypothesis is that B and T cells play a crucial role in the life cycle of the virus and that co-stimulation of these cellular subsets by super-antigens may enhance infection [62]. Infected B and T cells could benefit the viral life cycle by providing a reservoir of infectious virions which could enhance infection of the target organ, the mammary gland.
SUPER-ANTIGENSIN OTHER VIRUSES? The identification of endogenous super-antigens as retroviral gene products has, in part, resolved a puzzle that has perplexed immunologists for some time. However, it has also raised a number of questions. For example, why do these viruses express super-antigens and why do mice tolerate endogenous super-antigens?Another question is whether viruses other than MMTV carry super-antigen genes? On this last point, there are several exciting leads suggesting that viral super-antigens are a relatively common phenomenon. Hugin et al. have described a super-antigen which is probably encoded by a defective murine leukemia virus (MuLV) and which interacts with T cells expressing Vp5 elements [86]. Antibody inhibition studies have suggested that the gag protein of MuLV is responsible for these super-antigenic properties. More recently, work by Imberti et al. has suggested that the human immunodeficiency virus responsible for AIDS expresses a super-antigen which mediates the peripheral elimination of several families of T cells [87]. These authors and others have suggested that the depletion of CD4+ T cells in AIDS patients is mediated by a super-antigen which is constantly mutating and changing its Vp specificity [8, 871. Thomson and Nicholas have noted that the herpesvirus saimiri of primates encodes a gene homologous to the MMTV ORF [88]. Finally, Acha-Orbea and Palmer have suggested that a super-antigen may be involved in the pathogenic effects of the milk borne caprine encephalomyelitis virus which causes arthritis and encephalitis in goats [28]. The recent identification of viral super-antigens has provided exciting new insights into
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the workings of the immune system and raised a great many questions regarding the biological significance of super-antigens in general. In addition, the possibility that super-antigens may play a role in the pathogenesis of several viral-mediated diseases and autoimmunity will make these molecules the subject of intense study over the next few years. Acknowledgments
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The authors wish to thank Phuong Nguyen and Shem Surman for excellent technical assistance and Drs. Ron B. Corley and Mary Pat Happ for critically reviewing the manuscript. This work was supported by Cancer Center Support Grant CA21765 (D.L.W.), NIH grant AI31489 (M.A.B.), and a Bristol-Myers Cancer Grant Award (M.A.B.).
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Retroviral Super-Antigens and T Cells DAVID L. WOODLAND and MARCIA A. BLACKMAN Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA (Received December 16, 1991)
For many years immunologists have been intrigued by a series of potent antigens encoded in the murine genome. These antigens, originally termed minor lymphocyte stimulating (Mls) antigens, are capable of inducing extremely strong T cell proliferative responses when presented in the context of MHC class I1 molecules. Recently, Mls antigens have been shown to stimulate T cells bearing particular T cell receptor Vp elements, leading to the designation of super-antigens. The endogenous expression of these super-antigens in mice results in the clonal elimination of large numbers of T cells in order to maintain self-tolerance. In this review we discuss the recent identification of endogenous super-antigens as retroviral gene products. In addition, we analyze the role of class I1 MHC molecules in the presentation of endogenous super-antigens to T cells. Finally, we discuss the dramatic effect of retroviral super-antigens on the T cell repertoire. KEYWORDS: T cell, T cell receptor, super-antigen, MMTY Mls
INTRODUCTION T cells recognize foreign antigens which have been degraded into small peptides and presented on the cell surface complexed with Major Histocompatibility Complex (MHC) molecules. The T cell antigen receptor (Tcr) is thought to contact both the foreign antigen fragment and the self MHC molecule in this complex. Typically, less than one in 105 T cells bear the appropriate combination of T cell receptor elements required to interact with any given MHC/peptide complex [l, 21. Recently, a separate class of T cell antigens, termed super-antigens, has been described [3, 41. Super-antigens are distinguished from conventional antigens in three fundamental ways. First, they stimulate T cells with high frequency (usually one in ten T cells respond to a super-antigen). Second, T cell recognition of superantigens is mediated primarily through the Vp element of the Tcr and essentially independent of other elements of the Tcr. Since only a few Vp genes are present in the gene pool, each Vp element is represented at a relatively high frequency among T cells. Thus, a superantigen which interacts with several Vp elements is capable of stimulating a large proportion of the T cell repertoire. Third, super-antigens do not exhibit classical MHC restriction. Although they require presentation on class I1 molecules, they are promiscuous in terms of their haplotype specificity. Two classes of super-antigen have been described-bacterial and endogenous. The bacterial super-antigens are toxins secreted by several species of Gram positive bacteria, including Staphylococcus aureus and Streptococcus pyogenes . These toxins are responsible for a variety of diseases in man and animal, including toxic shock and food poisoning [ 5 ] . It 31 1
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has been shown that at least some of the pathogenic effects of these toxins are mediated by the hyper-stimulation of T cells [6]. The endogenous super-antigens are encoded by genes present in the genetic backgrounds of some strains of mice. Since endogenous superantigens are expressed as self antigens during T cell ontogeny, they have a profound effect on the T cell repertoire due to the clonal elimination of large numbers of potentially autoreactive T cells [7]. Endogenous super-antigens have recently been shown to be encoded by retroviruses [8].
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MULTIPLE ENDOGENOUS SUPER-ANTIGENSARE EXPRESSED IN MICE The Minor Lymphocyte Stimulating (Mls) antigens were originally characterized by their ability to induce extremely strong proliferative responses between MHC-matched murine lymphocytes [9, lo]. Analysis of proliferative responses between Mls-expressing and nonexpressing strains of mice revealed that the responding population was comprised predominantly of T cells, and that the major Mls-presenting cells were B cells. The stimulation of T cells was dependent on the expression of MHC class 11molecules on the presenting cells, but was promiscuous in terms of MHC haplotype 111-141. Despite the strong antigenicity of these gene products, it has proven to be surprisingly difficult to generate antibodies specific for them and to biochemically characterize them. Recently, several groups have established that the Mls series of antigens are in fact endogenous super-antigens which stimulate T cells bearing certain Vp elements. For example, the MIS-1 super-antigen, encoded on chromosome 1 of several strains of mice, stimulates the majority of T cells expressing Vp6+, Vp7+, Vp8.l+ and Vp9+ elements [13, 15-18]. In Mls-1-expressing strains of mice this antigen acts as a self molecule and mediates the clonal elimination of these subsets during T cell ontogeny in the thymus [13]. The elimination of these cells renders the animal tolerant to Mls-1, but at the expense of T cell diversity. Several Mls super-antigens have been described. Each of these super-antigens has essentially the same characteristics as Mls-1 described above, except that the Vp specificity varies [4]. The expression of several Mls genes in a given strain of mouse can result in a profound reduction in the T cell repertoire. In addition to the Mls family of super-antigens, studies from several laboratories have reported the existence of a second type of endogenous super-antigen which functions in association with MHC I-E molecules. The I-E associated super-antigens are discussed below.
ENDOGENOUS SUPER-ANTIGENSMEDIATE THE CLONAL ELIMINATION OF I-E REACTIVE T CELLS In 1987, Kappler et al., demonstrated that T cells bearing Vp17 elements of the Tcr tended to recognize I-E molecules, and that Vp17+ T cells were clonally deleted in the thymuses of I-E+ strains of mice [19,20]. This reactivity was apparently independent of other elements of the Tcr and was not self-MHC restricted, in that different alleles I-E were able to stimulate Vp17+ hybridomas. In later studies, several laboratories generated antibodies specific for Vp5+ and Vp11+ T cells and demonstratedthat these T cells were also clonally eliminated in most strains of mice which express functional I-E molecules [21-231. In addition, various other studies have suggested that Vp12+, Vp16+, Vp19a+, and Vp20+ T
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cells are I-E reactive, although there are currently no antibodies available to confirm these observations [24-291. A role for an endogenous super-antigen in the interaction between I-E molecules and I-Ereactive T cells was suggested by several observations. First, Marrack and Kappler demonstrated that Vp17+ hybridomas responded to I-E presented on B cell lines, but not macrophage lines, implicating a role for a B cell-specific gene product [30]. Second, studies by Tomonari’s laboratory suggested that not all I-E+ strains of mice clonally eliminated I-E reactive T cells [21]. For example, the C58 strain of mouse (I-Ek) expresses 10.8% VpII+ T cells, a level comparable to that found in I-E-negative strains of mice. Backcross analyses with this strain revealed a requirement for non-MHC gene products in the deletion of Vp11+ T cells [31,32]. Finally, Bill et al., and Woodland et al., demonstrated that several strains of recombinant inbred mice express high frequencies of V95+ and Vp11’- T cells, despite the fact that they express I-E molecules [22, 331. Taken together, these studies illustrated that I-E molecules were necessary, but not sufficient, to mediate the clonal deletion of these T cells and implicated a role for one or more I-E-associated super-antigens. In order to identify the super-antigens involved in the clonal deletion of I-E reactive T cells, Woodland et al. performed a detailed analysis of Vp5+ and VPll+ expression in C57BL/6 X DBN2 (BXD) recombinant inbred strains of mice [33, 34). Approximately half of the B X D strains had inherited the H-2b MHC locus from the C57BL/6 parent and expressed high frequencies of Vp5+ and VPll+ T cells (approximately 6-8%), typical of the C57BL/6 parent and consistent with the fact that the H-2b locus does not encode a functional I-E molecule. The remaining B X D strains expressed the I-Ed molecule encoded by the H-2d locus inherited from the DBN2 parent. The frequencies of Vp5+ and Vp11+ T cells in these strains varied widely from 1280t
>I280 11280
6 8
6
320 1280 160
2 8 13
80 320 80
11-40
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kMlsl3-11 bbml8- 19 bbml8- 109 3HB113 2HB79 5426
>1280
A20/2J
Etc- 1+ 1B4
lHlO
2C3
> I280
>1280 160
160
>1280 1280 >1280
*AU of the hybridomas, B cell lines and transfectantshave been described elsewhere [24,40,91]. LBB-1 is a B cell lymphoma which expresses MIS-1 and Etc-I. kMlsl3-11, bbm18-19 and bbm18-109 have all been shown to be strongly Mls-1 reactive. tHybridoma responses are presented as unitdml IL-2. Responses which were below detection threshold (< 10 U/ml)are shown as -. Stimulation assays were performed as described previously [33]. Xoncanavalin A was added to the responding hybridomas at 2pg/ml.
(controls)did not stimulate KE8. Table IV shows that the stimulation was specific for Vp5+ and Vp11+ Etc-1-reactive hybridomas. No IL-2 was produced by the hybridomas expressing non-Etc-1-reactive Vp elements, Vp2, 6, 8 and 13. These data confirmed that endogenous super-antigen expression, like that of infectious virus, is also controlled by the ORFs of integrated MMTV viruses. Surprisingly, transfectants generated with Mtv-8 constructs also stimulated the Vp5 + KE8 hybridoma (Table 111). This result was unexpected because there was no genetic evidence linking the deletion of Vp5+ T cells with the presence of the Mtv-8 integrant. It is currently unclear why this is the case. One possible explanation is that Mtv-8 is normally poorly expressed in most strains of mice and the observation that the Mtv-8 locus is heavily methylated in some strains is consistent with this [53]. An alternative explanation is that only a small fraction of VP5+ T cells recognize the Mtv-8 super-antigen, perhaps depending on contributions from the Tcr a chain (see below). Thus the deletion of a small fraction of Vp5+ T cells may not have been noticed, especially since Mtv-8 is present in almost all inbred strains of mice [54]. Studies by several groups have shown that Vp12+, Vp16+ and Vp17+ T cells are clonally eliminated in strains of mice which express functional I-E molecules [19, 20, 25-27]. The availability of MMTV transfectants made it possible to investigate whether this I-E reactivity is mediated by Etc-1. T cell hybridomas expressing Vp12, Vp16 and Vp17 elements were tested for their ability to recognize Etc-1 presented by B cell lines (A20/2J and CH12.LBK) transfected with Mtv-9 ORF DNA. The majority of Vp12+ and Vp17+ hybridomas responded to the transfectants (but not the parental cell lines), demonstrating that Etc-1 interacts with T cells bearing these Vps 140,551. In contrast, none of the Vp16+ hybridomas responded to the Etc-l+ transfectants. However, this result is inconclusive because most of the Vp16+ hybridomas did not express CD4 which is normally required for in vitro responses to Etc-1 (DLW, unpublished observations). In similar studies it has been shown that at least some Vp17+ hybridomas respond to the Mtv-8 encoded super-antigen [40].
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More recently, other endogenous Mtv 3’0RF genes have been successfully transfected into appropriate recipient cells. For example, the transfection of B cell lines with Mtv-7 3’LTR genes resulted in the expression of Mls-1 (personal communication, B. Hubex) and the transfection of B cell lines with Mtv-6 3’LTR resulted in the expression of Mls-3 (personal communication, A. Pullen).
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THE STRUCTURE OF RETROVIRAL SUPER-ANTIGENS The identification of endogenous super-antigens as retroviral gene products should allow the structure and function of these molecules to be rapidly determined. An examination of the known MMTV 3’LTR DNA sequences reveals several features [43]. First, there are several potential methionine translation starts which could result in the expression of proteins ranging in size from 37-39 kD. Second, a comparison between the known aminoacid sequences of MMTV ORFs reveals only 10-15% variation and suggests that the C-terminal region may control Vp specificity. Third, a 23 amino-acid hydrophobic region near the amino terminus suggests that the super-antigen is an integral membrane protein. Finally, there are multiple potential glycosylation sites. Recently, Choi et al. and Knight et al. have shown that MMTV super-antigen genes encode integral membrane glycoproteins which have the carboxy terminus exposed on the outside of the cell (type 11) [56, 571. This correlates with the suggestion that the carboxy terminus controls Vp specificity. Functional super-antigen is translated from the first methionine start codon and the protein is glycosylated at 4/5 potential sites.
REGULATED EXPRESSION OF RETROVIRAL SUPER-ANTIGENS The majority of data suggests that retroviral super-antigens are expressed predominantly on the surface of B cells [58-621. However, it is not clear how this expression is regulated during B cell development. Woodland et al. and Gollob and Palmer have shown that a 24 hour pre-treatment of splenic B cells with LPS and IL-4 greatly enhances Etc-1 expression [33, 611. The enhanced presentation of Etc-1 is not due simply to the increased levels of class I1 molecules, since IL-4 alone (which also upregulates surface class II), enhances Etc-1 expression only weakly [61]. If the LPS/IL-4 pretreatment is extended beyond 24 hours, the expression of Etc-1 drops off quickly despite the fact that I-E expression remains elevated for some time (Woodland and Blackman, unpublished observations). Corley and colleagues have measured the level of Mtv-9 transcription and Etc-1 cell-surface expression upon activation of two B cell lines, CH12 and BCL-1, and shown that various combinations of lymphokines regulate Etc-1 expression [62, 631. The expression of Etc-1 under these conditions generally correlated with the density of class I1 molecules on the surface of the presenting cell, suggesting that the regulation is at the level of class I1 expression. However, treatment of CH12 and BCL-1 with LPS resulted in the virtual elimination of super-antigen activity, despite the fact that Mtv-9 transcription was strongly upregulated and surface class U density was only moderately reduced. Since LPS is thought to drive these cells to a more mature phenotype, Lund et al. have suggested a model in which super-antigens can be posttranscriptionallyregulated by (i) class 11expression and (ii) an unknown mechanism which is correlated with B cell development [63]. This model is consistent with the finding that LPS treatment of normal spleen cells induces the transient expression of Etc-1.
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An alternative mechanism for regulating the expression of retroviral super-antigens has been suggested from studies in which multiple super-antigens were expressed in B cell tumor lines [40]. The A20/2Jline contains the proviral integrants Mtv-6, Mtv-8 and Mtv-9, but northern analysis has revealed that only the Mtv-6 locus is transcriptionallyactive. This line also functionally expresses the Mtv-6 linked super-antigen and stimulates Vp3+ hybridomas. Transfection of A20/2J with the Mtv-9 ORF gene (to convert it into an Etc-I+ line) resulted in a substantial reduction in the ability of A20/2J to stimulate Vp3+ T cells. These observationssuggest that endogenous super-antigens may under some circumstances compete with each another for functional expression.
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THE ROLE OF MHC CLASS II MOLECULES? Several models have been proposed to account for the interaction between T cells and retroviral super-antigens. However, current data is consistent with the model originally proposed by Janeway in which the super-antigen acts as a cross-linking molecule by binding directly to the Tcr and class II MHC molecule [64]. This model is based partly on analogy with bacterial super-antigens and partly on a molecular analysis of super-antigen/Tcr interaction. First, experiments with the functionally analogous, bacterial super-antigens have demonstratedthat the toxins bind directly to class II molecules and are fully functional in association with fixed presenting cells, indicating that no antigen processing is required for presentation [4, 65-67]. Second, in vitro mutagenesis of bacterial toxins has identified amino-acid residues which are involved in the interaction with either the Tcr or the class I1 molecule (J. Kappler, personal communication) [68, 691. Third, mutational analyses have revealed sites on both the human and the murine Tcr which seem to be involved in binding both bacterial and retroviral super-antigens, but which are distinct from the MHC/peptide binding site [70-731. For the purposes of this review we will discuss data in the context of the Janeway model; however, it should be borne in mind that this model remains hypothetical. Class II molecules vary significantlyin their ability to present retroviral super-antigens to T cells. For example, several super-antigens (such as Etc-1 encoded by the Mtv-9 OW) appear to be presented exclusively in association with I-E molecules [23]. Similarly, class I1 molecules vary in their ability to present Mls-1 to T cells (H-2k and H-2d are generally the strongest and H-2s the weakest) [13, 741. In terms of the Janeway model, the simplest explanation for these data is that different class II molecules vary in their ability to bind, and thus present, retroviral super-antigens to T cells. Thus, the I-E restriction of Etc-1 would be due to the inability of this super-antigen to bind to I-A. However, recent data suggest that this explanation is invalid. We have analyzed the response of an Etc-1 (Mtv-9 OW) reactive hybridoma, 5412 [33,34], to spleen cells from a number of different strains of mice (Table V). This hybridoma clearly responded to Etc-1 presented in the context of I-E molecules from several different haplotypes. However, this hybridoma also responded to C57BW6 presentor cells which express I-Ab but not I-E molecules and this response could be inhibited by antibodies specific for I-A (D.L. W and E. palmer, unpublished observations). This reactivity was not a generic I-Ab alloreactivity since not all H-2b spleen cells stimulated 5412. For example, the D1.LP strain (H-2b on a DBA/2 genetic background) does not stimulate 5Q12 suggesting a role for a non-MHC gene product. In order to investigate this further, we analyzed the ability of H-2b B x D FU strains to stimulate 5Q12 (Table V). Although only a limited number of strains have been analyzed, there is a perfect correlation between the stimulation of 5Q12 and the presence of the Mtv-9 retroviral
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TABLE V Response of the Etc-1 Reactive Hybndoma, 5412, to Spleen Cells from Recombinant Inbred Strains of Mice ~~
MHC
MW-9
5412 response* (IL-2 secreted)
B10.D2 DBN2
d d
+
16CI
C51BLl6 D1.LP
b b
+
6CI
-
-
BxD 2 BxD 14 BxD 8
b
+ +
33 87
Strain
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~~~
Concanavalin A
b b
-
-
-
-
160
*Stimulation assays were performed as described by Woodland, et al. [33]. Responses are presented as units/ml IL-2. Responses which were below detection threshold (< 10 Ulml) are shown as -.
integrant. These data suggest that Etc-1 is able to bind I-A molecules and stimulate at least some T cells bearing appropriate Tcr. Thus, the correlation between the clonal deletion of Vp5+ and Vp11+ T cells and I-E expression cannot be due to a failure of Etc-1 to bind to I-A. One possible explanation may be that the recognition of the I-Msuper-antigen complex by Vp5+ and Vp11+ T cells requires contributions from other components of the Tcr (e.g. the Tcr a chain) and that very few cells express the appropriate receptors. Clonal deletion of only a small percent of cells would not be readily noticed. There is additional evidence that T cells distinguish between super-antigens presented on different class I1 molecules. First, we have found that Mls-1-reactive VP7+ T cells were clonally deleted in H-2d, but not H-2b, B x D strains of mice (D.L.W., M. I? Happ and E. Palmer, unpublished observations) [17]. In other words, these T cells were unable to recognize MIS-1 presented in the context of I-Ab molecules. This was not due to the failure of I-Ab molecules to present Mls-1 because Vp6+ T cells were efficiently deleted in the H-2b strains. Second, we have analyzed the response of Vp17+ hybridomas to the Etc-1 superantigen presented on B cell Mtv-9 3’LTR transfectants and shown that the hybridomas were able to distinguish between Etc-1 presented on different class I1 molecules [ 5 5 ] . For example, whereas some hybridomas recognized Etc-1 exclusively in the context of I-Ed, others recognized Etc-1 only in the context of I-Ek. A third group of hybridomas recognized Etc-1 in the context of both I-Ed and I-Ek. A summary of the reactivity of these hybridomas is presented in Table VI. (Interestingly, some of the Vf517+ hybridomas were also able to recognize Etc-1 presented in the context of I-Ak. This is consistent with the observations of Kappler et al. that genetic loci tightly linked to I-E were involved in the deletion of some Vf517+ T cells [75].) Third, several groups have shown that T cells can distinguish between bacterial super-antigens presented by different class I1 molecules [76-781. Taken together, these data do not support a simple bridging model for super-antigen-mediated T cell stimulation and argue for a more intimate role of class I1 in the interaction. The simplest model to explain these observations is that there is a direct interaction between the Tcr and the class IYpeptide complex during super-antigen-mediated T cell activation [ 5 5 , 77, 781. In other words, non-Vp elements of the Tcr might enhance or destabilize the interaction,
D. L. WOODLAND AND M. A. BLACKMAN
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TABLE VI
MHC Restricted Recognition of Etc-1 by a Panel of T Cell Hybridomas
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Restriction* Hybridomat
V$
Ek
Ed
KE8
5.1
11-40
11
5447 4443.5
12 12
+ + + +
2B23-10 2B23-11 2B23-12 2B23-40 2B23-53 2B23-18
17 17 17 17 17 17
+ + + + + + +
-
+
-
-
+ + +
*MHC restriction was determined by measuring the response of hybridomas presenting Etc-1 in the context of I-Ek (CHl2.LBK) or I-Ed (A20/2). The symbols + and - indicate the presence or absence of a response. +Allhybridomas and transfectants have been described elsewhere [33, 551.
depending on the haplotype of the class I1 molecule. There is some evidence to support the view that non-vp elements of the Tcr contribute to super-antigen reactivity [79]. For example, we have analyzed a Vp8.1+ Tcr p transgenic mouse which expresses the Tcr Vp8. 1 p transgene in association with a full repertoire of Tcr a chains [80]. Although this Vp8.l element is associated with reactivity to the super-antigen, Mls-1 (Mtv-7), the data suggested that the Tcr 01 chain had a drastic effect on the ability of T cells bearing the transgenic f3 chain to recognize Mls-I. The precise role of the a chain in the recognition of Mls-1 in this system is not clear. It is possible that this chain directly contacts the Mls molecule or the class IVpeptide complex. Alternatively, the 01 chain may indirectly affect Mls-1 reactivity by inducing a slight conformational twist in the p chain [81].
RETROVIRAL SUPER-ANTIGENS AND THE T CELL REPERTOIRE The primary target of infectious MMTV is the mammary gland. During lactation, enormous quantities of infectious virus are produced and transmitted via the milk to the pups. The virus then finds its way from the gut to the mammary gland where the cycle is repeated. Occasionally, the virus, along with its super-antigen gene, becomes integrated into the murine germline and expressed in permissive tissues [82]. Endogenous expression of retroviral super-antigens in mice has a dramatic effect on the T cell repertoire because they behave as self antigens and mediate the clonal deletion of large numbers of T cells during ontogeny. Analysis of both wild and inbred strains of mice has revealed that any one individual may carry up to ten or more proviral integrants which may result in the elimination of more than 50% of the entire T cell repertoire [54, 831. AchaOrbea and Palmer have suggested that super-antigen-mediated clonal deletion of T cells by endogenous MMTV is an immunological accident, and that the mouse survives this because of the redundancy of the T cell repertoire [28]. On the other hand, it has been
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proposed that endogenous super-antigen expression may confer a selective advantage on the mouse. For example, some Vp elements have been associated with autoimmune diseases and it has been suggested that the deletion of T cells bearing these Vp elements may confer a degree of protection from these diseases at the expense of repertoire diversity [84]. This would be especially significant if some autoimmune diseases were initiated by bacterial or viral super-antigens in the first place [85]. Alternatively, integrated MMTV proviruses may serve to protect the animal from the toxic effects of bacterial super-antigens, as has been suggested by Marrack et al. [6]. Indirect support for the idea that endogenous super-antigen genes confer a selective advantage in mice comes from two observations. First, the MMTV 3’0RFgenes, which encode the super-antigens,remain functional even when other genes in the virus are heavily mutated or deleted. The preservation of gene structure suggests that these genes are selectively maintained in the mouse genome. Second, both inbred and wild mice have been shown to maintain structural deletions of up to 50% of Vp genes in the Tcr p locus [83]. It is conceivable that this represents an alternative strategy for eliminating sets of potentially harmful Vp elements from the T cell repertoire. The advantage of retroviral (or bacterial) super-antigens for the microorganism is far from clear. These molecules presumably confer some selective advantage on the virus, although it is not immediately obvious how the non-specific stimulation of large numbers of T cells would benefit the virus. It has been suggested that the massive production of inflammatory lymphokines induced by super-antigen stimulated T cells may temporarily suppress the immune system at a critical stage of infection [4]. An alternative hypothesis is that B and T cells play a crucial role in the life cycle of the virus and that co-stimulation of these cellular subsets by super-antigens may enhance infection [62]. Infected B and T cells could benefit the viral life cycle by providing a reservoir of infectious virions which could enhance infection of the target organ, the mammary gland.
SUPER-ANTIGENSIN OTHER VIRUSES? The identification of endogenous super-antigens as retroviral gene products has, in part, resolved a puzzle that has perplexed immunologists for some time. However, it has also raised a number of questions. For example, why do these viruses express super-antigens and why do mice tolerate endogenous super-antigens?Another question is whether viruses other than MMTV carry super-antigen genes? On this last point, there are several exciting leads suggesting that viral super-antigens are a relatively common phenomenon. Hugin et al. have described a super-antigen which is probably encoded by a defective murine leukemia virus (MuLV) and which interacts with T cells expressing Vp5 elements [86]. Antibody inhibition studies have suggested that the gag protein of MuLV is responsible for these super-antigenic properties. More recently, work by Imberti et al. has suggested that the human immunodeficiency virus responsible for AIDS expresses a super-antigen which mediates the peripheral elimination of several families of T cells [87]. These authors and others have suggested that the depletion of CD4+ T cells in AIDS patients is mediated by a super-antigen which is constantly mutating and changing its Vp specificity [8, 871. Thomson and Nicholas have noted that the herpesvirus saimiri of primates encodes a gene homologous to the MMTV ORF [88]. Finally, Acha-Orbea and Palmer have suggested that a super-antigen may be involved in the pathogenic effects of the milk borne caprine encephalomyelitis virus which causes arthritis and encephalitis in goats [28]. The recent identification of viral super-antigens has provided exciting new insights into
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the workings of the immune system and raised a great many questions regarding the biological significance of super-antigens in general. In addition, the possibility that super-antigens may play a role in the pathogenesis of several viral-mediated diseases and autoimmunity will make these molecules the subject of intense study over the next few years. Acknowledgments
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The authors wish to thank Phuong Nguyen and Shem Surman for excellent technical assistance and Drs. Ron B. Corley and Mary Pat Happ for critically reviewing the manuscript. This work was supported by Cancer Center Support Grant CA21765 (D.L.W.), NIH grant AI31489 (M.A.B.), and a Bristol-Myers Cancer Grant Award (M.A.B.).
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