The xid gene controls Ia.W39-associated immune response gene function.

Immune response (Ir) genes are encoded for by the I region of the major histocompatibility complex (MHC). A class of serologically defined specificities, Ia antigens, is also encoded for by genes within this region. A new Ia specificity, Ia.W39, has recently been defined. It is private for I-Ab and its expression is controlled by a gene on the X-chromosome. Using different approaches, the role of Ia.W39 in the immune response of H-2b mice to beef insulin was examined in a macrophage-dependent T cell proliferation assay. It was found that beef insulin-related Ir gene function was associated with the expression of Ia.W39 by antigen-presenting macrophages and that control of this Ir gene function was X-linked (xid gene).

A class II gene conversion event defines an antigen-specific Ir gene epitope.

To assess the role of Ia epitopes in conferring specificity for the immune response to nominal antigen, we compared the insulin response of mice with a defined mutation in the I-Ab beta gene, the B6.C-H-2bm12 (bm12), with that of wild-type H-2b C57BL/6 (B6) mice. We report that the bm 12 mutation resulted in a selective alteration of the specificity of insulin recognition, such that bm 12 mice responded upon immunization with sheep but not beef insulin, which differ by only one amino acid at position 9 of the insulin A chain. Thus, the bm12 mutation allows for the definition of the actual nucleotide sequence coding for an Ia epitope that is responsible for controlling the specificity of immune recognition of insulin. Furthermore, we show that the sheep insulin response of H-2k mice is controlled by the E molecule and that sheep insulin can be recognized by primed bm12 and H-2k T cells in the context of either bm12, B10.A, or B10.A(5R) antigen-presenting cells. Our data suggest that the mechanism for the bm12 mutation was the intergenic transfer of a hypervariable region in the first domain that is identical in the I-Abm12 beta, I-Eb beta, and I-Ek beta genes.

Production and characterization of an Mls-1-specific monoclonal antibody.

Superantigens (SAGs) represent a new class of antigens, characterized as T cell receptor (TCR) V beta-reactive elements. Bacterial toxins constitute the major group of exogenous SAGs, while the mouse mammary tumor virus (MMTV)-encoded Mls molecules represent the endogenous SAGs. Mls-1 is the prototype of the latter SAGs, because it elicits a very potent T cell stimulatory response in vitro in unprimed T cells expressing the TCR V beta 6 or 8.1 chains. In vivo, Mls-1 causes deletion of immature T cells bearing the V beta 6, 7, 8.1, or 9 chains. Although Mls-1 was functionally discovered > 20 yr ago, it has not been possible to raise antibodies against this molecule. We have previously cloned and sequenced the Mtv-7 sag gene, which encodes Mls-1. Sequence comparisons with other MMTV sag genes suggested that the polymorphic 3' end encodes the TCR V beta specificity of these SAGs. We have, therefore, immunized hamsters with a 14-amino acid peptide from the deduced COOH-terminal sequence of the Mtv-7 sag gene. We describe here the production of a monoclonal antibody (mAb), 3B12, which is peptide specific and reacts with a recombinant baculovirus product of Mtv-7 sag. This mAb blocks Mls-1-specific T cell recognition and detects the Mls-1 protein on the surface of the B cell hybridoma LBB.A, but not on LBB.11, which is an Mtv-7 loss variant of LBB.A. Transfection of the Mtv-7 sag gene into LBB.11 renders this cell functionally Mls-1+ as well as positive for 3B12 binding, confirming the specificity of this mAb. It is well documented that B cells and CD8+ T cells express T cell stimulatory Mls-1 determinants, and we show here that this functional profile correlates with the expression of MMTV-specific mRNA. However, primary lymphocytes derived from Mls-1+ mice do not stain with 3B12, even after in vitro activation with mitogens or phorbol ester.

Mls-1-like superantigen in the MA/MyJ mouse is encoded by a new mammary tumor provirus that is distinct from Mtv-7.

Mls-1 is an endogenous superantigen that leads to in vivo deletion and in vitro stimulation of T cell receptor (TCR) V beta 6-, 7-, 8.1-, and 9-expressing cells. The MA/MyJ mouse deletes the identical set of TCR from its mature T cell repertoire; however, it does not contain Mtv-7, the murine mammary tumor provirus (MMTV), whose sag gene encodes Mls-1. Interestingly, the superantigen activity of this mouse strain segregates with a new mammary tumor provirus, Mtv-43, not seen in other inbred strains. The predicted amino acid sequence of the sag gene of Mtv-43 was compared with that of Mtv-7. Strikingly, the COOH terminus of the two molecules is very similar, while all other MMTV-encoded superantigens differ 100% in this segment.

B cells are essential for murine mammary tumor virus transmission, but not for presentation of endogenous superantigens.

Murine mammary tumor viruses (MMTVs) are retroviruses that encode superantigens capable of stimulating T cells via superantigen-reactive T cell receptor V beta chains. MMTVs are transmitted to the suckling offspring through milk. Here we show that B cell-deficient mice foster nursed by virus-secreting mice do not transfer infectious MMTVs to their offspring. No MMTV proviruses could be detected in the spleen and mammary tissue of these mice, and no deletion of MMTV superantigen-reactive T cells occurred. By contrast, T cell deletion and positive selection due to endogenous MMTV superantigens occurred in B cell-deficient mice. We conclude that B cells are essential for the completion of the viral life cycle in vivo, but that endogenous MMTV superantigens can be presented by cell types other than B cells.

Cholera toxin discriminates between T helper 1 and 2 cells in T cell receptor-mediated activation: role of cAMP in T cell proliferation.

CD4+ T helper (Th) clones can be divided into interleukin 2 (IL-2)-secreting Th1 and IL-4-secreting Th2 cells. We show in the present report that these two Th subsets have different activation requirements for lymphokine production and proliferation: namely, cholera toxin (CT) as well as forskolin inhibit T cell receptor (TCR)-mediated IL-2 production and proliferation in Th1 cells, while the same reagents fail to block IL-4 production and proliferation in Th2 cells. In addition, CT and forskolin differentially influence the proto-oncogene mRNA expression in Th1 vs. Th2 cells after stimulation with Con A. Since both reagents lead to elevated levels of intracellular cAMP, it is likely that Th1 and Th2 cells differ in their sensitivity to an increase in cAMP. Our results indicate that the two Th subsets use different transmission signal pathways upon TCR-mediated activation.

Human T cells respond to mouse mammary tumor virus-encoded superantigen: V beta restriction and conserved evolutionary features.

Mouse mammary tumor virus (MMTV)-encoded superantigens (SAGs) influence the murine T cell repertoire and stimulate a strong mixed lymphocyte response in vitro. These SAGs are encoded by the open reading frame of the 3' long terminal repeat of MMTV, termed MMTV SAGs. The T cell response to MMTV SAGs is V beta restricted and requires expression of the class II molecules of the major histocompatibility complex (MHC) on the presenting cells. While human T cells respond to bacterial SAGs, it is not known if human T cells or human MHC class II molecules can interact with MMTV SAGs. A fibroblastic cell line expressing the human MHC class II molecule HLA-DR1 and the Mtv-7 sag gene encoding Mls-1 was used to stimulate human T cells. We show here that human T cells efficiently proliferate in response to Mls-1 presented by HLA-DR1. This T cell response was inhibited by mAbs directed against CD4 or MHC class II molecules but not by mAbs specific for CD8 or MHC class I molecules. Moreover, the response to Mls-1 was limited to human T cells expressing a restricted set of T cell receptor V beta chains. Human T cells expressing V beta 12, 13, 14, 15, and 23 were selectively amplified after Mtv-7 sag stimulation. Interestingly, these human V beta s share the highest degree of homology with the mouse V beta s interacting with Mls-1. These results show a strong evolutionary conservation of the structures required for the presentation and the response to retrovirally encoded endogenous SAGs, raising the possibility that similar elements operate in humans to shape the T cell repertoire.

Binding sites for bacterial and endogenous retroviral superantigens can be dissociated on major histocompatibility complex class II molecules.

Bacterial and retroviral superantigens (SAGs) interact with major histocompatibility complex (MHC) class II molecules and stimulate T cells upon binding to the V beta portion of the T cell receptor. Whereas both types of molecules exert similar effects on T cells, they have very different primary structures. Amino acids critical for the binding of bacterial toxins to class II molecules have been identified but little is known of the molecular interactions between class II and retroviral SAGs. To determine whether both types of superantigens interact with the same regions of MHC class II molecules, we have generated mutant HLA-DR molecules which have lost the capacity to bind three bacterial toxins (Staphylococcus aureus enterotoxin A [SEA], S. aureus enterotoxin B [SEB], and toxic shock syndrome toxin 1 [TSST-1]). Cells expressing these mutated class II molecules efficiently presented two retroviral SAGs (Mtv-9 and Mtv-7) to T cells while they were unable to present the bacterial SAGs. These results demonstrate that the binding sites for both types of SAGs can be dissociated.

An Epstein-Barr virus-associated superantigen.

More than 90% of adults are latently infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease characterized by extensive T cell activation. Reactivation of this herpesvirus during immunosuppression is often associated with oncogenesis. These considerations led us to analyze the early events that occur after exposure of the immune system to EBV. Strong major histocompatibility complex (MHC) class II-dependent but not MHC-restricted, T cell proliferation was observed in vitro in response to autologous, lytically infected EBV-transformed B cells. By measuring the appearance of the early activation marker CD69 on individual T cell V beta subsets, we could demonstrate selective activation of human V beta 13- T cells. This was confirmed with murine T cell hybridomas expressing various human BV genes. While EBV- Burkitt's lymphoma cells were nonstimulatory, they induced V beta-restricted T cell activation after EBV infection. EBV specific activation was also demonstrated in cord blood cells, excluding a recall-antigen response. Thus, all of the characteristics of a superantigen-stimulated response are seen, indicating that induction of the EBV lytic cycle is associated with the expression of a superantigen in B cells. A model is presented proposing a role for the superantigen in infection, latency, and oncogenesis.

Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis.

Treatment-resistant Lyme arthritis is associated with immune reactivity to outer surface protein A (OspA) of Borrelia burgdorferi, the agent of Lyme disease, and the major histocompatibility complex class II allele DRB1*0401. The immunodominant epitope of OspA for T helper cells was identified. A homology search revealed a peptide from human leukocyte function-associated antigen-1 (hLFA-1) as a candidate autoantigen. Individuals with treatment-resistant Lyme arthritis, but not other forms of arthritis, generated responses to OspA, hLFA-1, and their highly related peptide epitopes. Identification of the initiating bacterial antigen and a cross-reactive autoantigen may provide a model for development of autoimmune disease.

Superantigens. Gazing into the crystal ball.

The recently determined crystal structures of complexes between bacterial toxin 'superantigens' and MHC class II molecules shed light on the nature of the interactions between these two molecules.

Mls genes and self-superantigens.

The Mls gene products, which have long been known for their potent T-cell stimulatory function, have recently come of age through two pivotal discoveries, revealing that they act as superantigens and originate from retroviruses. In addition, recent experiments suggest that two retroviruses, the murine B-type mammary tumor virus and the human lentivirus HIV, make use of the T-cell stimulatory capacity of a virally encoded superantigen for facilitating viral replication.

Potentiation of the immune response in HIV-1+ individuals.

T cells from HIV-1+ individuals have a defect in mounting an antigen specific response. HIV-1 Tat has been implicated as the causative agent of this immunosuppression. We have previously shown that HIV-1 Tat inhibits antigen specific proliferation of normal T cells in vitro by binding to the accessory molecule CD26, a dipeptidase expressed on the surface of activated T cells. We now demonstrate that the defective in vitro recall antigen response in HIV-1 infected individuals can be restored by the addition of soluble CD26, probably by serving as a decoy receptor for HIV-1 Tat. The restored response is comparable to that of an HIV-1- individual, suggesting that early in HIV infection there is a block in the memory cell response, rather than deletion of these cells.

Molecular analysis of antigen recognition by insulin-specific T-cell hybridomas from B6 wild-type and bm12 mutant mice.

Molecular analysis of the heterodimeric T-cell antigen receptor of insulin-specific class II-restricted T-cell hybridomas (THys) derived from C57BL/6 (B6) wild-type and B6.C-H-2bm12 (bm12) mutant mice revealed that such T cells use a diverse V gene repertoire. Analysis of three THys that use related V genes, however, showed a number of novel features. Two THys that share major histocompatibility complex restriction use V alpha genes that are 98.6% homologous. Two THys sharing the same antigen fine specificity use a particular germ line V beta D beta J beta combination. A 21-base-pair deletion in the 5' segment of the J beta gene occurs in one THy, suggesting a novel mechanism for generating diversity in T-cell antigen receptor beta genes. The first amino acid encoded by N sequences at the V-D junction is conserved in a pair of T cells which recognize identical antigenic epitopes. The implications of these findings for the structural mechanisms underlying major histocompatibility complex-restricted antigen-specific T-cell recognition are discussed.

Cytokine rescue from glucocorticoid induced apoptosis in T cells is mediated through inhibition of IkappaBalpha.

We previously reported that dexamethasone (DEX), a synthetic glucocorticoid, causes apoptosis in mature Th cell lines, and that this induction of cell death is prevented by specific cytokines, namely, by IL-2 in Th1 cells and by IL-4 in Th2 cells. We now show that this differential rescue by specific cytokines in Th cells correlates with the level of IkappaBalpha that is regulated by DEX and cytokines. In both cell types the cellular levels of IkappaBalpha mRNA and protein were evaluated by DEX treatment. Interestingly, the DEX-mediated IkappaBalpha induction was completely inhibited by IL-2, but not IL-4, in Th1 cells, while the reverse profile was seen in Th2 cells. In both cell types, the cytokine that inhibits the induction of IkappaBalpha by DEX, also rescues these cells from DEX-induced apoptosis, although the rescue cytokine is different in Th1 and Th2 cells. Our results imply that T cells need to maintain a certain level of NF-kappaB transcriptional activity in order to survive; up- or down-regulation of nuclear NF kappaB through modulation of IkappaBalpha expression by cytokines or DEX may lead to cell survival or cell death, respectively.

Tyrosine protein phosphorylation is required for protein kinase C-mediated proliferation in T cells.

We have studied the role of tyrosine kinase in PMA-stimulated T cells. Protein kinase C (PKC)-mediated D10A cell proliferation is inhibited by the specific inhibitor of tyrosine kinase, tyrphostin. This inhibitor selectively blocks the mRNA expression of the proto-oncogene c-myc in response to the phorbol ester, PMA. On the other hand, the same doses of this inhibitor do not affect the mRNA expression of the proto-oncogene c-fos in PMA-stimulated D10A cells. Phorbol esters induce in this T cell line the tyrosine phosphorylation of a unique protein of 42 kDa and the enzyme PKC is required for this activity.

Molecular characterization of Mls-1.

Recently a series of endogenous and exogenous superantigens have been described which have one common feature, namely, they lead to in vivo deletion and in vitro stimulation of T cells expressing particular T cell receptor V beta genes. The Mls antigens represent the prototypes of these molecules. We have mapped Mls-1 to the endogenous mammary tumor virus (MMTV) Mtv-7, while other SAG have also been associated with various MMTV. The open reading frame gene of the MMTV encodes the SAG. Thus, the new terminology MMTV sag has been proposed for this gene. Transfection experiments suggest that the expression of MMTV sag is tightly controlled, probably by a negative acting factor encoded within the open reading frame. Furthermore, a pronounced IL-4 effect is seen in the functional detection of the transfected Mtv-7 sag. Since this lymphokine does not influence the mRNA level of the endogenous or transfected MMTV genes, it is likely that it exerts its effect by increasing transcription of MHC class II genes, whose products are required for functional detection of Mls. We have identified one mouse strain, MA/MyJ, which has an Mls-1 phenotype but does not contain Mtv-7. The SAG activity of this strain was mapped to a new mammary tumor provirus, Mtv-43, not seen in other inbred strains. Sequence analyses revealed that the predicted amino acid sequences of the Mtv-7 and the Mtv-43 sag genes are very similar. This is particularly striking in the C-terminus, where all other MMTV sag sequences differ 100%. Thus, this region of the molecule seems to control the V beta specificity of SAG molecules. It is likely that the SAG expression provides an advantage for the infectious MMTV, probably by facilitating its transmission by T cells from the site of primary residence in the gut to its final destination, the mammary glands.