Binding of staphylococcal enterotoxin A to purified murine MHC class II molecules in supported lipid bilayers

The staphylococcal enterotoxins are a family of bacterial toxins that are thought to exert their pathogenic effects by the massive activation of T lymphocytes to produce lymphokines. Activation of T cells by these toxins is dependent on MHC class II+ APC. Recent studies from a number of laboratories have implicated MHC class II proteins as the APC surface receptor for a number of the staphylococcal enterotoxins. The present report shows that staphylococcal enterotoxin A, (SEA) binds to the purified murine MHC class II molecule I-Ed reconstituted in supported planar membranes, indicating that no other cell surface proteins are required for SEA binding. The Kd for SEA binding to I-Ed was determined to be 3.5 +/- 1.6 x 10(-6) M. Specific binding of SEA to I-Ad was also observed, but the interaction was of significantly lower affinity. Binding of SEA to purified I-Ed was blocked by antibodies against both the alpha- and the beta-chain of the I-Ed molecule, but not by antibodies specific for an unrelated MHC class II protein. Binding of SEA to I-Ad was blocked by an A beta d but not by an A alpha d-specific antibody. Planar membranes containing only lipid and purified I-Ed molecules were sufficient for activation of a V beta 1 expressing T hybrid by SEA. The T cells responded to as few as 180 toxin molecules per T cell.     

DNA-mediated transfer of major histocompatibility class II I-Ab and I-Abm12 genes into B lymphoma cells: molecular and functional analysis of introduced antigens

A20.2J B lymphoma cells have been co-transfected with the A alpha b, A beta b or with the A alpha b, A beta bm12 and neomycin resistance genes. The transfected cell lines constitutively express the I-Ab or I-Abm12 class II molecules at a level comparable with that of the endogenous I-Ad antigen. The I-Ab antigens expressed on three independently transfected B cell clones (A20.Ab.1, A20.Ab.2, and A20.Ab.3) are serologically and functionally indistinguishable from the I-Ab molecules expressed by control H-2bxd B hybridoma cells (LB cells). These transfected cell lines were potent I region-restricted antigen-presenting cells to a large panel of antigen-specific, autoreactive and alloreactive T cell hybridomas, as well as normal T cell clones. There were not significant differences in the efficiency of antigen presentation by the Ia molecules encoded by the transfected, as compared with the endogenous, I-A genes. The expression of a functional I-Ab antigen on the surface of cells transfected with A beta bm12 and A alpha b genes is consistent with previous work that implicated the A beta-chain alone in the bm 12 mutation. Furthermore, because the transfected A20.Ab and A20.Abm12 cells display the serologic and functional properties of normal spleen cells from the wild-type and mutant mouse strains, respectively, it is clear that class II genes do not undergo unexpected and unpredictable alterations after transfection in this system. This system permits us to investigate the structural requirements for interactions between class II major histocompatibility complex antigens, a foreign antigen, and the T cell receptor by in vitro site-directed mutagenesis coupled with DNA-mediated gene transfer.     

Tissue-specific expression of allogeneic class II MHC molecules induces neither tissue rejection nor clonal inactivation of alloreactive T cells

To analyze the control of self tolerance to tissue-specific Ag, we have constructed C57BL/6 (H-2b) transgenic mice that express allogeneic class II (I-Ad) molecules exclusively on pancreatic islet cells. By a number of criteria, including I-Ad mRNA, and tissue and cell surface I-Ad protein levels, the islet cells appear to be expressing levels of I-Ad similar to B lymphocytes. Although one of the transgenic lines that expresses only the beta-chain occasionally displays slightly elevated glucose levels, this hyperglycemia is not enhanced when alpha and beta are coexpressed, allowing for cell surface I-Ad expression. None of the mice examined has demonstrated any autoimmune reaction to the I-Ad+ islet cells. Despite this apparent lack of recognition of the I-Ad+ islet cells, these animals demonstrate no reduction in the in vitro MLR generated to the same MHC molecule. Therefore, these mice remain functionally tolerant to the transgene product without inactivating those T cells that can recognize this same MHC molecule in vitro.     

T cells that recognize peptide sequences of self MHC class II molecules exist in syngeneic mice.

T cell reactivity toward self MHC class II molecules has been recognized in syngeneic MLR in a number of studies, where the T cells are believed to recognize the combination of self/nonself peptide and self MHC molecule. We investigated the stimulation of T cell proliferation by synthetic peptides of sequences corresponding to the first polymorphic amino terminal domain of alpha- and beta-chains of self I-A molecules. Both unprimed and primed T cells responded to a number of peptides of alpha 1 and beta 1 domains of self I-Ad molecules. The response was dependent on the presentation of I-Ad peptides by syngeneic APC and was blocked by anti-class II MHC mAb. Upon further investigation it was observed that I-Ad peptides could inhibit the stimulation of Ag-specific MHC class II-restricted T cell hybridoma due to self presentation of peptides rather than to direct binding of free peptides to the TCR, further supporting their affinity/interaction with intact self MHC class II molecules. The peptide I-A beta d 62-78 showed high affinity toward intact self MHC II molecule as determined by the inhibition of Ag-specific T cell stimulation and yet was nonstimulatory for syngeneic T cells, therefore representing an MHC determinant that may have induced self tolerance. Thus we have shown that strong T cell proliferative responses can be generated in normal mice against the peptides derived from self MHC class II molecules and these cells are part of the normal T cell repertoire. Therefore complete tolerance toward potentially powerful immunodominant but cryptic determinants of self Ag may not be necessary to prevent autoimmune diseases.     

Functional effects of N-linked oligosaccharides located on the external domain of murine class II molecules.

To evaluate the potential functional role of the alpha- and beta-chain N-linked oligosaccharides we used site-directed mutagenesis to construct class II Ak alpha and Ak beta genes that encode polypeptides with altered N-linked oligosaccharide acceptor sites in the N-terminal domain of both polypeptides. The alpha 1 domain acceptor site at positions 82 to 84 was eliminated by substituting Gln for Asn at position 82. The beta 1 domain acceptor site at positions 19 to 21 was deleted by substituting Gln for Asn at position 19 or Ala for Thr at position 21. The mutant genes (Ak alpha* or Ak beta*) were transfected either individually (mutants T.19, T.21, and T.82) or together (mutant T.82-21) into class II cell surface negative B lymphoma cell lines. Quantitative immunofluorescence with a panel of Ak beta- or Ak alpha- reactive mAb demonstrated that although the oligosaccharide-deleted Ak alpha Ak beta molecules were serologically wild type, the Ad alpha serologic epitope defined by mAb K24-199 was eliminated in both the T.19 and T.21 Ak beta* Ad alpha molecules. Cloned cell lines expressing the T.19 or T.21 Ak beta* Ak alpha molecules exhibited limited functional Ag presentation defects. Cells expressing the T.82 Ak alpha* Ak beta molecules exhibited defects in Ag presentation function to nine of the ten T hybridomas tested. Surprisingly, cells expressing the mutant T.82-21 class II molecule stimulated a response that was equal to the wild-type response from three of the nine T hybrids and a response that was significantly greater than that of wild-type cells from five of nine T hybridomas. These functional and serological analyses also indicate that some of the observed Ag presentation defects may be due to altered secondary structure caused by either deletion of the oligosaccharide or the amino acid substitution used to delete the N-linked oligosaccharide acceptor site.     

Diabetes and tolerance in transgenic mice expressing class II MHC molecules in pancreatic beta cells

Insulin-dependent diabetes is caused by the loss of insulin-producing beta cells in pancreatic islets. It has been proposed that aberrant expression of Class II Major Histocompatibility Complex (MHC) molecules on beta cells stimulates an autoimmune attack against beta cell antigens. To test this hypothesis, we generated transgenic mice that express Class II MHC molecules (E alpha d/E beta b, or I-Eb) on beta cells. Diabetes was found in 100% of transgenic progeny from three expressing transgenic mouse lines, but without evidence for lymphocytic infiltrates. Furthermore, T lymphocytes appeared to be tolerant to the transgene I-Eb molecule, despite the absence of expression of I-Eb in the thymus or any other lymphoid tissue. The results suggest that novel expression of Class II MHC molecules on nonlymphoid cells is by itself insufficient to initiate autoimmune responses against tissue-specific antigens.     

Formation of complexes between self-peptides and MHC class II molecules in cells defective for presentation of exogenous protein antigens.

A vertebrate immune response is initiated by the presentation of foreign protein Ag to MHC class II-restricted T lymphocytes by specialized APC. Presentation of self-peptides in association with MHC class II molecules is also necessary for the induction of T cell tolerance. It is important to understand whether functionally divergent APC are responsible for delivering these distinct signals to class II-restricted T cells. Here we examine the ability of I-Ad surface molecules expressed in diverse cell types to stimulate I-Ad-restricted T cells. Recipients included J558L myeloma cells and EL4 lymphoma cells expressing barely detectable or undetectable levels of Ii chain mRNA. This allowed us to examine the influence of Ii expression on the presentation of intracellular Ag and thus test the hypothesis that Ii chain is necessary to prevent access of self-peptides to newly synthesized class II molecules. Ii chain expression did not restore the ability of transformants to process and present soluble protein Ag. A striking result was the finding that cells showing a defect in the exogenous class II presentation pathway were capable of functioning as stimulators when they expressed intracellular secreted but not signal-less V-CH3b Ag. Thus, so-called professional APC that can capture and process exogenous protein Ag may express a specialized set of proteins not required for the presentation of self-peptides.     

Simultaneous expression of allogenic class II MHC and B7.1 (CD80) molecules in A20 B-lymphoma cell line enhances tumor immunogenicity

We expressed the allogenic class II MHC antigen and B7.1 (CD80) co-stimulatory molecule in A20 beta-lymphoma cells in order to test their efficacy as immuno-stimulating adjuvant agents in inducing tumor-specific immunity. The transduction of the allogenic I-Ab alpha and beta chain genes into A20 cell resulted in a surface expression of the allogenic class II MHC molecules. The expression of the allogenic class II MHC antigen (I-Ab) in A20 cells enhanced the proliferation of T cells in a mixed lymphocyte tumor culture and in vitro cytotoxic T lymphocyte (CTL) generation against parental cells. The B7.1 gene, which is known to be a potent co-stimulatory molecule, was also transduced and expressed in A20 cells, either alone or in combination with I-Ab. The B7.1 transduction alone leads to a similar in vitro immune enhancing effect as I-Ab. When both the I-Ab and B7.1 genes were transduced, the in vitro immunostimulating capacity was further enhanced. Finally, we also tested the A20 cells that were transduced with I-Ab and/or B7.1 for their efficacy as preventive tumor vaccines in vivo. The results indicate that the A20 cells that express both the I-Ab and B7.1 have more potent vaccinating potential, compared to the cells that express only one of the molecules.     

Structural requirements for the interaction between peptide antigens and I-Ed molecules

We have analyzed the structural characteristics of the interaction between I-Ed molecules and their peptide ligands. It was found that unrelated good I-Ed binders share structurally similar "core" regions that were experimentally demonstrated to be crucial for binding to I-Ed molecules. Single amino acid substitution analogues of one good I-Ed binder, hen egg lysozyme 107-116, were analyzed for their capacity to bind to I-Ed molecules and to activate two different I-Ed-restricted T cell hybridomas. The results illustrate the great permissiveness of I-Ed-peptide interaction and the great specificity of T cell recognition. It was concluded from these analyses that basic residues on the peptide molecule play a crucial role in binding to I-Ed. This contrasts with the structural requirements for binding to the other Iad isotype, I-Ad, the crucial hydrophobic residues. Thus, different class II molecules of the same MHC haplotype may have rather distinct peptide binding specificities, thereby expanding the repertoire of possible immunogenic peptides presented for T cell recognition.     

An immune response defect due to low levels of class II cell surface expression. Analysis of antigen presentation and positive selection.

The effects of quantitative differences in class II cell surface expression have been difficult to address in intact animals. This study uses several lines of H-2s/s mice carrying an A beta k transgene that differ significantly in terms of class II cell surface expression. Due to inefficient chain pairing, mice carrying 60 to 65 copies of this transgene express only low levels of A alpha s/A beta k on the cell surface, and cell surface expression of the endogenous A alpha s/A beta s complex (and total Ia) is severely reduced (to 7-15% control levels). The significant decrease in class II cell surface expression in the thymic cortex of these mice did not affect the frequency of peripheral T cells expressing at least 10 distinct TCR V beta chains. However, T cell proliferative responses to the A alpha s/A beta s-restricted peptide MBP 89-101 were abrogated in high copy number A beta k mice. Experiments using bone marrow chimeras demonstrated that both inefficient Ag presentation and failure to positively select appropriate T cells contributed to this lack of response. Inefficient Ag presentation was clearly the dominant defect, and the density of class II cell surface expression required for positive selection appeared to be quite low.     

The expression of a tissue-specific self-peptide is required for allo-recognition

We have compared the functional properties of I-Ad expressed on different cell types. Specifically, we have transfected I-A alpha d and I-A beta d cDNA into a panel of T cell thymomas of various phenotypes. Excellent class II surface expression was achieved in all T cell tumors, equivalent in level to that found on the B cell lymphoma A20. Interestingly, however, two allo-I-Ad-specific Thy differed in their recognition of the transfected tumor cells: whereas the 42H11 T cell hybridoma (THy) was stimulated very efficiently by all transfectants, the RK38.2.2 Thy did not react to any of them. Both THy responded equally well to I-Ad on A20 B lymphoma cells. Purified macrophages isolated from various sources were also differentially recognized by the two THy, although there was only a quantitative difference in stimulation. Taken together, these results are best interpreted to show that the TCR of the RK38.2.2 THy is specific for I-Ad in the context of a B cell-specific determinant, possibly a self-peptide that is naturally associated with Ia. A cross-reactive molecule could be expressed by macrophages and COS-1 cells, but not by T cells.     

Generation of functional I-Ed variants from an antigen-presenting macrophage cell line

We report the ability of the BAC1.2 macrophage cell line to present antigen both to populations of antigen-primed lymph node cells and to T cell hybridomas. We also describe the selection of I-Ed variants derived from this cell line. These variants have lost the ability to present antigen to an I-Ed plus ovalbumin-specific T cell line, but retain the ability to stimulate an I-Ad plus ovalbumin-restricted T cell line, and a T cell line reactive with I-Ed alone. These variants should aid in the elucidation of structural elements of the I-E molecule involved in the interaction with helper T cells.     

Analysis of the molecular specificities of anti-class II monoclonal antibodies by using L cell transfectants expressing HLA class II molecules

Expressible HLA class II alpha- and beta-chain cDNA were used for DNA-mediated gene transfer to produce L cell transfectants expressing single types of human class II molecules. Cloned transfectants expressing nine different class II molecules were isolated: DR alpha: DR1 beta I, DR alpha: DR4 beta I, DR alpha: DR5 beta I, DR alpha: DR5 beta III (DRw52), DR alpha: DR7 beta I, DR alpha: DR4/7 beta IV (DRw53), DQ7 alpha: DQw2 beta, DQ7 alpha: DQw3 beta, and DPw4 alpha: DPw4 beta. These class II-expressing transfectants were used to analyze by flow cytometry the molecular specificities of 20 anti-class II mAb. These analyes indicate that some mAb are more broadly reactive than was previously thought based on immunochemical studies. In contrast, the narrow molecular specificities of other anti-class II mAb were confirmed by this approach. Transfectants expressing human class II molecules should be valuable reagents for studies of B cell and T cell defined epitopes on these molecules.     

How MHC class II molecules reach the endocytic pathway.

We have examined trafficking of major histocompatibility complex (MHC) class II molecules in human B cells exposed to concanamycin B, a highly specific inhibitor of the vacuolar H(+)-ATPases required for acidification of the vacuolar system and for early to late endosomal transport. Neutralization of vacuolar compartments prevents breakdown of the invariant chain (Ii) and blocks conversion of MHC class II molecules to peptide-loaded, SDS-stable alpha beta dimers. Ii remains associated with alpha beta and this complex accumulates internally, as ascertained biochemically and by morphological methods. In concanamycin B-treated cells, a slow increase (> 20-fold) in surface expression of Ii, mostly complexed with alpha beta, is detected. This surface-disposed fraction of alpha beta Ii is nevertheless a minor population that reaches the cell surface directly, or is routed via early endosomes as intermediary stations. In inhibitor-treated cells, the bulk of newly synthesized alpha beta Ii is no longer accessible to fluid phase endocytic markers. It is concluded that the majority of alpha beta Ii is targeted directly from the trans-Golgi network to the compartment for peptide loading, bypassing the cell surface and early endosomes en route to the endocytic pathway.     

Selective expression of class II E alpha d gene in transgenic mice

Class II genes of the MHC must be expressed by APC for activation of CD4+ T cells and efficient delivery of T cell help to B lymphocytes. Class II genes have restricted tissue expression and are under complex regulation. By using various deletion constructs of the class II E alpha d gene in transgenic mice we have mapped different 5' flanking regions which control E alpha d gene expression in distinct cell types. We demonstrate dissociate expression of E alpha d within the macrophage lineage as well as within the B cell lineage, and present evidence for a repressive element operative in B cells and macrophages. We describe the generation of novel transgenic lines with limited constitutive and inducible E alpha mRNA and I-E protein.     

Somatic cell hybrids between macrophages from Bcgr and Bcgs mice: characterization of MHC class II expression

In order to gain a better understanding of the regulation of MHC class II expression related to the Bcg gene, we have produced macrophage-macrophage somatic cell hybrids by fusing the RAW 309 macrophage cell line derived from BALB/c.Bcgs mice with peritoneal macrophages from Bcgr C3H/HeN mice. The differential screening of the hybrids was based on the differential sensitivity of Ia expression to suppression with cycloheximide. We found that most of the hybrids expressed Ia without further stimulation with rIFN-gamma. Cycloheximide suppressed the expression of Ia by some of the hybrids. Treatment of these cells with rIFN-gamma resulted in a cycloheximide resistant Ia expression of both parental haplotypes. The macrophage hybrids produced IL-1 beta, IL-1 alpha, and TNF-alpha when stimulated with LPS. There was no correlation between the levels of monokines produced and the persistence of Ia expression. The results of this investigation indicate that the product of the Bcg gene contributed by macrophages from C3H/HeN mice will affect the expression of the I-Ad glycoprotein that is normally transiently expressed by the RAW 309 cell line.     

Cellular and subcellular distribution of PBP72/74, a peptide-binding protein that plays a role in antigen processing

A 72/74-kDa peptide binding protein (PBP72/74) was previously described which plays a role in the processing and/or presentation of Ag, possibly by facilitating the association of processed Ag with the MHC class II molecules. PBP72/74 was recently shown to be related to the 70-kDa family of heat shock proteins (hsp70), whose members show the general characteristic of binding to denatured or inappropriately folded proteins. Here we describe the cellular and subcellular distribution of PBP72/74. By flow cytometry with PBP72/74-specific rabbit antisera, PBP72/74 is detected on the surfaces of mouse Ig+ B cells and MAC-1+ macrophages. PBP72/74 74 was not detected on the surfaces of Thy-1+ T cells or NK1.1+ NK cells. The cell surface expression of PBP72/74 does not require MHC class II expression. Indeed, the Ia- variant B cell lymphoma cell line, M12.C3, expresses PBP72/74 at levels equivalent to that of the Ia+ parent cell line, M12.4.1, from which it was derived. Furthermore, the fibroblast L cell line, DAP.3, shows no cell surface expression of PBP72/74, nor do DAP.3 lines transfected with and expressing genes encoding the alpha- and beta-chain of the I-Ad and I-Ed molecules. Moreover, treatment of B cells with either IL-4 or LPS, which increases Ia expression severalfold, does not affect PBP72/74 expression. Thus, PBP72/74 cell surface expression appears to be a property of B cells and macrophages, independent of Ia expression. In addition, the B cell surface expression of PBP72/74 is not altered by stress in the form of heat shock. Thus, PBP72/74 appears to be a constitutive noninducible member of the hsp70 family. By immunoelectron microscopy, PBP72/74 is detected in approximately 36% of early endocytic vesicles into which surface Ig is internalized after binding to anti-Ig antibodies. This compartment was previously shown to contain class II en route to the cell surface associated with invariant chain and the proteases cathepsin B and D and is suggested to be a subcellular site of antigen processing. PBP72/74 is also found associated with the plasma membrane, endoplasmic reticulum, and membranes proximal to the Golgi stacks. The cellular and subcellular distribution of PBP72/74 is consistent with its playing a role in the processing of presentation of Ag with the MHC class II molecules.     

Two distinct class II molecules encoded by the genes within HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating and restriction molecules

By using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we investigated the difference in the HLA class II molecule between HLA-Dw2 and Dw12, both of which are typed as HLA-DR2 serologically. The anti-HLA-DR framework monoclonal antibody (MoAb) HU-4 precipitated an alpha-chain and two beta-chains of human class II molecules from both Dw2 and Dw12 homozygous B lymphoblastoid cell lines. It was demonstrated clearly that an alpha-chain (alpha 1) and one of the beta-chains (beta 1) showed no difference in mobility in the 2D-PAGE between Dw2 and Dw12, but that another beta chain (beta 2) of Dw2 was distinct from that of Dw12 in the 2D-PAGE profile. Thus, MoAb HU-4 precipitated alpha 1 beta 1 and alpha 1 beta 2 molecules from Dw2 and Dw12, and the alpha 1 beta 1 molecule appears to be an HLA-DR2 molecule. The alpha 1 beta 2 molecule, on the other hand, is a class II molecule distinct from those precipitated with anti-DR2, anti-DQw1 (DC1, MB1, MT1), or anti-FA MoAbs. MoAb HU-4 completely inhibited the mixed lymphocyte culture reaction (MLR) between Dw2 and Dw12, but anti-DR2 MoAb HU-30, which reacts only with the alpha 1 beta 1 molecule, did not show an inhibitory effect on the MLR between Dw2 and Dw12. The alpha 1 beta 2 molecule is therefore the molecule which elicits MLR between Dw2 and Dw12. An IL 2-dependent T cell line established from an HLA-Dw12/D blank heterozygous high responder to the streptococcal cell wall antigen (SCW) clearly distinguished the Dw2 specificity from Dw12 specificity expressed on the antigen-presenting cell (APC). Moreover, MoAb HU-4 markedly inhibited the cooperation between the T cell line and APC to respond to SCW. These observations indicate that the alpha 1 beta 2 molecule is recognized as a restriction molecule by the T cell line at the antigen presentation of SCW through APC MoAb HU-30 on the other hand partially inhibited the MLR between Dw2 or Dw12 homozygous cell as a stimulator cell and non DR2 cell as a responder cell. It markedly inhibited the proliferative response of the Dw12/D- heterozygous T cell line to SCW, presented by Dw2+ but Dw12- allogeneic APC, and the peripheral response of Dw2 or Dw12 homozygous peripheral blood lymphocytes to SCW. Thus, two distinct class II molecules encoded by the genes within the HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating molecules in the MLR and as restriction molecules in the antigen presentation by APC.