Characterization of the interaction of a TCR alpha chain variable domain with MHC II I-A molecules.

The alphabeta TCR recognizes peptides bound to MHC molecules. In the present study, we analyzed the interaction of a soluble TCR alpha chain variable domain (Valpha4.2-Jalpha40; abbreviated to Valpha4.2) with the MHC class II molecule I-Au. Valpha4.2 bound specifically to I-Au expressed on the surface of a transfected thymoma cell line. Modifications in the amino acid residues located within the three complementarity-determining regions (CDRs) of the Valpha domain did not markedly affect this interaction. However, mutation of glutamic acid to alanine at position 69 of the fourth hypervariable region (HV4alpha) significantly increased the binding. Antibody inhibition studies suggested that the binding site was partly contributed by a region of the beta chain of I-Au. Furthermore, the binding of Valpha4.2 to the MHC molecule was dependent on the nature of the peptide bound in the groove. Soluble Valpha4.2 specifically inhibited the activation of TCR transfectants by I-Au-expressing cells pulsed with an N-terminal peptide of myelin basic protein. Valpha4.2 also bound to MHC class II-expressing spleen cell populations from mice of the H-2(u) and H-2(d) haplotypes. The binding of Valpha4.2 to I-A molecules might explain the immunoregulatory effects reported previously for TCR alpha chains. This Valpha4.2 interaction may also be relevant to models of antigen presentation involving the binding of intact proteins to MHC class II molecules followed by their processing to generate epitopes suitable for T cell recognition.     

Hb(64-76) epitope binds in different registers and lengths to I-Ek and I-Ak

The nature of peptide binding to MHC molecules is intrinsically degenerate, in what, one given MHC molecule can accommodate numerous peptides which are structurally diverse, and one given peptide can bind to different alleles. The structure of the MHC class II molecules allows peptides to extend out of the binding groove at both ends and these residues can potentially influence the stability and persistence of peptide/class II complexes. We have previously shown that both I-E(k) and I-A(k)-restricted T cell hybridomas could be generated against the Hb(64-76) epitope. In this study, we characterized the binding register of the Hb(64-76) epitope to I-A(k), and showed that it was shifted by one residue in comparison to its binding to I-E(k), and did not use a dominant anchor residue at P1. This conclusion was further supported by the modeling of the Hb(64-76) epitope bound to I-A(k), which revealed that all of its putative anchor residues fit into their corresponding pockets. We identified the naturally processed Hb epitopes presented by both I-E(k) and I-A(k), and found that they consisted of different species. Those associated with I-A(k) being 20-22 residues long, whereas, those found to I-E(k) contained 14-16 residues. These findings suggested that the lack of a dominant P1 anchor could be compensated by the selection of longer peptides. Overall, these studies revealed the Hb(64-76) epitope bound to I-E(k) and I-A(k) in distinct registers and lengths, demonstrating the plasticity MHC molecules have in generating distinct TCR ligands from the same amino acid sequence.     

A role for the P1 anchor residue in the thermal stability of MHC class II molecule I-Ab

The thermal stability of the murine MHC class II molecule, I-A(b), in complex with invariant chain-derived peptide (CLIP) and an antigenic peptide derived from the alpha subunit of the I-E molecule (Ealpha) at mildly acidic and neutral pH were analyzed using circular dichroism (CD). The stability of I-A(b)-CLIP was increased by a single amino acid substitution in the P1 anchor residue, from Met of CLIP to Phe of Ealpha, similar, in this respect, to I-A(b)-Ealpha. This indicates that hydrophobic interaction in the P1 pocket is critical and plays a primary role in the stability of the complex. The structural models of I-A(b)-peptides based on the crystal structure of I-A(d) might explain the increased stability and the preference for hydrophobic residues in this site. Taken together with what is known of the resident stability at a mildly acidic pH, the difference in stability would closely correlate with the ability of MHC class II to exchange peptides from CLIP to antigenic peptides in the endosome.     

A novel single chain I-A(b) molecule can stimulate and stain antigen-specific T cells

Multimers of soluble major histocompatibility complex class I and II molecules have proven to be useful reagents in quantifying and following specific T cell populations. This study describes the design, generation, and characterization of a novel, single chain I-A(b) molecule which utilizes a unique linker derived from the murine invariant chain. A fragment of the invariant chain, residues 58-85, binds to a region proximal to the class II peptide binding groove and stabilizes occupancy of the class II invariant chain-associated peptide. We have utilized this fragment, replacing CLIP with the Ealpha peptide sequence, to lock the attached peptide into the class II binding groove. The single chain I-A(b) molecule was recognized by a panel of conformation-sensitive, I-A(b)-specific, monoclonal antibodies. Membrane-bound and soluble forms of the single chain I-A(b) stimulated an antigen-specific T cell hybridoma, and tetramers made from soluble monomers stained these cells. The unique features of this molecule may be useful in the design of recombinant T cell receptor ligands containing peptides with low affinity for MHC.     

Steric recognition of T-cell receptor contact residues is required to map mutant epitopes by immunoinformatical programmes

MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues. Immunoinformatical programmes based on the binding affinity to MHC class I molecules are not sufficient for the accurate prediction of CD8 T-lymphocyte epitopes. The predictive capacity is further improved to distinguish mutant epitopes from the non-mutated epitopes if the peptide-TCR interface is integrated into the computing simulation programme.     

Limited plasticity in T cell recognition of modified T cell receptor contact residues in MHC class II bound peptides

The balance between specific and degenerate T cell recognition of MHC class II bound peptides is crucial for T cell repertoire selection, and holds important implications for protective immunity versus autoimmunity. To investigate the degree of degeneracy in T cell recognition, we applied selected modifications to T cell receptor (TCR) contact residue amino acids in the MHC class II bound epitope gpMBP72-85. By using glycosylated amino acids, as an example of a posttranslational modification, large alterations were applied. Small modifications were accomplished by exchanging an arginine residue for a citrulline or an ornithine residue. Finally, the unmodified TCR contact residue side chains were shifted one atom position to the left, using peptoid residues. Both these large and subtle changes in the wild type (WT) peptide caused lack of recognition by WT peptide specific monoclonal and polyclonal T cells. Furthermore, T cells specific for the modified peptides did not cross recognize the WT peptide. Using a set of additional compounds, we investigated the specificity of these T cell populations into detail. Our data reveal a strongly limited plasticity in T cell recognition, and a high specificity for TCR contact residue side chains.     

Modulation of T cell responses with MHC-derived peptides

T cells are activated by an interaction of their TCRs with a complex made up of antigenic peptide bound to the interhelical groove of MHC molecules. The helices lining the antigen binding groove of MHC molecules are felt to contribute several contact residues for TCR binding. Peptides derived from the amino acid sequences of these helices may be capable of modulating immune responses and aiding in the dissection of immune recognition. These studies address the effects of a peptide derived from the sequence of amino acids 68-83 of the IAk beta 1 domain (IAk 68-83) predicted to represent a portion of an antigen-binding helix on the IAk molecule. The IAk 68-83 peptide is bound by a monoclonal anti-IAk antibody and inhibits its binding to IAk-bearing cells. The IAk 68-83 peptide inhibits antigen-dependent activation of the IAk+con-albumin restricted T cell clone D10.G4, and this effect is more pronounced at lower doses of antigen-presenting cells. The free peptide has a small effect in limiting binding of anticlonotypic antibodies to D10.G4, and a multivalent form bound to BSA has a more pronounced effect in this regard. The BSA-peptide conjugate, when fluoresceinated, specifically stained D10.G4 cells, and this was specifically competed by unfluoresceinated IAk 68-83 peptide-BSA conjugate, as well as by anticlonotype. These results suggest that peptides derived from the predicted helical region of MHC class II molecules may have a direct interaction with T cell receptors. Such peptides may be capable of modulating immune responses in a physiologically significant manner.     

MHC Class II-Dependent Peptide Antigen Versus Superantigen Presentation to T Cells

T lymphocytes expressing the CD4 coreceptor can be activated by two classes of major histocompatibility complex (MHC) class II-bound ligands. The elaboration of a conventional T-cell mediated immune response involves recognition of an antigenic peptide bound to the MHC class II molecules by a T-cell receptor (TCR) specific to that particular antigen. Conversely, superantigens (SAgs) also bind to MHC class II molecules and activate T cells, leading to a completely different functional outcome; indeed, SAg-responsive T cells die through apoptosis following stimulation. Superantigens are proteins that are secreted by various bacteria. They interact with the TCR using molecular determinants that are distinct from the residues involved in the recognition of nominal antigenic peptides. Despite the similarities between the recognition of the two classes of ligands by the TCR, considerable structural difference is observed. Here, we discuss the current knowledge on the presentation of SAgs to T cells and compare the different aspects of the SAg response with the recognition of antigenic peptide/MHC complexes.     

Permissive recognition of a mycobacterial T-cell epitope: localization of overlapping epitope core sequences recognized in association with multiple major histocompatibility complex class II I-A molecules.

Most T-cell epitopes are recognized in the context of a single or limited number of major histocompatibility complex (MHC) class II molecules. We have shown previously, however, that the immunodominant p61-80 epitope from the Mycobacterium tuberculosis 19,000 MW protein is recognized in a genetically permissive manner. In this study, permissive recognition of p61-80 was analysed in three murine MHC haplotypes (H-2b,d and k) with respect to: (i) T-cell-epitope core structure; (ii) I-A/I-E class II MHC restriction; and (iii) the identification of critical amino acid residues within the core region. Overlapping epitope core sequences composed of 6 to 8 amino acids were identified for each of the three H-2 haplotypes by T-cell epitope scanning (PEPSCAN) using peptide-specific T-cell lines. The epitope core sequences recognized by peptide and 19,000 MW protein-specific T cells were similar. In all three haplotypes, responses to p61-80 were restricted by class II MHC I-A molecules. To identify residues within the epitope core critically required for recognition, single substitution (alanine or leucine) analogue peptides were tested for their capacity to stimulate p61-80-specific T-cell hybridomas. A heterogeneous pattern of reactivity was observed, even among individual hybridomas derived from the same H-2 haplotype. Although every core residue could be defined as critical for at least one hybridoma, only one critical substitution (74Val-->Ala) was common to all hybridomas. The identification and structural analysis of genetically permissive epitopes of mycobacteria may be a useful strategy for the rational design of peptide-based vaccines for tuberculosis.     

H-2-associated effects of flanking residues on the recognition of a permissive mycobacterial T-cell epitope.

Previously we have identified an immunodominant, eight-residue, epitope core sequence (TAAGNVNI) from the 19,000 MW protein of Mycobacterium tuberculosis, which is recognized in the context of multiple H-2 I-A molecules. In this study, the role of residues flanking this T-cell epitope core was examined, using a series of 20 mer analogue peptides in which the native flanking residues were progressively replaced with L-alanine. Analogue peptides were tested for their capacity to stimulate a CD4+ 19,000 MW protein-specific T-cell line, revealing that all but one N-terminal flanking residue could be replaced collectively by alanine without significant loss of stimulatory activity. However, clear H-2-associated differences in the requirement for flanking residues were demonstrated with peptide-specific T-cell hybridomas. In particular, H-2d-derived hybridomas were much more stringent in their requirement for flanking residues than were H-2b hybridomas. All polyalanine-substituted peptides bound I-Ab molecules, with affinities similar to the native unsubstituted peptide. In contrast, significantly reduced binding to I-Ad was observed with several analogue peptides, although without a clear relationship to the degree of substitution. Furthermore, in H-2b mice, neither immunogenicity nor cross-reactivity with the native peptide showed a clear inverse relationship with respect to the degree of alanine substitution. The results presented in this paper indicate that flanking residues can influence T-cell specificity and that these effects may be controlled by major histocompatibility complex (MHC) haplotype.     

Site of nonrestrictive binding of SEA to class II MHC antigens

We have used the synthetic peptide approach to show that the N-terminal 45-amino acids of staphylococcal enterotoxin A (SEA), SEA(1-45), constitute an important part of its binding site on class II major histocompatibility complex (MHC) molecules. SEA(1-45) and to a lesser extent SEA(1-27) were able to displace SEA from HLA-DR on Raji cells as assessed by flow cytometry and to compete with radiolabeled SEA for interaction with HLA-DR in a direct binding assay. Specific binding of SEA to Ia on murine A-20 cells could be inhibited by the same peptides [i.e. SEA(1-45) greater than SEA(1-27)] that blocked binding to HLA-DR. Therefore, different class II MHC molecules associate with the same functional site on SEA. Further, an ELISA system was used to demonstrate that SEA(1-45) is able to directly bind to a mouse synthetic I-A beta b peptide, I-A beta b (65-85), which contains a binding site of the class II MHC molecule involved in SEA presentation to T cells. Thus, we have localized a site on SEA that is involved in selective surface association with class II MHC antigens and identified the region on the class II MHC antigen to which that site binds.     

Unconventional cytotoxic T lymphocyte recognition of synthetic peptides corresponding to residues 1—23 of Ras protein

A peptide corresponding to amino acids 1 through 23 of Ras protein containing a mutation at position 12 was used to induce cytotoxic T lymphocytes (CTL) in mice. Although the CTL were CD8⁺ and expressed α, β T cell antigen receptors (TCR), their major histocompatibility complex (MHC)-restriction was unconventional. They recognized peptide-treated murine cells of different H-2 haplotypes, but not MHC class I-negative cells. Human HLA class I molecules did not present Ras peptides and hybrid human/mouse MHC molecules revealed that all three extracellular domains α1, α2 and α3 were required for recognition by peptide-specific CTL. Shortening the 23-mer peptide by 5 residues at either the amino or carboxy terminus resulted in loss of CTL recognition. This demonstrates an unusual form of antigen recognition by mouse CTL in which peptide presentation requires murine H-2 class I molecules but is not class I allele restricted, and the peptides recognized are much larger than peptides in conventional class I-restricted responses.     

Characterization of MHC- and TCR-binding residues of the myelin oligodendrocyte glycoprotein 38-51 peptide

Myelin oligodendrocyte glycoprotein (MOG) is a major experimental autoimmune encephalomyelitis (EAE) antigen in H-2b mice and a potential autoantigen in multiple sclerosis. How well MOG peptides bind to MHC and how TCR recognize the peptide/MHC complex have important implications for thymic selection as well as T cell activation in the periphery. In this study, we have characterized amino acids in the MOG(38-51) peptide important for peptide binding to I-Ab, and for TCR recognition of the peptide/MHC complex. We found that the amino acids R41, F44, R46 and V47 constituted the major TCR contact residues, as alanine substitution at these positions abrogated T cell responses without decreasing their binding affinity to I-Ab. In addition, G38 and W39 were found to be minor TCR contact residues. Finally, substituting tyrosine for alanine at position 40 decreased binding to I-Ab by approximately 50% and prevented induction of T cell responses in C57BL/6J mice upon immunization. Thus, Y40 is the dominant MHC-binding residue of the MOG(38-51) peptide and most likely occupies the p1 pocket of I-Ab. Our results could be useful to design peptides with altered agretopes and epitopes of the MOG(38-51) peptide to study their therapeutic potential in the EAE model.     

Deviant trafficking of I-Ad mutant molecules is reflected in their peptide binding properties.

I-Ad molecules harboring single amino acid changes in the conserved 80-82 region of the beta-chain show altered trafficking in invariant chain (Ii)-negative cell lines. Since residues beta81 and beta82 form hydrogen bonds with the backbone of bound peptide, alterations in this region may result in distinct MHC class II conformers that are targeted aberrantly. We examined the assembly and peptide binding properties of the mutant I-Ad molecules generated by in vitro translation. Indeed, loss of a single hydrogen bond at beta81, or of two hydrogen bonds at beta82, is sufficient to render I-Ad incapable of stable interaction with CLIP and other antigenic peptides, despite normal assembly with intact invariant chain. These results suggest that stable interaction of MHC class II molecules with peptide requires the integrity of the H-bond network between residues in the MHC class II alpha-helices and bound peptide, and that conformational features revealed by stable peptide binding are critical for MHC class II intracellular transport.     

Allogeneic core amino acids of an immunodominant allopeptide are important for MHC binding and TCR recognition

The indirect alloimmune response seems to be restricted to a few dominant major histocompatibility complex (MHC)-derived peptides responsible for T-cell activation in allograft rejection. The molecular mechanisms of indirect T-cell activation have been studied using peptide analogues derived from the dominant allopeptide in vitro, whereas the in vivo effects of peptide analogues have not been well characterized yet. In the present study, we generated allochimeric peptide analogues by replacing the three allogeneic amino acids 5L, 9L, and 10T in the sequence of the dominant MHC class I allopeptide P1. These allochimeric peptide analogues were used to define the allogeneic amino acids critical for the MHC binding and TCR recognition. We found that position 5 (5L) of the dominant allopeptide acts as an MHC-binding residue, while the other two allogeneic positions, 9 and 10, are important for the T-cell receptor (TCR) recognition. A peptide containing the MHC-binding residue 5L, as the only different amino acid between donor (RT1.A(u)) and recipient (RT1.A(l)) sequences, did not induce proliferation of lymph node cells primed with the dominant peptide and prevented dominant peptide-induced acceleration of allograft rejection. Identification of MHC and TCR contact residues should facilitate the development of antigen-specific therapies to inhibit or regulate the indirect alloimmune response.     

The relative energetic contributions of dominant P1 pocket versus hydrogen bonding interactions to peptide:class II stability: implications for the mechanism of DM function

Peptides are bound to MHC class II molecules by an array of hydrogen bonds between conserved MHC class II protein side-chains and the peptide backbone and through interactions between MHC protein pockets and peptide side-chain anchors. The crystal structure of murine I-A(k) protein with peptide shows a network of electrostatic interactions with the P1 aspartic acid anchor and an arginine in the P1 pocket that are thought to constitute the major stabilizing interaction between peptide and MHC. In this paper, have explored the relative energetic contribution of this dominant P1 pocket interaction with that made by a genetically conserved hydrogen bond which is formed by the beta 81 histidine residue and the main chain of the bound peptide. We have performed peptide dissociation experiments using antigenic peptides or variants that have altered side-chain interactions with the I-A(k) P1 pocket using either native I-A(k) or I-A(k) proteins mutated to disrupt the N-terminal hydrogen bond. The results demonstrate that the N-terminal hydrogen bonds in I-A(k) complexes make highly significant energetic contributions to the kinetic stabilities comparable to or greater than the energetic contribution of highly favorable P1 pocket interactions. Hence, we conclude that the kinetic stability of MHC class II:peptide complexes critically depends on two quite distinct molecular interactions between peptide and MHC located at the peptide's amino terminus. We discuss these results in light of the proposed mechanism for DM function.     

Short peptide sequences mimic HLA-DM functions

HLA-DM (DM) plays a critical role in Ag presentation to CD4 T cells by catalyzing the exchange of peptides bound to MHC class II molecules. It is known that DM interaction with MHC II involves conformational changes in the MHC II molecule leading to the disturbance of H-bonds formed between the bound peptide and the MHC II groove leading to peptide dissociation. The specific region of the DM molecule that induces this peptide dissociation is not defined. In this study, we describe three short peptides (helper peptides) that accelerate DM-catalyzed peptide exchange. Kinetic studies presented here demonstrate that these peptides act similarly to DM in; (a) enhancing peptide binding to HLA-DR1; (b) dissociation of complexes of peptide-DR1; and (c) maintaining a receptive conformation of empty DR1. We further report that helper peptides are effective in increasing peptide binding to DR1 expressed on B cells in vitro, and, when mixed with peptide and adjuvant, cause enhanced T cell priming in HLA-DR1 Tg mice. We suggest that helper peptides might interact with the same critical residues on MHC class II that is targeted by DM.     

The structural basis of T-cell allorecognition

Foreign allogeneic major histocompatibility complex (MHC) class I and class II molecules elicit an exceptionally vigorous T-cell response. A small component of the alloresponse comprises CD4+ T cells that recognize allogeneic MHC indirectly after processing into peptide fragments that are bound and presented by self-MHC class II. The majority of alloreactive T cells directly recognize intact allogeneic MHC molecules expressed on foreign cells. Some alloreactive T-cell interactions with allogeneic MHC molecules are indifferent to the bound peptide, but evidence suggests that most show specificity to peptide. The vigor and diversity of the direct alloreactive T-cell response can therefore be explained by summation of numerous responses to each of the peptides in the novel set bound by allogeneic MHC molecules. Structural studies definitively show that the overall mechanism of T-cell receptor (TCR) recognition of self-MHC and allogeneic MHC molecules is similar. Many alloreactive T cells recognize several different combinations of MHC and bound peptide that do not necessarily possess structural homology. Flexibility within the TCR structure allows adaptation to the different contact surfaces. Crossreactivity seems to be an intrinsic property of the TCR required, because a single TCR must possess the ability to interact with both self-MHC during positive selection and at least one combination of foreign antigenic peptide presented by self-MHC. Recognition of allogeneic MHC molecules is an inadvertent consequence of the need for TCR crossreactivity.     

Conformation of MHC class II I-A(g7) is sensitive to the P9 anchor amino acid in bound peptide

Type I diabetes is a chronic autoimmune disease resulting in the destruction of insulin-producing beta cells in the pancreas. In humans, disease incidence is linked to expression of specific MHC class II alleles and in mice type I diabetes is associated with the class II allele I-A(g7). I-A(g7) contains a polymorphism that is shared by human class II alleles associated with the disease, at position 57 in the beta chain, in which aspartic acid is changed to a serine. The P9 pocket in the peptide-binding groove is in part shaped by beta57, and therefore the structure of this pocket is modified in I-A(g7). Using mAbs, we have previously determined that alternative conformations of I-A(g7) form in response to peptide binding. In this study, we have extended these findings by examining how peptides induce I-A(g7) molecules to adopt different conformations. By mutating the amino acid in the P9 position of either class II-associated invariant chain peptide (CLIP) or glutamic acid decarboxylase (GAD) 65 (207-220), we have determined that the chemical nature of the P9 anchor amino acid, either acidic or small hydrophobic, affects the overall conformation of the I-A(g7) class II molecule. T cell hybridomas specific for GAD 65 (207-220) in the context of I-A(g7) were also examined for recognition of I-A(g7) bound to GAD 65 (207-220), in which Glu(217) in the P9 position was changed to alanine. We found that although some TCRs were able to recognize both peptides in the context of I-A(g7), and thus both class II conformations, approximately one-third of the T cells tested were not able to recognize the alternate class II conformation formed with the mutated peptide. These results indicate that the I-A(g7) conformations may affect functional activation of T cells, and thus may play a role in autoimmunity.     

Structural analysis of two HLA-DR-presented autoantigenic epitopes: crucial role of peripheral but not central peptide residues for T-cell receptor recognition

Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.