Functionally distinct agretopic and epitopic sites. Analysis of the dominant T cell determinant of moth and pigeon cytochromes c with the use of synthetic peptide antigens

The dominant T cell determinant on moth and pigeon cytochromes c in B10.A (E beta k:E alpha k) mice is located in the C-terminal portion of the protein, contained within residues 93-103 or 93-104. Thirty-seven antigen analogs, containing single amino acid substitutions at positions 98, 99, 101, 102, 103, and 104, were synthesized. The effects of the substitutions on in vitro antigenicity and in vivo immunogenicity were determined. Functional assays with T cell clones identified residues 99, 101, 102, and 103 as critical, based on their effect on antigenic potency. Peptides containing substitutions at residues 99, 101, and 102 were capable of eliciting unique clones upon immunization of B10.A mice. This was consistent with the identification of these residues as part of the epitope, the site on the antigen that interacts with the T cell receptor. Immunization with peptides substituted at residue 103, however, failed to elicit clones with unique specificity for the immunogen. When these peptides were tested for their ability to stimulate the T cell clones with antigen-presenting cells from B10.A(5R) mice expressing the E beta b:E alpha k Ia molecule, a consistent change in the relative antigenic potency was observed with 50% of the peptides. The effect of the Ia molecule on the antigenic potency ruled out the possibility that residue 103 nonspecifically affected antigen uptake or processing and identified residue 103 as part of the agretope, the site that interacts with the Ia molecule. The locations of the agretope and the epitope on this antigenic determinant appear to be fixed, even in the presence of large numbers of amino acid substitutions. However, some substitutions were found to affect both the agretope and the epitope, placing limits on the functional independence of the two sites. The results are discussed in terms of the trimolecular complex model of T cell activation and the implications of these data for antigen-Ia molecule interactions.     

Alteration of a non-polymorphic residue in a class II E beta gene eliminates an antibody-defined epitope without affecting T cell recognition

The interaction between the clonally selected T cell receptor, antigen, and Ia molecule is poorly understood at the molecular level. A cell line bearing an altered E beta k molecule has been examined to provide more information about the relationship between Ia structure and function. The cell line, 2B1, was derived from the TA3 B cell hybridoma through a series of negative and positive immunoselection steps. The 2B1 mutant lacked the binding site recognized by the 17.3.3 monoclonal antibody (mAb) but presented antigen normally to all I-Ek-restricted T cell hybridomas and clones examined. Sequence analysis of the mutant E beta k gene showed a single base transition (G----A) that resulted in an arginine to a histidine substitution at amino acid 49 of the beta 1 domain. This mutation demonstrates that residue 49 is not involved in antigen presentation to T cells but can be involved in B cell recognition (mAb binding).     

The molecular basis of class II MHC allelic control of T cell responses.

To identify the molecular basis for the effects of MHC molecule polymorphism on T cell responses, we have combined functional T cell response testing with measurements of peptide binding to the class II MHC molecules on transfected cells. Our studies identify a small subset of spatially localized polymorphic residues of the E alpha E beta dimer (strand residue beta 29, and helix residues beta 72 and beta 75) regulating cytochrome c peptide presentation by two distinct mechanisms. The first effect is on quantitative control of net peptide binding. The replacement of the valine found at position beta 29 in E beta k with the glutamic acid found in E beta b results in a selective loss of pigeon cytochrome peptide but not moth cytochrome peptide binding to the resultant mutant E alpha E beta k molecule. Reciprocally, the replacement of glutamic acid at beta 29 in E beta b with valine results in a gain of pigeon peptide binding. These changes in binding parallel changes in T cell responses in vitro to these peptide-E alpha E beta combinations and mirror the in vivo immune response gene phenotypes of mice expressing E alpha E beta k and E alpha E beta b. E alpha E beta s molecules, which have a beta 29 glutamic acid, are nevertheless able to bind and present pigeon cytochrome peptides, and this is due to changes in helix residues beta 72 and beta 75 that compensate for the negative effect of the beta 29 glutamic acid. The second activity is a critical change in the conformation of the peptide bound to the same extent by distinct MHC molecules, as revealed by changes in T cell responses to moth cytochrome peptides presented by two E alpha E beta molecules differing only at position beta 29. Both of these effects can be ascribed to a single polymorphic residue modeled to be inaccessible to TCR contact (beta 29), providing a striking demonstration of how MHC molecule polymorphism can modify T cell-dependent immune responses without direct physical participation in the receptor recognition event.     

Mutational analysis of simian virus 40 large T antigen DNA binding sites.

We have tested the effects of various mutations within SV40 T antigen DNA recognition sites I and II on specific T antigen binding using the DNase footprint technique. In addition, the replication of plasmid DNA templates carrying these T antigen binding site mutations was monitored by Southern analysis of transfected DNA in COS cells. Deletion mapping of site I sequences defined a central core of approximately 18 bp that is both necessary and sufficient for T antigen recognition; this region contains the site I contact nucleotides that were previously mapped using methylation-interference and methylation-protection experiments. A similar deletion analysis delineated sequences that impart specificity of binding to site II. We find that T antigen is capable of specific recognition of site II in the absence of site I sequences, indicating that binding to site II in vitro is not dependent on binding of T antigen at site I. Site II binding was not diminished by small deletion or substitution mutations that perturb the 27-bp palindrome central to binding site II, whereas extensive substitution of site II sequences completely eliminated specific site II binding. Analysis of the replication in COS7 cells of plasmids that contain these mutant origins revealed that sequences both at the late side of binding site I and within the site II palindrome are crucial for viral DNA replication, but are not involved in binding T antigen.     

Interactions between the amino-terminal domains of MHC class II molecules have a profound effect on serologic and T cell recognition: an analysis using recombinant HLA-DR/H-2E molecules.

A series of transfected L cell lines were generated expressing the products of wild-type or recombinant HLA-DR1/H-2Ek beta-chain-encoding genes paired to DR alpha or E alpha. The recombinant genes were created by reciprocal exchange of the gene segments encoding the amino (NH2)-terminal and carboxy (COOH)-terminal halves of the beta 1 domain and the beta 2 domain. The majority of the serologic determinants, predicted from the genetic composition of the class II dimers, were expressed indicating that no gross conformational changes were induced by the creation of the interspecies recombinant molecules. Subtle conformational variation was detected by the anti-H-2Eb,k,s mAb Y17. Epitope expression was dependent on the presence of the E alpha-chain and NH2-terminal sequence from the beta 1 domain of H-2Ek. Substitution of DR1 sequence in either region led to loss of recognition by Y17. This pattern of reactivity maps the Y17 epitope either to the E alpha-chain or to an exposed sequence on the fourth strand of the beta sheet of the beta 1 domain. If the Y17 epitope is located on the E alpha-chain this raises the interesting possibility that the conformation of this chain, which is invariant by sequence, may vary according to the beta-chain with which it is coexpressed. The ability of the recombinant class II dimers to present Ag to the pigeon cytochrome c-specific, H-2Ek-restricted T cell hybridoma 2B4 was assessed. Transfected L cells expressing E beta k paired to E alpha or DR alpha presented Ag with equal efficiency, and the beta 2 domain of H-2Ek could be substituted with the equivalent region from DR1 without any loss of response. Wild-type DR1 failed to function as a restriction element, however, substitution of the COOH-terminal portion of the beta 1 domain with the equivalent sequence from H-2Ek was sufficient to produce a partial recovery of Ag recognition. Cells expressing a recombinant beta 1 domain comprising the COOH-terminal sequence from H-2Ek and the NH2-terminal sequence from DR1 presented Ag when paired to DR alpha but failed to do so when paired to E alpha. This indicates that a subtle conformational disturbance caused by mismatching of the NH2-terminal region of the beta-chain and the alpha-chain can have pronounced effects on T cell recognition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional Mapping of the DNA Binding Domain of Bovine Papillomavirus E1 Protein

Bovine papillomavirus type 1 (BPV-1) requires viral proteins E1 and E2 for efficient DNA replication in host cells. E1 functions at the BPV origin as an ATP-dependent helicase during replication initiation. Previously, we used alanine mutagenesis to identify two hydrophilic regions of the E1 DNA binding domain (E1DBD), HR1 (E1(179-191)) and HR3 (E1(241-252)), which are critical for sequence-specific recognition of the papillomavirus origin. Based on sequence and structure, these regions are similar in spacing and location to DNA binding regions A and B2 of T antigen, the DNA replication initiator of simian virus 40 (SV40). HR1 and A are both part of extended loops which are supported by residues from the HR3 and B2 alpha-helices. Both elements contain basic residues which may contact DNA, although lack of cocrystal structures for both E1 and T antigen make this uncertain. To better understand how E1 interacts with origin DNA, we used random mutagenesis and a yeast one-hybrid screen to select mutations of the E1DBD which disrupt sequence-specific DNA interactions. From the screen we selected seven single point mutants and one double point mutant (F175S, N184Y/K288R, D185G, V193M, F237L, K241E, R243K, and V246D) for in vitro analysis. All mutants tested in electrophoretic mobility shift assays displayed reduced sequence-specific DNA binding compared to the wild-type E1DBD. Mutants D185G, F237L, and R243K were rescued in vitro for DNA binding by the replication enhancer protein E2. We also tested the eight mutations in full-length E1 for the ability to support DNA replication in Chinese hamster ovary cells. Only mutants D185G, F237L, and R243K supported significant DNA replication in vivo which highlights the importance of E1DBD-E2 interactions for papillomavirus DNA replication. Based on the specific point mutations examined, we also assigned putative roles to individual residues in DNA binding. Finally, we discuss sequence and spacing similarities between E1 HR1 and HR3 and short regions of two other DNA tumor virus origin-binding proteins, SV40 T antigen and Epstein-Barr virus nuclear antigen 1 (EBNA1). We propose that all three proteins use a similar DNA recognition mechanism consisting of a loop structure which makes base-specific contacts (HR1) and a helix which primarily contacts the DNA backbone (HR3).     

Altered binding and transport of vitamin B12 resulting from insertion mutations in the Escherichia coli btuB gene

The BtuB protein of Escherichia coli is a multifunctional outer membrane receptor required for the binding and uptake of vitamin B12, bacteriophage BF23, and the E colicins. The btuB gene was mutagenized by the insertion of 6-base pair linkers into each of ten HpaII sites distributed throughout the coding region. Receptor function was measured with the mutated genes present in single or multiple copies. All of the mutant proteins were found in the outer membrane in similar amounts, although two of them were susceptible to cleavage by endogenous proteolytic activity. The vitamin B12 transport activity mediated by five of the mutants was essentially identical to that of the wild type. Four mutations (insertions after amino acids 50, 252, and 412, and a duplication of residues 434-472) reduced uptake activity to less than 2% of parental, whereas insertions at residues 343 and 434 had less severe effect. The insertions at residues 50 and 252 appeared to slow the rate of cobalamin binding to the receptor; the defect in the former mutant was partially corrected by elevated calcium levels. The insertion at residue 412 did not affect the rate of substrate binding but slowed its release from the receptor. Most of the receptors conferred susceptibility to phage BF23 and the E colicins, although several mutants were altered in the degree of their sensitivity to the lethal agents. None of the mutations affected the entry of only one type of ligand. Thus, several receptor domains have been implicated in substrate binding and energy coupling.     

Generation of auxotrophic mutants of Enterococcus faecalis.

A 22-kb segment of chromosomal DNA from Enterococcus faecalis OG1RF containing the pyrimidine biosynthesis genes pyrC and pyrD was previously detected as complementing Escherichia coli pyrC and pyrD mutations. In the present study, it was found that the E. faecalis pyrimidine biosynthetic genes in this clone (designated pKV48) are part of a larger cluster resembling that seen in Bacillus spp. Transposon insertions were isolated at a number of sites throughout the cluster and resulted in loss of the ability to complement E. coli auxotrophs. The DNA sequences of the entire pyrD gene of E. faecalis and selected parts of the rest of the cluster were determined, and computer analyses found these to be similar to genes from Bacillus subtilis and Bacillus caldolyticus pyrimidine biosynthesis operons. Five of the transposon insertions were introduced back into the E. faecalis chromosome, and all except insertions in pyrD resulted in pyrimidine auxotrophy. The prototrophy of pyrD knockouts was observed for two different insertions and suggests that E. faecalis is similar to Lactococcus lactis, which has been shown to possess two pyrD genes. A similar analysis was performed with the purL gene from E. faecalis, contained in another cosmid clone, and purine auxotrophs were isolated. In addition, a pool of random transposon insertions in pKV48, isolated in E. coli, was introduced into the E. faecalis chromosome en masse, and an auxotroph was obtained. These results demonstrate a new methodology for constructing defined knockout mutations in E. faecalis.     

Analysis of a protein-binding domain of p53.

The tumor suppressor protein p53 was first isolated as a simian virus 40 large T antigen-associated protein and subsequently was found to function in cell proliferation control. Tumor-derived mutations in p53 occur predominantly in four evolutionarily conserved regions spanning approximately 50% of the polypeptide. Previously, three of these regions were identified as essential for T-antigen binding. We have examined the interaction between p53 and T antigen by using Escherichia coli-expressed human p53. By a combination of deletion analysis and antibody inhibition studies, a region of p53 that is both necessary and sufficient for binding to T antigen has been localized. This function is contained within residues 94 to 293, which include the four conserved regions affected by mutation in tumors. Residues 94 to 293 of p53 were expressed in both wild-type and mutant forms. T-antigen binding was unaffected by tumor-derived mutations which have been associated with the wild-type conformation of p53 but was greatly reduced by mutations which were previously shown to alter p53 conformation. Our results show that, like T-antigen binding to the Rb tumor suppressor protein, T antigen appears to interact with the domain of p53 that is commonly mutated in human tumors.     

Selection of H-2 molecules for the context of antigen recognition by T lymphocytes

T lymphocytes recognize the synthetic polypeptides GA and GLT and the natural antigen LDH_B and are thereby stimulated to proliferate in vitro. Simultaneously with the antigen, T cells recognize class II MHC molecules of the antigen-presenting cell and the T-cell proliferation can therefore be inhibited by the addition of monoclonal antibodies specific for either A (AA ) or E (EF ) molecules. Antibody blocking of in vitro responses thus provides an opportunity to test the rules governing the selection of class II molecules (A versus E) in the recognition of different antigens. To determine these rules we tested T cells for some 40 strains (classical inbred strains and B10.W lines) carrying H-2 haplotypes derived from wild mice) for their proliferative response to GA, GLT, and LDH_B. Strains that responded were then tested in the antibody-blocking assay to determine the class II context of the response. The response to GA occurred always in the context of the A molecule; no single instance was found of the response being channelled through the E molecule. Of the 19 different A molecules (A allomorphs) that could be tested, nine (47 percent) were able to provide the context for GA recognition (and hence conferred responsiveness), while the rest failed to do so (conferred nonresponsiveness). Of the 17 informative cases tested for the response to LDH_B, 14 channelled the response through the A molecule, while, in the remaining cases, the cells failed to respond altogether. And again, there was no case where the response was channelled through the E molecule. However, in two instances (of 14) the E molecule provided the context for the stimulation of suppressor T cells, which then suppressed the response of helper T cells occurring in the context of the A molecule. Of the 19 cases tested for the response to GLT, eight channelled the response through the E molecule and two through the A molecule. The two cases of an E A switch were those in which the strains failed to express cell-surface E molecules as a result of a mutation in one of the E-encoding loci. These data indicate a remarkable but puzzling consistency in the channelling of the response to a given antigen via either A or E molecules. This consistency may be a hint that there is a link between the specificity of antigen (nonself) and MHC (self) recognition by T lymphocytes.Abbreviations used in this paper APC antigen-presenting cell - GA poly (Glu⁴⁰Ala⁶⁰) - GLT poly (Glu⁵¹Lys³⁴Tyr¹⁵) - Ir immune response - LDH_B lactate dehydrogenase 134 - MHC major histocompatibility complex - T_H T helper (cell) - T_S T suppressor (cell)     

T cell recognition of Mlsc. I. Influence of MHC gene products in Mlsc-specific T cell recognition

Monospecific T cell clones have been proven to be powerful tools for the characterization of T cell recognition in many Ag-specific as well as allo-specific T cell responses. In this report, in order to elucidate the mechanism of T cell recognition of minor stimulating locus Ag (Mlsc) determinants, Mlsc-specific cloned T cells were employed together with primary T cell responses to clarify the role of MHC-gene products in Mlsc-specific T cell recognition. The results indicated that T cells recognize Mlsc determinants in conjunction with I-region MHC gene products. Moreover, certain MHC haplotypes (e.g., H-2a and H-2k) appear to function efficiently in the "presentation" of Mlsc, whereas other haplotypes (e.g., H-2b and H-2q) function poorly if at all in presenting Mlsc. Experiments with the use of stimulators derived from F1 hybrids between the low stimulatory H-2b, Mlsc strain, C3H.SW, and a panel of Mlsb, H-2-different or intra-H-2 recombinant strains strongly suggested that expression of E alpha E beta molecules on stimulators plays a critical role for Mlsc stimulation. The functional importance of the E alpha E beta product in Mlsc recognition was further demonstrated by the ability of anti-E alpha monoclonal antibody to inhibit the response of cloned Mlsc-specific T cells. Inhibition of the same Mlsc-specific response by anti-A beta k antibody suggests that the A beta product may also play a role in T cell responses to Mlsc.     

Activation of the human epsilon- and beta-globin promoters by SV40 T antigen.

We have studied the effect of the SV40 T antigen on expression from human globin promoters fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and compared its effect with the SV40 enhancer and the adenovirus E1A protein. We have observed that expression of p epsilon GLCAT and p beta GLCAT (the epsilon-globin or beta-globin promoter linked to the CAT gene) was significantly stimulated when cotransfected with a cloned T antigen plasmid into CV-1 cells, indicating that trans-activation of the globin promoters was mediated by SV40 T antigen. Transfection of the p beta GLCAT-SV (p beta GLCAT containing the SV40 enhancer element) into CV-1 cells resulted in a 50-60-fold increase in CAT activity as compared to p beta GLCAT (no enhancer). However, cotransfection of the p beta GLCAT-SV with the cloned T antigen resulted in an additional increase of CAT expression, which suggests that T antigen and the SV40 enhancer activate globin gene expression independently. We found that T antigen but not E1A could further stimulate the expression of an enhancer-containing plasmid in CV-1 cells; whereas E1A but not T antigen could further stimulate p epsilon GLCAT expression in COS-1 cells which constitutively express the SV40 T antigen. These results suggest that T antigen and E1A also act independently. Deletion analysis showed that the minimum sequence required for a detectable level of stimulation of the epsilon-globin promoter by T antigen is 177 bp 5' to the cap site, suggesting that the target sequences for response to T antigen do not reside in the canonical 100 bp promoter region, but rather reside in sequences further upstream, and therefore the cellular factors interacting with T antigen are not the TATA or CAT box binding proteins, but the proteins interacting with upstream regulatory sequences.     

Hydrophilic residues at the apical domain of GroEL contribute to GroES binding but attenuate polypeptide binding

The GroES binding site at the apical domain of GroEL, mostly consisting of hydrophobic residues, overlaps largely with the substrate polypeptide binding site. Essential contribution of hydrophobic interaction to the binding of both GroES and polypeptide was exemplified by the mutant GroEL(L237Q) which lost the ability to bind either of them. The binding site, however, contains three hydrophilic residues, E238, T261, and N265. For GroES binding, N265 is essential since GroEL(N265A) is unable to bind GroES. E238 contributes to rapid GroES binding to GroEL because GroEL(E238A) is extremely sluggish in GroES binding. Polypeptide binding was not impaired by any mutations of E238A, T261A, and N265A. Rather, these mutants, especially GroEL(N265A), showed stronger polypeptide binding affinity than wild-type GroEL. Thus, these hydrophilic residues have a dual role; they help GroES binding on one hand but attenuate polypeptide binding on the other hand.     

Casein kinase II phosphorylation of the human papillomavirus-18 E7 protein is critical for promoting S-phase entry.

The human papillomavirus type 18 E7 protein subverts the pRb/E2F pathway to promote S-phase reentry by postmitotic, differentiated primary human keratinocytes in support of viral DNA amplification. We prepared a panel of HPV-18 E7 mutations in pRb binding or in casein kinase II (CKII) phosphorylation. Our results showed that the ability of E7 binding to pRb correlated with the activation of DNA polymerase alpha or cyclin E to various extents in differentiated keratinocytes of organotypic cultures but was insufficient to induce the proliferating cell nuclear antigen. Proteins mutated in the CKII recognition sequence or in one or both serine substrates (S32 and S34) bound pRb in vitro, but only those with negative charges at these two residues induced proliferating cell nuclear antigen effectively. Nevertheless, unscheduled cellular DNA synthesis occurred very inefficiently relative to the wild-type E7, if at all. Thus, both pRb binding and CKII phosphorylation of E7 are critical for activating cellular genes essential for S-phase entry.     

The nucleotide sequence of the murine I-E beta b immune response gene: evidence for gene conversion events in class II genes of the major histocompatibility complex.

We have determined the DNA sequence of the murine I-E beta b immune response gene of the major histocompatibility complex (MHC) of the C57BL/10 mouse and compared it with the sequence of allelic I-E and non-allelic I-A genes from the d and k haplotypes. The polymorphic exon sequences which encode the first extracellular globular domain of the E beta domain show approximately 8% nucleotide substitutions between the E beta b and E beta d alleles compared with only approximately 2% substitutions for the intron sequences. This suggests that an active mechanism such as micro gene conversion events drive the accumulation of these mutations in the polymorphic exons. The fact that several of the nucleotide changes are clustered supports this hypothesis. The E beta b and E beta k genes show approximately 2-fold fewer nucleotide substitutions than the E beta d/E beta b pair. The A beta bm12, a mutant I-A beta b gene from the C57BL/6 mouse, has been shown to result from three nucleotide changes clustered in a short region of the beta 1 domain, which suggests that a micro gene conversion event caused this mutation. We show here that the E beta b gene is identical to the non-allelic A beta bm12 DNA sequence in the mutated region and suggest, therefore, that the E beta b gene was the donor sequence for this intergenic transfer of genetic information. Diversity in class II MHC genes appears therefore to be generated, at least in part, by the same mechanism proposed for class I genes: intergenic transfer of short DNA regions between non-allelic genes.     

Immortalization of rat embryo fibroblasts by mutant polyomavirus large T antigens deficient in DNA binding.

We have identified a putative DNA-binding domain in polyomavirus large T antigen. Mutations introduced into the gene between amino acids 290 and 310 resulted in proteins that no longer bound to the high-affinity binding sites on the polyomavirus genome, showed no detectable nonspecific DNA binding, and were not able to initiate DNA replication from the viral origin. These mutant T antigen genes were introduced into rat embryo fibroblasts together with the neomycin resistance gene to allow selection for growth in the presence of G418. All the mutations tested facilitated the establishment of these cells in long-term culture at an efficiency indistinguishable from that of the wild-type protein.     

The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein

The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.     

A T cell clone expresses two T cell receptor alpha genes but uses one alpha beta heterodimer for allorecognition and self MHC-restricted antigen recognition

All of the T cell receptor alpha- and beta-chain rearrangements present in a dual reactive T cell clone were characterized. This clone exhibits allelic exclusion of its beta-chain genes in that only one of the two alleles is productively rearranged. Unexpectedly, it displays two productive V alpha-gene rearrangements, which are both transcribed into 1.5 kb mRNA. The contribution of each of the two productive alpha genes to the dual recognition was analyzed by gene transfer. To this end, each of the two alpha genes was separately transfected with the single productively rearranged beta gene. Transfer of only one of the two alpha beta combinations restored both allogeneic MHC recognition and self MHC-restricted antigen recognition. Thus, T cell dual recognition results from the cross-reactive recognition of an allo-MHC product by a single antigen-specific and MHC-restricted alpha beta T cell receptor. Furthermore, the presence of two productively rearranged alpha-chain genes in a T cell clone raises questions concerning the level at which allelic exclusion operates in T cells.     

The effects of MHC gene dosage and allelic variation on T cell receptor selection

In a T cell receptor transgenic mouse model of thymic selection, the efficiency of selection of the transgenic alpha beta heterodimer is significantly enhanced in animals that express higher densities of the relevant major histocompatibility complex molecule (I-Ek/b). These results imply that there is a stochastic component to positive selection in the thymus. Allelic variants of the original selecting I-Ek molecule are either less efficient (E alpha k:E beta b) or incapable (E alpha k:E beta s and I-Ed) of mediating the selection of transgenic alpha beta + T cells. Two of these three I-E variants appear to differ from I-Ek in amino acid residues of the peptide binding site and not in residues capable of contacting the T cell receptor, suggesting that specific peptides, or conformations of peptides, play a role in positive selection. In contrast, mice transgenic for only the beta chain of this T cell receptor show selection for CD4+ T cells in the presence of all four I-E variants tested.     

Clonal analysis of B and T cell responses to Ia antigens. IV. Proliferative T cell clones recognizing E beta and/or E alpha allodeterminants

The allospecific T cell recognition of the I-Ek molecule was assessed by using eight A. TH anti-A. TL proliferative T cell clones, all of which expressed the Thy-1-2+, Lyt-1+, Lyt-2-, Ia-, and p94,180+ cell surface phenotype. The use of panels of stimulating cells from homozygous of F1 hybrid strains indicated each T cell clone exhibited specificity for distinct alloactivating determinants including: i) a private E beta k-controlled determinant expressed in cis- or trans-complementing E beta kE alpha strains; ii) an apparently nonpolymorphic E alpha determinant resembling the serologic specificity Ia.7, i.e., present in all strains carrying E alpha and E beta expressor alleles; and iii) a series of conformational I-E determinants, the expression of which required a precisely defined combinatorial association of E beta plus E alpha chains. Two clones were found to be reactivated by cis- but not trans-complementing E beta k E alpha k strains, and another recognized an allodeterminant shared by the I-Ab molecule. Various I-Ek-reactive monoclonal antibodies (mAb) directed to epitopes presumably expressed on either E alpha (epitope clusters I and II) or E beta (epitope cluster III) chains inhibited the proliferative responses of seven clones recognizing private E beta k or unique E beta E alpha conformational activating determinants. By contrast, the restimulation of the clone directed to a nonpolymorphic E alpha determinant was selectively blocked by anti-Ia.7 mAb defining epitopes on the E alpha chains but not by those directed to the E beta chain. On the basis of these data, it was concluded that the recognition sites of most anti-I-Ek proliferative T cells were expressed on the E beta chain or the E beta plus E alpha interaction products, and that a minority of such alloreactive T cells could be activated through recognition of the E alpha chain per se.