Regulation of major histocompatibility complex class-II genes: X, Y and other letters of the alphabet.
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Animals↗
Biomedical subjects
D Mathis
Publications and source records attributed to D Mathis.
Positive selection of the T cell repertoire: where and when does it occur?
The T cell repertoire is shaped by both positive and negative influences. T lymphocytes that express the V beta 6 variable region are positively selected in the thymus by cells expressing major histocompatibility complex (MHC) class II E molecules. To identify these cells, we have quantitated V beta 6+ T lymphocytes in a set of transgenic mice showing variant patterns of E expression in the thymus. We demonstrate that class II molecules must be expressed on epithelial cells of the cortex for positive selection to occur. Using a direct assay of unmanipulated thymocytes, we show that positive selection is manifest only as a rather late event in thymocyte differentiation, after the maturation of cortical double-positives into single-positives.
Animals↗
Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.
Transgenic mice expressing a T cell receptor heterodimer specific for a fragment of pigeon cytochrome c plus an MHC class II molecule (I-Ek) have been made. We find that H-2k alpha beta transgenic mice have an overall increase in the number of T cells and express a 10-fold higher fraction of cytochrome c-reactive cells than H-2b mice. Surface staining of thymocytes indicates that in H-2b mice, T cell development is arrested at an intermediate stage of differentiation (CD4+8+, CD310). Analyses of mice carrying these T cell receptor genes and MHC class II I-E alpha constructs indicate that his developmental block can be reversed in H-2b mice by I-E expression on cortical epithelial cells of the thymus. These data suggest that a direct T cell receptor-MHC interaction occurs in the thymus in the absence of nominal antigen and results in the enhanced export of T cells, consistent with the concept of "positive selection".
Animals↗
Functional sites on the A alpha-chain. Polymorphic residues involved in antigen presentation to insulin-specific, Ab alpha:Ak beta-restricted T cells.
The interaction between the clonally selected TCR, the processed Ag peptide and the Ia molecule is not fully understood in molecular terms. Our study intended to delineate the residues of Ab alpha molecules that function as contact sites for Ag and for the TCR of a panel of T cells specific for the A chain of insulin in combination with mixed haplotype Ab alpha:Ak beta molecules. Multiple L cell transfectants expressing alpha,beta-heterodimers composed of wild-type A beta- and chimeric or mutant A alpha-chains served as antigen presenting cells. The recombinant A alpha-chains had been generated by an exchange of allelically hypervariable regions (ahv) or amino acids. The results point out a broad spectrum of b sequence requirements for the bovine insulin-specific activation of the various T cell populations. Activation of some T cells seemed quite permissive, requiring b-haplotype amino acids in any one of the three ahv, while others had strict requirements, demanding b-haplotype sequence in all three ahv. Our data stress the role of ahvII and especially ahvIII in T cell activation. Interestingly, single amino-acid substitutions in ahvII or ahvIII of Ak alpha were sufficient to bring up full stimulation potential for two T cell hybridomas. We also found that some ahv permutations influenced the Ag preference (beef insulin versus pig insulin) of some T cells. These data suggest a critical role for the three-dimensional structure of the complex formed by Ia and the processed Ag peptide. The stability of the trimolecular complex essential for T cell activation is envisioned as being the sum of the interactions between Ag/I-A, TCR/Ag, and TCR/I-A, each variable in strength and compensated for by the others.
Animals↗
I-Ak polymorphisms define a functionally dominant region for the presentation of hen egg lysozyme peptides.
The class II molecules of the MHC not only bind processed antigenic peptides but also interact with the TCR. This latter interaction is thought to be the basis for allele specific "restriction" of Ag presentation to T cells. The specificity of this interaction is likely due to amino acid differences in a small number of polymorphic or "hypervariable" regions located in the amino terminal domains of the alpha- and beta-chains. We have explored the functional significance of these polymorphic regions in an I-Ak-restricted, hen egg lysozyme specific Ag presentation system in which the measurement of IL-2 production by T cell hybridomas was used as the indicator of TCR recognition of the I-A/Ag complex. Chimeric I-A molecules, in which b allelic residues were substituted in one or more of the polymorphic regions of the A alpha k chain or in which d allelic residues were substituted in one or more of the polymorphic regions of the A beta k chain, were used to examine the contribution of each polymorphic region of the molecule to its function. The results obtained demonstrate that the regions between residues 69 to 76 of the A alpha k chain and the regions between residues 63 to 67 and 75 to 78 of the A beta k-chain exert a dominant effect on the presentation of lysozyme peptides by I-Ak to the T cell hybridomas in our panel. These observations were confirmed and extended by the analysis of Ag presentation by seven serologically selected mutants, all of which have amino acid interchanges in or around the dominant polymorphic regions. The results suggest that the serologically selected mutants fail to present Ag not because they fail to bind the peptide Ag but because the amino acid substitutions destabilize the interaction between the Ia/peptide complex and the TCR. Use of the recently published hypothetical model for class II structure to interpret the Ag presentation results suggests that the dominant polymorphic regions lie across from one another near one end of the alpha-helices that form the two walls of the proposed Ag-binding cleft located on the top surface of the class II molecule. Furthermore, the majority of the amino acids which have been changed in the serologically selected mutants have side chains which are postulated to point up toward the exterior of the molecule and would, therefore, be potential contact residues for the TCR.
Amino Acid Sequence↗
Transgenic mice with I-A on islet cells are normoglycemic but immunologically intolerant.
Insulin-dependent diabetes mellitus (IDDM) is caused by a specific loss of the insulin-producing beta cells from pancreatic Langerhans islets. It has been proposed that aberrant expression of major histocompatibility complex (MHC) class II molecules on these cells could be a triggering factor for their autoimmune destruction. This proposal was tested in transgenic mice that express allogeneic or syngeneic class II molecules on the surface of islet cells at a level comparable with that normally found on resting B lymphocytes. These animals do not develop diabetes, nor is lymphocyte infiltration of the islets observed. This immunological inactivity does not result from tolerance to the "foreign" class II molecules.
Animals↗
I-A alpha polymorphic residues that determine alloreactive T cell recognition.
An individual's T lymphocytes are highly reactive to allogeneic MHC molecules. As a step in deciphering the mechanism of allorecognition by T lymphocytes, we have attempted to identify the TCR's target on MHC class II molecules, in particular the polymorphic residues that determine the specificity of recognition. We have generated a panel of Ak-reactive, Ab-nonreactive T cell hybridomas, and sets of L cell transfectants displaying A alpha A beta molecules with wild-type, chimeric or single site-mutated A alpha chains, with reciprocal interchanges between Ak and Ab. We then measured the stimulation of the T hybridomas in response to the transfectants. The results indicate that the hybridomas recognize diverse and complex determinants, with contributions from both A alpha and A beta chains, and from several regions or amino acids of the A alpha chain. The data are most consistent with a model in which alloreactivity results from the presentation of peptides to the T cell by an allogeneic MHC molecule, peptides that cannot be presented by the responder's own MHC complexes. The specificity of allorecognition seems to be imparted mainly by peptide/MHC molecule rather than TCR/MHC molecule contacts.
Animals↗
Restricted assembly of MHC class II molecules in transgenic mice.
Studies on cell lines transfected with MHC class II genes have revealed important limitations on the assembly of haplotype-mismatched A alpha:A beta complexes. These findings led to the speculation that pairing restrictions, if applied in a cell type-specific fashion, might be involved in various autoimmune phenomena. We have investigated pairing restrictions in vivo by analyzing transgenic mice that carry an Ak alpha chain, an Ak beta chain, or the Ak alpha:Ak beta complex on an H-2b or H-2s background. Our conclusion is that the assembly of haplotype-mismatched A alpha:A beta complexes is limited in vivo, and that this is equally true for all cell types examined, regardless of their role in the immune response.
Alleles↗
Sperm cells as vectors for transferring DNA into mouse eggs: a breakthrough for immunologists?
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Allergy and Immunology↗
Fine-tuning of MHC class II gene expression in defined microenvironments.
Strict control of major histocompatibility complex class II gene expression is essential for proper functioning of the immune system. Recent transgenic mouse studies have revealed an intricate fine-tuning of class II gene transcription in microenvironments such as the germinal centers and thymic cortex and medulla.
Animals↗
T-cell recognition of superantigens: inside or outside the groove?
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Animals↗
Anatomy of a new B-cell-specific enhancer.
The major histocompatibility complex class II molecules, like the immunoglobulins, are prominent B-lymphocyte markers. Herein, we describe a B-cell-specific enhancer associated with the murine class II gene, Ek alpha. This enhancer has a complex anatomy that suggests interactions between remotely spaced elements. Of particular interest is the finding that two CCAAT boxes spaced one kilobase apart are important for enhancer activity. Somewhat surprisingly, the E alpha and immunoglobulin enhancers seem to show little resemblance.
Animals↗
New B-lymphocyte-specific enhancer-binding protein.
We report the discovery of a new B-lymphocyte-specific enhancer-binding protein. A series of gel retardation assays using fragments that scan the -2172 to -1180 region of the major histocompatibility complex class II gene E alpha reveal a site (W) that serves as the recognition sequence for two nuclear proteins, one B-cell restricted and the other ubiquitously occurring. Certain characteristics of the NF-W1 and NF-W2 pair recall the OTF-2/NF-A2 and OTF-1/NF-A1 pair that binds to the immunoglobulin octamer, but we demonstrate that the two protein pairs are distinguishable by several criteria. NF-W1 and NF-W2 interact differentially with their common GTTGCATC binding site, display a different affinity for it, and have molecular weights that differ by about 20,000. Yet, proteolysis experiments and cross-linking analyses indicate that the two W complexes show structural relatedness.
Animals↗
Slot-machine mutagenesis of a polymorphic residue on the A kappa alpha-chain.
This study explores the limitations on variability at a polymorphic position of an MHC class II molecule. Using a convenient and rapid method termed "slot-machine mutagenesis," we have converted Glu75 on the A kappa-chain to 15 alternative amino acids. This residue is of interest because it is an immunodominant site on the A kappa alpha chain and because it participates in certain T cell epitopes. The wild-type and mutant A kappa alpha cDNA were transfected into L cells (together with the A kappa beta cDNA and a selection marker), and transfectants displaying high surface levels of the A kappa complex were selected and expanded. We sought to examine three questions: what is the effect of these mutations on the expression and overall conformation of the A alpha: A beta complex? How do these diverse mutations influence mAb epitopes for which Glu75 makes a direct contribution to specificity? Do such substitutions affect T cell recognition of the A kappa alpha:A kappa beta complex? The answers to these three questions are quite different. Position 75 of the A alpha chain can accommodate essentially all chemically divergent amino acids without major consequences for expression and overall A alpha:A beta structure. In contrast, mAb that recognize Glu75-dependent epitopes are extremely particular about the amino acid residing at this position. T cells are less fastidious: those that are affected by the mutations still recognize a number of substitutions. These data emphasize the tolerance of MHC molecules to evolutionary tampering.
Amino Acid Sequence↗
An enhancer factor defect in a mutant Burkitt lymphoma cell line.
RJ 2.2.5 is an immunoselected mutant of the Burkitt lymphoma line Raji. It fails to display MHC class II antigens at the cell surface due to a transcriptional defect. We have identified the function of a regulatory factor that is defective in RJ 2.2.5 cells; this factor is absolutely required for the activity of an MHC class II gene enhancer.
DNA-Binding Proteins↗
Compartmentalization of MHC class II gene expression in transgenic mice.
A set of transgenic mouse lines carrying Ek alpha genes with promoter region deletions was created in an attempt to compartmentalize MHC class II gene expression. Fine immunohistological analyses established that one transgenic line is essentially devoid of E complex in the thymic cortex, another displays almost no E in the thymic medulla or on peripheral macrophages, and two lines display no E on greater than 98% of B cells. We have assayed these mice for immune function: E-dependent tolerance, antigen presentation, T cell priming, and antibody response. Certain of the findings are difficult to reconcile with currently popular hypotheses, e.g., tolerance induction to E molecules in the virtual absence of E complex in the thymic medulla and efficient antibody responses to E-restricted antigens when almost all B cells are E-.
Animals↗
The enhancer in an MHC class II gene, in vitro and in mouso.
The E alpha class II gene of the major histocompatibility complex is expressed in a variety of immunocompetent cells. Part of the control of tissue-specific expression is mediated by a block of sequences found far upstream of the transcriptional startsite; this stretch is necessary for expression in the B lymphocytes of transgenic mice, but largely dispensable elsewhere. We review the evidence for the role of this region in E alpha transcription in transgenic animals, as well as data from transfections into tissue-culture cells, which indicate that this region has non-specific enhancer activity. We discuss possible models to explain how a non-specific enhancer can participate in cell-specific control.
Animals↗
B-cell control region at the 5' end of a major histocompatibility complex class II gene: sequences and factors.
Transcription of major histocompatibility complex class II genes is elaborately regulated. Mouse class II genes are transcribed primarily in B cells, peripheral macrophages and interdigitating cells, and thymic cortical and medullary cells. In this study, we began to identify the DNA sequences and protein factors that control expression of a class II gene in B cells, addressing in particular how closely they resemble those that regulate immunoglobulin gene expression. We describe a region upstream of the E alpha gene that is crucial for its transcription in the B cells of transgenic mice but is less important in cultured B-cell lines. The sequence of this region reveals several familiar motifs, including a second X-Y pair reminiscent of that residing in the promoter-proximal region of all class II genes, a B motif strikingly homologous to that associated with the immunoglobulin kappa gene enhancer, several Ephrussi motifs, and a Pu box-like sequence very similar to that implicated in simian virus 40 and lymphotrophic papovavirus expression in B cells. Careful study of the proteins that bind specifically to these different motifs prompts us to suggest that major histocompatibility complex class II and immunoglobulin genes rely on quite different factors to achieve B-cell-specific expression.
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