Nobel lecture in physiology or medicine--1987. Somatic generation of immune diversity.
Explore the source record for details and available documents.
Allergy and Immunology↗
Biomedical subjects
S Tonegawa
Publications and source records attributed to S Tonegawa.
Early expression of a T-cell receptor beta-chain transgene suppresses rearrangement of the V gamma 4 gene segment.
beta transgenic mice have a T-cell receptor beta-chain gene that is prematurely expressed on the surface of CD4- CD8- thymocytes and paired with an uncharacterized non-T-cell receptor alpha-chain polypeptide. The rearrangement of the T-cell receptor variable region gamma chain gene segment V gamma 4, a component of the gamma-chain gene that is rearranged and expressed preferentially on thymocytes of normal adult mice, is severely repressed in beta transgenic mice. Consequently no gamma delta T-cell receptor heterodimers are detectable on the surface of adult thymocytes or splenic T cells. These results indicate that cells expressing alpha beta or gamma (V gamma 4)-delta TCRs originate from a common precursor in which the first productive rearrangement of either the beta or gamma locus determines the further differentiation pathway into either alpha beta or gamma delta T cells. The repression of V gamma 4 rearrangement by a preexisting beta-chain gene may be indicative of one of several mechanisms which ensure that gamma delta and alpha beta receptors do not as a rule appear on the surface of the same cell.
Alleles↗
Cloning and sequencing of murine T3 gamma cDNA from a subtractive cDNA library.
The coding sequences of the murine and human T3 gamma chains are of identical length (182 amino acids) and contain a remarkable conservation of residues. The most striking observation is the high degree of homology between the murine and human cytosolic domains (89%), suggesting that the effector function of the T3 complex may be extremely similar or identical within human and murine lymphocytes. Both murine and human T lymphocytes can express two T3 gamma mRNA transcripts, suggesting that a second polyadenylation signal is present downstream. A poly(A) tail is not found in the 3' untranslated region of the murine gamma presented here, indicating that the murine clones analyzed represent mRNA generated by reading through the overlapping poly(A) signals at position 850-860 and possibly terminating at a position that would produce the 1.0 kb transcript.
Animals↗
Expression of the T-cell receptor gamma-chain gene products on the surface of peripheral T cells and T-cell blasts generated by allogeneic mixed lymphocyte reaction.
The gamma-chain genes of the T-cell receptors form a family of related genes that are specifically expressed and somatically rearranged in T cells. Using poly- and monoclonal anti-gamma antibodies, we studied the cell-surface expression of the gamma-chain gene products in mouse peripheral T cells as well as in the T-cell blasts generated by allogeneic mixed lymphocyte reactions. The gamma chains are expressed in the Lyt2-,L3T4- subsets of these T-cell populations as disulfide-linked heterodimers. Whereas the electrophoretic mobility and the N-glycosylation of the spleen and lymph-node gamma chains are indistinguishable from those of the reported thymocyte gamma chain, a minor fraction of the T blasts generated by allogeneic stimulation of B10 lymph-node T cells with B10.BR spleen cells seems to express gamma chains with distinct properties. This suggests that the mixed lymphocyte culture conditions exert a selective effect on the expression of gamma chains among peripheral T-cell populations.
Animals↗
T-cell gamma gene is allelically but not isotypically excluded and is not required in known functional T-cell subsets.
The T-cell gamma genes, structurally related to immunoglobulin genes and the T-cell antigen-receptor alpha- and beta-chain genes, undergo somatic rearrangement in T-lineage cells. However, the role of the T-cell gamma genes has not yet been determined. To determine the potential for gamma gene expression in a set of well-characterized, cloned T-cell lines, we cloned all of the rearranged gamma genes from each cell line. The genes were sequenced to determine if the junction of the variable and joining regions maintained the proper translational reading frame. We then attempted to correlate the presence of an in-frame gamma gene with a T-cell subset. We were unable to establish such a correlation. We found evidence, however, that allelic exclusion influences the rearrangement of the gamma gene. This is consistent with the idea that the gamma gene product participates in establishing a clonally diverse population of T cells recognizing a polymorphic ligand. Isotypic exclusion does not apply to the gamma gene, however, suggesting different roles for the different gamma gene isotypes.
Alleles↗
Close linkage of the mouse and human CD3 gamma- and delta-chain genes suggests that their transcription is controlled by common regulatory elements.
Antigen receptors on the T-cell surface are noncovalently associated with at least four invariant polypeptide chains, CD3-gamma, -delta, -epsilon, and -zeta. The mouse CD3-gamma gene, consisting of seven exons, was found to be highly homologous to the CD3-delta gene described earlier. Both the high level of sequence homology and the exon/intron organization indicate that the CD3-gamma and -delta genes arose by gene duplication. Surprisingly, murine and human genomic DNA clones could be isolated that contained elements of both the CD3-gamma and CD3-delta genes. In fact, the putative transcription start site of the mouse CD3-gamma gene is less than 1.4 kilobases from the transcription initiation site of the mouse CD3-delta gene. Common elements that regulate the divergent transcription of the two genes are therefore proposed to be located in the intervening 1.4-kilobase DNA segment. This might contribute to the coordinate expression of the CD3-gamma and -delta genes during intrathymic maturation of T lymphocytes.
Amino Acid Sequence↗
Structure of a cytotoxic T-lymphocyte-specific gene shows a strong homology to fibrinogen beta and gamma chains.
Using a subtractive cDNA cloning method, we isolated a number of T-lymphocyte-specific genes. One of these genes, represented by the cDNA clone pT49, is expressed in cytotoxic T lymphocytes but not in helper T lymphocytes or B lymphocytes. The protein structure deduced from the nucleotide sequence showed a high degree of homology to fibrinogen beta and gamma subunits. This might indicate that evolutionarily fibrinogen has its origin in lymphocytes. In spite of the strong homology of pT49 protein to the fibrinogen subunits, the positions of the introns in the pT49 gene are totally different from those of the fibrinogen genes.
Amino Acid Sequence↗
[Molecular biology of immunologic recognition].
Explore the source record for details and available documents.
Animals↗
A phosphorylated, disulfide-linked membrane protein in murine cytotoxic T lymphocytes.
The previously determined sequence of the murine T-cell gamma gene and its transcription in cloned T lymphocytes suggests that the polypeptide encoded by this gene is generally present in cytotoxic T cells as a 33-kDa monomer in a disulfide-bonded dimer. The gamma chain is also expected to be phosphorylated because a sequence in its cytoplasmic domain is homologous to an active site for serine phosphorylation in the regulatory subunit of cAMP-dependent protein kinase. We describe here a cytotoxic-T-cell-associated phosphorylated protein, many of whose properties suggest that it may be the product of the T-cell gamma gene. Its phosphorylation is greatly enhanced by interleukin 2 stimulation.
Amino Acids↗
Secondary, tertiary, and quaternary structure of T-cell-specific immunoglobulin-like polypeptide chains.
To explore the possibility that the difference in antigen recognition between B and T cells derives from a structural difference in their respective antigen-specific receptors (immunoglobulins on B cells and immunoglobulin-like molecules on T cells), we compared the extracellular segments of the T-cell receptor alpha, beta, and gamma polypeptide chains and the N-terminal segment of the T-cell T8 (Lyt-2) antigen chain with the corresponding regions of immunoglobulins whose three-dimensional structures are known. The results indicate that the four T-cell polypeptide chains are organized into immunoglobulin-like domains consisting of multistranded antiparallel beta-sheet bilayers. Invariant amino acid side chains that are conserved in diverse immunoglobulins, including those that mediate domain-domain interactions and form a constant scaffold for antibody binding sites, are also conserved in the chains encoded by the T-cell receptor genes and in the N-terminal domain of T8 (Lyt-2). It appears that the binding sites of the antigen-specific T-cell alpha beta-chain receptors and of antibodies are very similar in their overall dimensions and geometry: a T-cell alpha beta receptor molecule probably has an antigen-specific binding site that is fundamentally no different than the conventional binding site of an antibody.
Amino Acid Sequence↗
Chromosomal locations of the murine T-cell receptor alpha-chain gene and the T-cell gamma gene.
Two independent methods were used to identify the mouse chromosomes on which are located two families of immunoglobulin (Ig)-like genes that are rearranged and expressed in T lymphocytes. The genes coding for the alpha subunit of T-cell receptors are on chromosome 14 and the gamma genes, whose function is yet to be determined, are on chromosome 13. Since genes for the T-cell receptor beta chain were previously shown to be on mouse chromosome 6, all three of the Ig-like multigene families expressed and rearranged in T cells are located on different chromosomes, just as are the B-cell multigene families for the Ig heavy chain, and the Ig kappa and lambda light chains. The findings do not support earlier contentions that genes for T-cell receptors are linked to the Ig heavy chain locus (mouse chromosome 12) or to the major histocompatibility complex (mouse chromosome 17).
Animals↗
B lineage--specific interactions of an immunoglobulin enhancer with cellular factors in vivo.
The mouse heavy chain immunoglobulin gene contains a tissue-specific enhancer. The enhancer and flanking sequences were studied in vivo by carrying out dimethyl sulfate protection experiments on living cells, in combination with genomic sequencing. Relative to reactions on naked DNA, there are changes (protections and enhancements) in the reactivity of guanine residues to dimethyl sulfate within the enhancer sequence in myeloma, B, and early B cells, whereas virtually no alterations appear in cells of non-B lineage. Most of the affected residues are in four clusters, in sequences homologous to the octamer 5'CAGGTGGC 3' (C, cytosine; A, adenine; G. guanine; T, thymine). The alterations in the pattern of G reactivity are consistent with the tissue-specific binding of molecules to the mouse immunoglobulin heavy chain enhancer.
Animals↗
Structure, organization, and somatic rearrangement of T cell gamma genes.
We present the initial characterization of a novel family of genes that rearrange in T cells, but do not encode either of the defined (alpha/beta) subunits of the clone-specific heterodimer of the T cell receptor. The family comprises at least three variable (V) gene segments, three constant (C) gene segments, and three junction (J) gene segments. In a cloned cytolytic T lymphocyte, 2C, one of each of these fragments has productively rearranged to yield an expressed VJC transcription unit, which shows no evidence for somatic mutation. Short sequences similar to those implicated in immunoglobulin gene and T cell receptor beta chain gene rearrangement flank the V and J segments of this family. The linkage of two of the three V gene segments has been determined: the segments lie approximately 2.5 kb apart, and are arranged head-to-head. The inverted arrangement may cast light upon the mechanisms utilized by lymphocytes for gene rearrangement.
Animals↗
The molecules of the immune system.
Explore the source record for details and available documents.
Animals↗
Regulation of T-cell receptor gene expression in human T-cell development.
A cDNA clone encoding the alpha chain of the human T-cell antigen receptor was isolated by screening a library from the human T-cell line Jurkat with a mouse alpha-chain cDNA clone. This human alpha-chain clone, together with a human antigen receptor beta-chain cDNA clone, was used to determine the stage of T-cell development at which antigen receptor mRNAs first appear. Blot-hybridization analysis of mRNA isolated from a panel of human thymic tumor lines clearly demonstrated that beta-chain transcripts could be detected in all T-lineage cells. However, alpha-chain transcripts were only found in the most phenotypically mature lines, which express the antigen receptor-associated molecule T3. Furthermore, beta-chain transcripts were abundant in RNA prepared from purified T3-negative thymocytes, whereas alpha-chain transcripts were virtually absent. From these results we conclude that alpha-chain expression occurs later in thymic ontogeny than that of the beta chain and propose that it controls surface expression of the antigen receptor-T3 complex.
Amino Acid Sequence↗
The beta 1 domain of the mouse E beta chain is important for restricted antigen presentation to helper T-cell hybridomas.
We have constructed a hybrid E beta gene by replacing the second exon of the Ed beta gene (which encodes the majority of the beta 1 protein domain) with the corresponding exon from the Eb beta gene. The hybrid gene has been introduced into a d haplotype host, the lymphoma beta-cell line A20-2J, and an E alpha E beta dimer composed of the endogenous Ed alpha chain and the product of the hybrid Ed beta/Eb beta gene was immunoprecipitated from extracts of transfected cells with an Ed alpha Eb beta-specific monoclonal antibody. Transfected cells have acquired the ability to present antigen to Ed alpha Eb beta-restricted helper T-cell hybridomas, indicating that the second exon of the gene for the E beta chain encodes sequences required for the restricted recognition of the antigen-presenting cell by the class II-restricted responder T-cell.
Animals↗
An unusual clonotypic determinant on a cytotoxic T lymphocyte line is encoded by an immunoglobulin heavy chain variable region gene.
We have detected, in a CTL clone, an mRNA homologous to an immunoglobulin heavy chain variable region (VH) gene segment. A full-length copy of this mRNA was cloned and sequenced, revealing that it is the transcript of an authentic, unrearranged VH gene. The predicted protein product of this expressed VH gene is an approximately 12-kilodalton polypeptide, with secretory signal peptide leader and no membrane-anchoring sequences. Using immunologic reagents generated against a synthetic peptide representing the carboxyl terminus of the VH protein, we detect this protein as a clonotypic cell surface molecule. Strikingly, the anti-peptide reagents also exert effects on CTL function clonotypically. Immunoprecipitation experiments suggest that the VH protein may be associated noncovalently with the consensus, major histocompatibility complex-restricted, antigen-specific T cell receptor alpha- and beta-chains on the cell surface of this CTL. We detect weakly cross-reactive material similar to the VH protein in electrophoretic mobility in other CTL clones and suggest the possibility that small VH-like molecules may constitute a novel class of receptor components with variable determinants involved in the binding of nominal antigen and contributing to overall receptor diversity.
Animals↗
Somatic mutation creates diversity in the major group of mouse immunoglobulin kappa light chains.
Using a cloned cDNA of a mouse immunoglobulin kappa light chain synthesized in a myeloma MOPC321 (V kappa-21 subgroup C) as a probe we could detect 13 germ line V kappa gene segments. 11 of these were isolated. Using a set of overlapping cloned segments, we showed that nine of these germ line V kappa genes are arranged in two linkage clusters and that they all have the same transcriptional orientation (11, 12, 22). These two clusters occupy 90 and 30 kb of chromosomal DNA and contain six and three V kappa's, respectively. We determined the complete nucleotide sequences of five germ line V kappa's and showed that three of them encode the prototype sequence of V kappa-21 subgroups B, C, and E. None of these five germ line V kappa's encodes the variant amino acid sequences of known V kappa-21 subgroups. We thus conclude that, as in the lambda 1 light chains, the variant V regions are encoded by gene segments derived by a few somatic mutations from the corresponding germ line DNA. Such somatic mutations are not restricted to sequences encoding the hypervariable regions: they also occur in sequences encoding framework regions.
Amino Acid Sequence↗