Genomic DNA encoding rabbit T cell receptor beta-chains: isotypes and allotypes of C beta

We have discovered sequence differences in DNA encoding the first exon of rabbit T cell receptor beta-chains from unrelated rabbits that probably reflect allelic C beta 1 allotypes. Rabbit I was from a colony bred to maintain the K1-expression mutation Basilea, and rabbit II was from a colony bred to maintain the K1b9 allotype. Genomic DNA from rabbits I and II also exhibit restriction fragment length polymorphism of C beta on Southern blots. In addition, several different restriction enzyme digests of DNA from rabbit I give three bands, whereas DNA from rabbit II gives two when probed with C beta. An approximately 14-kb cloned genomic DNA fragment from rabbit I has two copies of C beta exon 1 and a 6-kb fragment has a third copy, suggesting that rabbit I has three different C beta genes. The DNA sequence of a germ-line genomic DNA fragment encoding the first exon of the beta-chain constant region from rabbit I also has an open reading frame encoding 140 amino acids immediately 5' of the C beta sequence. A corresponding sequence had previously been found in a cDNA clone from the second rabbit (rabbit II).     

Interactions between autologous T cell clones

A human CD4 clone (Mx9/9) using the V beta 8 receptor was used as antigen to generate autologous clones (termed anti-Mx9/9 clones) which proliferate in response to this clone, but not other autologous clones. This was used as an experimental model to explore the specific interactions between autologous T cells. Anti-HLA-DR monoclonal antibodies inhibited the response of the anti-Mx9/9 clones, suggesting that these clones recognize their target antigen in association with HLA-DR. Because of the specificity of the anti-Mx9/9 clones for the initiating clone (Mx9/9), but not any other autologous V beta 8- or V beta 8+ CD4 clones, the target antigen seems to be part of the T cell receptor, but not V beta 8 itself. However, the anti-Mx9/9 clones responded also to the autologous EBV line, and thus the target antigen is not known. The regulatory activity of the anti-Mx9/9 clones was assayed by coculture with their target clone. A variety of responses were seen, both inhibitory and stimulatory, which varied depending on the "conditions" of the T cell used. These results suggest that T cells interact in a complex network, perhaps as complex as the regulatory interactions between antibody molecules and B cells.     

Functional consequences of a T cell receptor D beta 2 and J beta 2 gene segment deletion

The TCR beta-chain locus of NZW mice carries an 8.8-kb deletion which encompasses the C beta 1, D beta 2, and all six J beta 2 gene segments. On a theoretical basis, the absence of D beta 2 and J beta 2 gene segments in this strain should result in a 70% reduction of the diversity of the TCR repertoire. To experimentally assess the effects of this deletion, we bred the NZW TCR beta-chain allele onto a BALB/c background and tested the ability of this new congenic strain to respond to a panel of 22 random Ag. T cells from BALB/c.beta NZW mice responded to all 22 Ag tested but the magnitude of the response to a large proportion of these Ag (11 of 22) was markedly reduced when compared with T cells from BALB/c mice. Responses to the remaining Ag were either comparable (9 of 22) or occasionally even enhanced (2 of 22) compared with BALB/c mice. In addition, we found that the frequency of V beta 6- and V beta 8.1-bearing T cells was increased by approximately 20% in BALB/c.beta NZW mice. These results suggest that D beta 2 and J beta 2 gene segments are required to maintain a diverse T cell repertoire and that their deletion from the genome may confer a significant selective disadvantage in the wild.     

The human T cell antigen receptor is encoded by variable, diversity, and joining gene segments that rearrange to generate a complete V gene

A cDNA clone YT35 , synthesized from poly(A)+ RNA of the human T cell tumor Molt 3, exhibits homology to the variable (V), joining (J), and constant (C) regions of immunoglobulin genes. We have isolated and sequenced the germ-line V and J gene segment counterparts to YT35 from a human cosmid library, and these failed to encode 14 nucleotides of the cDNA clone between the V and J regions. We postulate that these 14 nucleotides are encoded by a third gene segment analogous to the diversity (D) gene segments of immunoglobulin heavy chain genes. This T cell antigen receptor V gene appears to be assembled from three gene segments, V, D, and J, and accordingly most closely resembles immunoglobulin heavy chain V genes.     

The T cell receptor V alpha 3 gene segment is associated with reactivity to p-azobenzenearsonate

The murine T cell receptor V alpha 3 gene segment is associated with reactivity to p-azobenzenearsonate, as indicated by several independent lines of evidence. First, three out of four arsonate-reactive T cell clones tested (two I-Ad-and one I-Ak-restricted) utilized V alpha 3. Second, bulk splenic cultures enriched for arsonate/H-2d and arsonate/H-2k responsive T cells showed increased expression of V alpha 3 mRNA. Third, a V alpha 3-containing alpha chain (Ar-5: arsonate/I-Ad) transferred arsonate responsiveness to an appropriate recipient T cell (O3: ovalbumin/I-Ad). Fourth, an independently derived V alpha 3-expressing T cell clone (2C: alloreactive to Ld) showed a response to arsonate/Ld. Thus, a V alpha 3 gene segment, in conjunction with at least two different J alpha segments (J alpha 20'(Ar-5) and J alpha pHDS58(2C)) and at least three different beta chains (V beta 2(Ar-5), V beta 6(O3), and V beta 8(2C], confers reactivity to arsonate in association with at least three different MHC proteins (I-Ad, I-Ak, and Ld). We suggest that V alpha 3 encodes a protein sequence with a binding site for the arsonate hapten.     

The T cell receptor V beta 6 domain imparts reactivity to the Mls-1a antigen

A monoclonal antibody secreting hybridoma was established by fusing spleen cells from a rat immunized with a murine T cell clone, OI11, which has I-Ab restricted specificity for the male H-Y antigen and unrestricted specificity for the minor lymphocyte stimulating antigen, Mls-1a, to the mouse myeloma P3X63AG8.653 and screening for the capacity of the hybridoma supernatants to stimulate the OI11 T cell clone. An antibody (RR4-7) was found to be specific not only for the immunizing T cell clone but virtually for all T cells using the V beta 6 TCR gene product as part of their surface antigen receptor. When the expression of the V beta 6 gene in various strains of mice was analyzed, it was found that strains expressing the Mls-1a antigen contained few T cells expressing V beta 6-encoded TCRs. The majority of T cell hybridomas which expressed V beta 6-encoded TCRs were found to be reactive to the Mls-1a antigen. These data confirm the finding of H. R. MacDonald et al. (Nature (London) 332, 40, 1988) that most TCRs encoded by the V beta 6 gene have a biased specificity for the Mls-1a antigen.     

Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants.

The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.     

Rearranged beta T cell receptor genes in a helper T cell clone specific for lysozyme: no correlation between V beta and MHC restriction

The helper T cell clone 3H.25 is specific for hen egg white lysozyme and the class II MHC molecule I-Ab. This TH cell has three rearrangements in the beta-chain gene family-a V beta-D beta-J beta 1 and a D beta 2-J beta 2 rearrangement on one homolog and a D beta 1-J beta 2 rearrangement on the other. These observations demonstrate that this functional T lymphocyte expresses only a single V beta gene segment and, accordingly, exhibits allelic exclusion of beta-chain gene expression. The rearranged 3H.25 V beta gene segment is the same as that expressed in a T helper cell specific for cytochrome c and an I-Ek MHC molecule. Thus, there is no simple correlation between the V beta gene segment and antigen specificity or MHC restriction.     

Protection against experimental encephalomyelitis. Idiotypic autoregulation induced by a nonencephalitogenic T cell clone expressing a cross-reactive T cell receptor V gene.

The recovery process in experimental autoimmune encephalomyelitis (EAE) in Lewis rats is characterized by an increasing diversity of T cell clones directed at secondary epitopes of myelin basic protein. Of particular interest, residues 55 to 69 of guinea pig basic protein could induce protection against EAE. A nonencephalitogenic T cell clone, C455-69, that was specific for this epitope transferred protection against both active and passive EAE. Clone C4 was found to express V beta 8.6 in its Ag receptor, and residues 39 to 59 of the TCR V beta 8.6 sequence were found to be highly crossreactive with the corresponding residues 39 to 59 of TCR V beta 8.2, which is known to induce protective anti-idiotypic T cells and antibodies. Like the TCR V beta 8.2 peptide, the V beta 8.6 sequence induced autoregulation and provided effective treatment of established EAE. Thus, the EAE-protective effect of the guinea pig basic protein 55-69 sequence was most likely mediated by T cell clones such as C4 that could efficiently induce anti-TCR immunity directed at a cross-reactive regulatory idiotope.     

Chimeric immunoglobulin-T cell receptor proteins form functional receptors: implications for T cell receptor complex formation and activation

We constructed chimeric receptor chains in which an immunoglobulin heavy chain variable region (VH) from a phosphorylcholine-specific antibody is substituted for T cell receptor (Tcr) alpha and beta V regions. We demonstrate that the VH region joined to either the C alpha or the C beta region can form stable chimeric proteins in EL4 T cells. Both chimeric receptor chains associate with CD3 polypeptides in functional receptor complexes and respond to phosphorylcholine coupled to Sepharose beads. The VH-C alpha chimeric chain associates with the EL4 beta chain, while the VH-C beta chimeric protein appears to form either a homodimer or a heterodimer with the native EL4 beta chain. Thus, functional receptor complexes can be formed using two C beta regions, and the C alpha region may not be required for CD3 association and surface expression of Tcr complexes.     

Rearrangement and expression of T-cell antigen receptor genes in human T-lymphocyte tumor lines and normal human T-cell clones: evidence for allelic exclusion of Ti beta gene expression and preferential use of a J beta 2 gene segment.

The gene encoding the beta chain of the human T-cell receptor for antigen is composed of variable (V), diversity (D), joining (J), and constant (C) gene segments which undergo specific rearrangements during T-lymphocyte ontogeny. Southern blot analyses of seven human T-cell tumor lines and normal human T-lymphocyte clones revealed that most of these T-cell lines rearrange their Ti beta genes differently. The T-cell tumor line HPB-MLT rearranges and transcribes both of its Ti beta genes. Cloning and sequencing of the Ti beta cDNAs corresponding to these rearrangements revealed that one of the rearranged Ti beta genes is defective, while the other is functional and corresponds to the Ti beta protein expressed on the surface of these cells. Thus, this cell line displays a pattern of allelic exclusion of Ti beta gene expression. A comparison of four C beta 2-containing Ti beta cDNAs from three different cell lines revealed that three of the four utilize the same J beta 2.5 gene segment joined to different D beta and V beta genes, suggesting that there may be preferential use of this J gene during J beta 2 rearrangements. Hybridization analyses with probes for the alpha and beta genes of the T-cell receptor and the T-cell-specific T gamma gene revealed that HPB-MLT cells appear to express approximately equivalent amounts of RNA corresponding to each of the rearranged Ti alpha and Ti beta genes. However, they express a much lower level of T gamma RNA.     

Murine AIDS superantigen reactivity of the T cells bearing V beta 5 T cell antigen receptor.

A B cell line, B6-1710, that expresses the defective virus known to induce murine AIDS stimulates a large fraction of nonprimed splenic T cells. Analysis of the T cell population responding to the B6-1710 for TCR V beta-chain usage revealed that, in addition to the previously reported V beta 5-chain-positive T cells, T cells bearing V beta 11 and V beta 12 are also specifically enriched. We have established V beta 5+ T cell lines, clones, and hybridomas expressing identical TCR with different CD4/CD8 phenotypes and demonstrated that T cell reactivity to B6-1710 is, although not absolute, dependent on the presence of CD4 molecules. Further analysis of T cell hybridomas with known J beta-chain usage revealed that D beta- and J beta-chain usage do not play crucial roles in T cell reactivity to B6-1710 B cells. However, T cell hybridomas derived from TCR-V beta gene transgenic mice were found to be heterogeneous for their reactivity to B6-1710, suggesting that the V alpha-chains associating with the transgenic V beta-chain determine T cell responsiveness to B6-1710. These data clearly demonstrate that T cell reactivity to a murine AIDS virus expressing B cell line resembles that previously reported for Mls-like superantigens.