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.     

Alloantigen-specific cytotoxic clones bearing the alpha,beta T cell antigen receptor but not CD4 or CD8 molecules

The vast majority of circulating lymphocytes that express the alpha,beta TCR in association with CD3 also express either CD4 or CD8 molecules, which are thought to act as important accessory structures in HLA class II- and I-restricted T cell functions, respectively. In the current study alpha,beta TCR+ clones devoid of detectable CD4 or CD8 were generated by repeated stimulation of fresh CD3+,CD4-,CD8- cells with an allogeneic lymphoblastoid cell line in the presence of conditioned medium containing IL-2. Except for the absence of CD4 and CD8, which was associated with undetectable levels of CD4 and CD8 mRNA, the clones were phenotypically indistinguishable from classical CD3+,alpha,beta TCR+ cells. Furthermore, they mediated potent cytolysis of their specific stimulator line but did not kill irrelevant LCL or NK-sensitive targets. mAb to CD3 and the alpha,beta TCR inhibited cytolysis, suggesting that the clones use the TCR/CD3 complex to recognize and respond to their targets. mAbs to CD2 and CD11a also inhibited cytolysis, indicating that the clones use these accessory molecules to interact with their targets. Finally, cytolysis was inhibited by an HLA-A,B,C framework-specific mAb (W6/32) as well as a mAb (MA2.1) specific for an HLA-A2 epitope. These results demonstrate that CD3+,alpha,beta TCR+,CD4-,CD8- cytotoxic clones can be generated from the peripheral blood of healthy adults, and use their TCR/CD3 complexes to function in an HLA class I-restricted manner.     

Expression of functional alpha beta T cell receptor gene rearrangements in suppressor T cell hybridomas correlates with antigen binding, but not with suppressor cell function

We have examined the expression of TCR genes in 4-hydroxy-3-nitrophenyl-acetyl (NP)-specific Ts cell hybridomas. Each of three independently isolated hybridomas expressed in-frame TCR alpha-chain rearrangements derived from the original suppressor Ts cell. Different V alpha and J alpha gene segments were rearranged and expressed in each Ts cell line. The only TCR beta-chain expressed in these cells was derived from the BW5147 fusion partner. Expression of the BW5147 beta-chain was found to correlate with cell surface Ag binding, inasmuch as subclones derived from one of the original Ts lines expressed greatly reduced levels of beta-chain mRNA and no longer bound to NP-coupled RBC. Subclones that continued to express beta-chain mRNA did bind to NP-coupled RBC. This suggests that the Ag receptor on Ts hybridomas is a TCR-alpha beta dimer composed of a unique alpha-chain and the BW5147 beta-chain. Ag binding could be modulated by preincubation of Ts hybridoma cells with anti-TCR-alpha beta antibody, thereby supporting this conclusion. Suppressor factor activity was measured in the conditioned media of Ts subclones that differed by 250-fold in levels of beta-chain mRNA expression. No difference in suppressor factor activity was found; conditioned media from these subclones suppressed both plaque-forming cell responses and delayed-type hypersensitivity responses at approximately equivalent dilutions. Suppressor factor activity in the conditioned media of both a beta-chain negative subclone and a beta-chain positive subclone could be absorbed with an antibody that recognizes the TCR alpha-chain, but not with an antibody that recognizes the TCR beta-chain. We conclude that suppressor factor activity in the conditioned media of these Ts hybridomas is not derived from surface TCR-alpha beta receptors, although it does share TCR alpha-chain determinants.     

Cloned natural suppressor cell lines express the CD3+CD4-CD8- surface phenotype and the alpha, beta heterodimer of the T cell antigen receptor

Several cloned lines of natural suppressor (NS) cells were studied for their expression of the TCR complex. Almost all bore the CD3+CD4-CD8- surface phenotype with the alpha, beta TCR as judged by immunofluorescent staining. Immunoprecipitation experiments showed a spot on two-dimensional gels which is characteristic of the TCR heterodimer, but neither gamma- nor delta-chains could be precipitated with the appropriate antibodies. NS cells were stimulated to proliferate in vitro with anti-CD3 antibodies and PMA in the presence of irradiated spleen cells. However, anti-CD3 antibodies did not inhibit the suppressive activity of the NS cells. The role of the TCR complex in the suppressive function of these cells remains to be elucidated.     

Random length assortment of human and mouse T cell receptor for antigen alpha and beta chain CDR3.

In view of the recently determined three-dimensional structures of complexes formed by the T cell receptor for antigen (TCR), the processed peptide and the MHC class I molecule, it is expected that the combined configuration formed by the third complementarity determining regions (CDR3) of TCR alpha and beta chains will be very restricted in size and shape due to the limited length variations of the processed peptides. Thus, the combined TCR alpha and beta chain CDR3 lengths should have a fairly narrow distribution. This feature can be due to the selective association of long alpha chain CDR3 with short beta chain CDR3 and vice versa or due to random assortment of alpha and beta chain CDR3 of even narrower length distribution. Based on existing translated amino acid sequence data, it has been found that the latter mechanism is responsible.     

The T cell receptor: the alpha and beta chains define idiotype, and antigen and MHC specificity

Three independent T cell hybridomas were isolated that have identical specificities for antigen and products of the major histocompatibility complex (MHC). All three react with the same clone-specific antireceptor antibody, and Southern blots show all three contain the same rearranged alpha and beta genes. Variants of one of these hybridomas, DO-11.10, were isolated that had lost the ability to respond to antigen plus MHC. These proved to have lost the DO-11.10-specific alpha or beta genes or both. Fusion of alpha-loss variants to beta-loss variants restored reactivity. These results indicate that the specific recognition of antigen plus MHC is determined solely by the alpha/beta-containing T cell receptor.     

Identification of a novel T cell surface disulfide-bonded dimer distinct from the alpha/beta antigen receptor

Hybridomas were prepared from the spleen of a BALB/c mouse immunized with EL-4 T lymphoma cells. One, designated A1, was found to secrete a monoclonal antibody that reacted with two T lymphoma cells of C57BL origin, EL-4 and C6VLB, but not with normal C57BL/6 splenocytes or thymocytes, C57BL/6 T cell clones, or other T or B lymphomas by complement-mediated cytotoxicity or indirect immunofluorescent staining. Monoclonal antibody (MAb) A1 precipitated a protein that migrated at 85 kD under nonreducing and 43 kD under reducing conditions. The fact that the antigen defined by MAb A1 was a disulfide-linked dimer, together with the essentially clone-specific distribution of the reactive epitope, raised the possibility that the antibody defined an epitope of the antigen receptor. However, several additional observations revealed that the antibody defined a distinct and novel T cell surface structure. MAb 124-40, previously shown to react with the antigen receptor of C6VLB cells, reacted with variants of C6VLB that failed to express the A1 epitope. Sequential immunoprecipitation indicated that MAb A1 and MAb 124-40 reacted with distinct molecular species on C6VLB cells. Endoglycosidase digestion showed that the structure reactive with MAb A1 was not derived from that reactive with MAb 124-40 by addition of N-linked oligosaccharide residues. Two-dimensional gel electrophoretic analysis of precipitates obtained from radioiodinated C6VLB cells with MAb 124-40 resolved the alpha and beta subunits of the antigen receptor. Similar analysis of precipitates obtained with MAb A1 revealed only a single basic chain under reducing conditions, although anomalous mobility suggestive of a second, more acidic chain was observed under nonreducing conditions. Two-dimensional maps of tyrosine-containing chymotryptic peptides of the proteins isolated with MAb A1 and MAb 124-40 were completely different, suggesting that the molecules shared no peptides and were distinct in primary structure. Finally, cross-linking studies performed with a cleavable reagent indicated that the A1 molecule, unlike the antigen receptor defined with MAb 124-40, was not associated with additional, T3-like structures on the surface of C6VLB cells. Although the MAb A1 was unreactive with normal cells in cytotoxicity or staining assays, a molecule of the appropriate size was immunoprecipitated in small amounts from lysates of radioiodinated normal spleen and thymus cells. These data indicate that MAb A1 defines a novel disulfide-linked T cell surface molecule distinct from the antigen receptor.     

Depletion of mouse alpha beta T cell antigen receptor bearing lymphocytes by neonatal monoclonal antibody treatment.

Neonatal treatment with a monoclonal antibody specific for the alpha beta TCR results in mice with a long term, severe depletion in the number of alpha beta T cells in the periphery. Significant numbers of T cells reappear in the periphery about age 65 days, but these cells tend to lack expression of CD4 or CD8. Splenocytes of antibody-treated mice are less sensitive to mitogen stimulation or stimulation with MHC allogeneic cells. The level of serum IgG but not IgM was decreased by the treatment. Anti-alpha beta TCR antibody treatment decreased single-positive T lymphocytes that express high levels of the CD3/alpha beta TCR complex from the thymus, suggesting that the treatment could act in part by affecting negative selection of alpha beta TCR+ thymocytes. This treatment does not, however, detectably affect either the homing or the numbers of gamma delta T cells which are abundant in the intestinal epithelium, but which remain a minor population in the spleen and lymph nodes. This supports the hypothesis that gamma delta T cells are developmentally autonomous from alpha beta T cells. These mice provide an excellent model system for assessing the developmental and functional role of gamma delta T lymphocytes in vivo.     

The joining of germ-line V alpha to J alpha genes replaces the preexisting V alpha-J alpha complexes in a T cell receptor alpha, beta positive T cell line

To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCR alpha and beta genes in the progeny of the CD3+, CD4-/CD8- M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing V alpha-J alpha rearrangements by joining an upstream V alpha gene to a downstream J alpha segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCR beta genes are stable. These secondary recombinations are usually productive, and new forms of TCR alpha polypeptides are expressed in these cells in association with the original C beta chain. Developmental control of this V alpha-J alpha replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.     

Exclusion and inclusion of alpha and beta T cell receptor alleles.

Exclusion and inclusion of T cell receptor (TCR) genes were analyzed in alpha beta TCR transgenic mice. Both transgenes are expressed unusually early on the surface of CD4-8-, HSA+, IL-2R- thymocytes. These progenitor cells give rise to progeny, which at the single-cell level contains endogenous alpha but not beta TCR-RNA as well as protein, in addition to products encoded by the transgenes. Thus, the surface expression of an alpha beta TCR does not prevent further alpha TCR rearrangement in immature thymocytes that still transcribe RAG-1 and RAG-2 genes. Reduced levels of RAG-1 and RAG-2 RNA are detectable only in CD4+8+ TCR high cells, which result from positive selection in the thymus. The results suggest that a developing T cell may try different alpha beta TCRs for binding to thymic MHC ligands, and that recombination at the alpha locus ceases only after positive selection.     

Cross-reactive antigen recognition by an encephalitogenic T cell receptor. Implications for T cell biology and autoimmunity.

The dominant immune response to rat myelin basic protein in H-2u mice is directed against the acetylated, N-terminal peptide Ac1-11 (AcASQKR-PSQRHG). This peptide causes encephalomyelitis on injection into mice of the H-2u haplotype. Only two residues of the peptide are required for ligation of the TCR from an Ac1-11-specific T cell hybridoma. Proline at position 6 could not be substituted by any other L-amino acid, whereas glutamine at position 3 could be replaced by phenylalanine, histidine, methionine, or tyrosine. Cross-reactive recognition of these residues appears to be specific, because increasing the affinity of each analogue for its MHC restriction element, by replacing lysine with tyrosine at position 4, did not alter the pattern of cross-reactivity. For the majority of substitutions at this position, a lack of stimulation could not be explained by failure to bind to I-Au. However, competition binding studies showed that introduction of proline at position 3 reduced the efficacy of binding to I-Au. Cross-reactive analogues of Ac1-11 were injected into H-2u mice to test the extent to which cross-reactive T cell activation might lead to autoimmune disease in this model. An analogue containing methionine at position 3 caused clinical experimental autoimmune encephalomyelitis in a small percentage of H-2u mice.     

Not one odour but two: A new model for nestmate recognition

Recognition systems play a key role in a range of biological processes, including mate choice, immune defence and altruistic behaviour. Social insects provide an excellent model for studying recognition systems because workers need to discriminate between nestmates and non-nestmates, enabling them to direct altruistic behaviour towards closer kin and to repel potential invaders. However, the level of aggression directed towards conspecific intruders can vary enormously, even among workers within the same colony. This is usually attributed to differences in the aggression thresholds of individuals or to workers having different roles within the colony. Recent evidence from the weaver ant Oecophylla smaragdina suggests that this does not tell the whole story. Here I propose a new model for nestmate recognition based on a vector template derived from both the individual’s innate odour and the shared colony odour. This model accounts for the recent findings concerning weaver ants, and also provides an alternative explanation for why the level of aggression expressed by a colony decreases as the diversity within the colony increases, even when odour is well-mixed. The model makes additional predictions that are easily tested, and represents a significant advance in our conceptualisation of recognition systems.     

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.