T cell antigen receptors in autoimmunity

Three mAb to variable region determinants of the alpha/beta-chain TCR were used to detect discrete populations of peripheral blood T cells. T cells sharing a TCR determinant defined by such an antibody presumably use the same or similar TCR V or J genes for their alpha- or beta-chains. Thus analysis with these mAb provides a tool to investigate TCR gene usage and expression. Since autoantigen specific T cells may play an important role in initiating autoimmune diseases, TCR were analyzed in different autoimmune diseases and control groups including rheumatoid arthritis, Graves disease, idiopathic thrombocytopenic purpura, psoriasis, SLE, insulin-dependent diabetes mellitus, and in nonautoimmune control diseases and normals. Purified T cells were stained by indirect immunofluorescence with three mAb to TCR variable regions: mAb S511 stains 1.8 +/- 0.9% (mean +/- 2 SD), mAb C37 stains 3.4 +/- 1.5% and mAb OT145 stains from 0 to 6% of T cells from normal donors. Several individuals were identified with expanded subsets of positive T cells. One patient with adult ITP followed during a 12-mo period consistently had elevated percentages of T cells staining with the mAb OT145 (15.9 to 24.5%). These cells were found to be exclusively CD8+. By Southern blotting DNA prepared from these OT145+, CD8+ cells, but not DNA from the patient's OT145- T cells, revealed a clonal rearrangement using a beta-chain C region probe. Thus this patient had a monoclonal expansion of CD8+, OT145+ cells. Hyperexpression of a TCR variable region, as defined by the available mAb, could not be associated with any of the diseases studied. Examination of T cells at the site of autoimmunity, such as T cells from rheumatoid arthritis synovial fluid, revealed normal percentages of cells staining with these mAb. Immunoperoxidase staining of psoriatic lesional skin showed no striking enrichment of T cells bearing one or the other TCR type.     

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.     

Characterization and expression of the human alpha beta T cell receptor by using a framework monoclonal antibody

A monoclonal antibody (mAb) with framework reactivity against the T cell receptor (TCR) alpha beta complex is characterized. The mAb, beta Framework 1 (beta F1) is capable of immunoprecipitating the TCR alpha beta complex from 125I-labeled human T cell tumors, immunocompetent T cell clones, and peripheral blood lymphocytes (PBL). beta F1 recognizes the separated TCR beta subunit in Western blotting. Because it does not bind to the surface of viable T cells but does react with the plasma membrane form of the TCR after treatment with membrane solubilizing agents, the beta F1 mAb reacts with a "hidden" determinant on the TCR beta subunit. After solubilization with 70% ethanol, the TCR alpha beta complex is shown to exist on greater than 92% of T3+ human PBL, whereas 2 to 8% of T3+ PBL do not react with the mAb. The beta F1 mAb demonstrates the existence of differently glycosylated surface 125I-labeled TCR alpha-chains (alpha, alpha', alpha") in association with a common TCR beta-chain on the HPB-MLT T cell leukemia. Reactivity of the beta F1 mAb on thymus tissue sections is similar to that of anti-Leu-4 (anti-T3). The beta F1 mAb should prove useful as a research tool for both the immunochemical characterization and isolation of virtually any alpha beta T cell receptor, whether from individual T cell clones or polyclonal populations of T lymphocytes. Recognition of T cell receptors in histologic tissue sections suggests that the beta F1 mAb may be useful in the clinical diagnosis of T cell lineage neoplasms. In failing to recognize all T3+ lymphocytes, it allows the identification of novel populations of T3+ lymphocytes that may express non-alpha, non-beta T cell receptors.     

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis (MAM). VIII. Selective activation of T cells expressing distinct V beta T cell receptors from various strains of mice by the "superantigen" MAM

Mycoplasma arthritidis T cell mitogen (MAM), in association with its MHC ligand, is recognized by T cells that express TCR-alpha/beta assembled with a product(s) of the V beta 8 gene family. We show here that lymphocytes from mice which fail to express V beta 8 products can also be activated by MAM and the resulting cultures exhibit a marked increase in V beta 6 TCR-bearing cells. Evidence was also obtained that MAM can activate T cells that express all three V beta 8 TCR. The mAb, F23.1, which recognizes all V beta 8 gene products, was strongly inhibitory for MAM-induced proliferation of CBA cells whose T cell repertoire for MAM consists of T cells that express V beta 8.2 and 8.3 TCR. In contrast, the F23.1 mAb was only weakly inhibitory for BALB/c splenocytes which express V beta 6 TCR in addition to all three V beta 8 TCR. Involvement of V beta 8.1, 8.2, 8.3, and V beta 6 in MAM-induced proliferation was confirmed by expanding lymphocyte cultures in the presence of MAM and phenotyping the activated cells for expression of individual V beta TCR. There was also evidence for a selective activation of T cells bearing specific V beta TCR because BALB/c T cell populations expanded with MAM were comprised of 46.2% V beta 8.2+ cells, 18.6% V beta 8.1+ cells, 7.6% V beta 8.3+ cells and 6.7% V beta 6+ cells. Recent studies suggest that the newly described "superantigens" including the staphylococcal enterotoxins and the self minor lymphocyte-stimulating Ag activate T cells in a manner similar to that described earlier for MAM. The discovery of shared recognition of these proteins by specific V beta TCR strongly suggests that MAM belongs to the superantigen protein family, the members of which may share cross-reactive epitopes. Inasmuch as MAM is produced by an organism which induces chronic joint disease, our findings provide the basis for a new model to study the role of superantigens in the development of chronic autoimmune type diseases.     

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.     

Schistosoma mansoni: incorporation and metabolism of protein amino acids in vitro

1. The amino acid composition of total proteins in six stages of the life cycle of Schistosoma mansoni was determined by routine autoanalysis of acid hydrolysates. Aspartate, glutamate and glycine were consistently the most abundant protein amino acids in all stages. 2. Incorporation of each of the protein amino acids into adult and egg proteins was determined using 72 hr cultures in complex media. Incorporation rates varied widely and there was no correlation between abundance in protein and the rate of incorporation. 3. Only five amino acids were interconverted to other amino acids which were themselves incorporated into worm and egg proteins (ala, arg, asp, gly, ser); of these only two (glu from ala and pro from arg) appeared to be of quantitative significance. Exogenous glucose yielded only three protein amino acids (ala, asp, glu). 4. The data are considered in the light of differences in egg and adult protein synthesis and with particular regard to potential chemotherapy at this level.     

Characterization of a rat monoclonal antibody specific for a determinant encoded by the V beta 7 gene segment. Depletion of V beta 7+ T cells in mice with Mls-1a haplotype.

We have generated a rat mAb, TR310, which recognizes a determinant encoded by the murine V beta 7 gene segment of the TCR. TR310 immunoprecipitates TCR from cell lysates, co-modulates with CD3, and can be used for immunofluorescence staining of T cells. By using this antibody, we found that the average percentage of V beta 7+ peripheral T cells in Mls-1b mice was 3.8%, but only 0.8% in Mls-1a mice. A similar difference was also observed in the mature TCRhi thymocyte subsets, suggesting that V beta 7+ T cells are deleted during intrathymic maturation in Mls-1a mice. TR310 should prove to be a valuable reagent in further studies of the TCR repertoire and the analysis of factors which alter it.     

Staphylococcal enterotoxin-dependent lysis of MHC class II negative target cells by cytolytic T lymphocytes.

The enterotoxins of Staphylococcus aureus (SE) are extremely potent activators of human and mouse T lymphocytes. In general, T cell responses to SE are MHC class II dependent (presumably reflecting the ability of SE to bind directly to MHC class II molecules) and restricted to responding cells expressing certain T cell receptor beta-chain variable (TCR V beta) domains. Recently we demonstrated that CD8+ CTL expressing appropriate TCR V beta could recognize SE presented on MHC class II-bearing target cells. We now show that MHC class II expression is not strictly required for T cell recognition of SE. Both human and mouse MHC class II negative target cells could be recognized (i.e., lysed) in a SE-dependent fashion by CD8+ mouse CTL clones and polyclonal populations, provided that the CTL expressed appropriate TCR V beta elements. SE-dependent lysis of MHC class II negative targets by CTL was inhibited by mAb directed against CD3 or LFA-1, suggesting that SE recognition was TCR and cell contact dependent. Furthermore, different SE were recognized preferentially by CTL on MHC class II+ vs MHC class II- targets. Taken together, our data raise the possibility that SE binding structures distinct from MHC class II molecules may exist.     

The human T cell receptor beta-chain gene complex contains at least 57 variable gene segments. Identification of six V beta genes in four new gene families.

The human TCR beta-chain gene complex includes at least 57 variable (V) gene segments, a number estimated using a combination of Southern blots of conventional and pulsed field gels, sequence analysis of cDNA clones, and from the analysis of genomic cosmid and phage clones. This number includes six TCR beta-chain V genes in four new families identified here by sequence analysis of clones derived from a human TCR beta-chain specific cDNA library. Comparison of the sequences of the new V beta genes with previously reported V beta sequences reveals predicted similarities but less than 75% nucleic acid identity that establishes them as new V beta families. One of the new V beta gene families includes three genes and the other three are single member families. Identification of these six new V beta genes falling into four V beta families brings the total number of transcribed human V beta families to 24 and makes it possible to refine the estimate of the total number of human TCR V beta genes to 57.     

Expansion of murine T cells bearing a unique T cell receptor beta-chain in Friend virus-induced tumor in situ.

Heterogeneity of V alpha 1+ and V beta 10+ TCR alpha beta-chains, which are predominantly used in anti-FBL-3 CTL clones established in vitro, was investigated at a nucleotide level in FBL-3 tumor-infiltrating lymphocytes (TIL) in vivo. The majority (90%) of V beta 10+ beta-chains dominated in TIL used homogeneous V beta 10D beta 2.1 sequences identical to that used in the T cell clones with cytotoxic functions. The homogeneous TCR beta-chain expression was dominant and found to be about 10% of the total TCR beta-chains in the TIL population, which was a greater than 300-to 900-fold increase than in the regional lymph nodes. This is in good agreement with the in vitro data showing that about 11% CTL clones used the homogeneous V beta 10D beta 2.1+ beta-chain. However, the J beta segment does not seem to contribute greatly to the recognition and selection of this TCR because some of homogeneous VD+ beta-chains were associated with J beta segments other than J beta 2.7 of the CTL clones. The frequency of the V alpha 1J alpha 112-2+ alpha-chain expression of the CTL type was much less (3- to 80-fold increase compared to that of lymph node) and also varied in sample materials, indicating the lower contribution of the alpha-chain for the oligoclonality of the TCR. The results were also confirmed by quantitative PCR and RNase protection assays. This suggests that the dominant expression of the homogeneous TCR beta-chain is due to the expansion of the particular anti-FBL-3 CTL in the tumor in situ. Also, the TCR beta-chain, especially the V beta D beta region, rather than alpha-chain is more important for the recognition and selection of the anti-FBL-3 TIL with cytotoxic functions.     

Beta-chain broadens range of CD8 recognition for MHC class I molecule.

It is known that the alpha-chain of CD8 binds to a negatively charged loop composed of residues 223 to 229 on MHC class I Ag and that binding of CD8 alpha enhances Ag recognition of T cells. We have recently shown that the mouse CD8 alpha homodimer does not bind to either the HLA class I alpha 3 domain or a mutant of H-2Kb Ag containing a substitution of glutamine for methionine at residue 224, which brings this residue toward the human consensus. Here we report a complementary study of the CD8 beta-chain. The functional role of the CD8 beta-chain was analyzed by using four T cell hybridoma lines expressing mouse CD8 alpha and transfected with the mouse CD8 beta gene. As compared with the lines expressing only CD8 alpha, allorecognition of the chimeric H-2Kb Ag that contains the HLA class I alpha 3 domain was enhanced in lines expressing both CD8 alpha and -beta. This enhancement was blocked by either anti-CD8 mAb or anti-HLA class I alpha 3 domain mAb. In addition, we show that CD8 alpha beta binds the H-2Kb mutant Ag at residue 224. These results suggest that the beta-chain allows the CD8 alpha beta heterodimer to recognize the chimeric H-2Kb Ag. A model for the role of the beta-chain is presented.     

Cutting edge: Evidence of direct TCR alpha-chain interaction with superantigen

Superantigens are known to activate a large number of T cells. The SAg is presented by MHC class II on the APC and its classical feature is that it recognizes the variable region of the beta-chain of the TCR. In this article, we report, by direct binding studies, that staphylococcal enterotoxin (SE) H (SEH), a bacterial SAg secreted by Staphylococcus aureus, instead recognizes the variable alpha-chain (TRAV27) of TCR. Furthermore, we show that different SAgs (e.g., SEH and SEA) can simultaneously bind to one TCR by binding the alpha-chain and the beta-chain, respectively. Theoretical three-dimensional models of the penta complexes are presented. Hence, these findings open up a new dimension of the biology of the staphylococcal enterotoxins.     

I-E-independent deletion of V beta 17a+ T cells by Mtv-3 from the nonobese diabetic mouse.

Analysis of TCR beta-chain V region (V beta) frequency among NOD lymphocytes reveals a profound depletion of V beta 3+ T cells, and a recent study has linked this phenomenon to the Mtv-3 insertion on chromosome 11. When the V beta 17a gene segment is introduced into mice with an nonobese diabetic mouse background, T cells bearing the TCR encoded by this gene segment are also dramatically reduced in frequency. Deletion of V beta 17a+ T cells segregates with deletion of T cells bearing V beta 3 and occurs in the absence of I-E, which had been shown in previous studies to be a major deleting element for V beta 17a+ thymocytes.     

Mapping of distinct serologic and T cell recognition epitopes on an HLA-DR beta-chain.

A new DR beta-chain allele is defined that is identical to the previously described DR6b molecule except for the first hyperpolymorphic region, where the new allele displays the same polymorphisms found on DR8 and DR12 genes. Two distinct epitopes have been mapped on this new allele. The polymorphism in common with DRw8 and DRw12 is recognized by mAb GS313-9D11. However, alloreactive T cell clones specific for DR6b cells (Dw9) recognize this allele, whereas Dw8-specific T cell clones do not. The mAb determinant maps to the first beta-sheet and probably involves a polymorphic residue lying outside the helix. The binding of mAb 9D11 to this region does not interfere with TCR binding. Alloreactive T cell recognition is associated with polymorphisms located predominantly on the alpha-helical portion of the molecule.     

Cloned suppressor T cells derived from mice tolerized with conjugates of antigen and monomethoxypolyethylene glycol. Relationship between monoclonal T suppressor factor and the T cell receptor

Cloned Ts cells specific for the Ag, human monoclonal (myeloma) IgG, were derived from spleen cells of mice that had been immunosuppressed by treatment with a tolerogenic conjugate of HIgG and monomethoxypolyethylene glycol. The cloned Ts cells (clone 23.32) suppressed in vitro antibody responses in an Ag-specific and MHC-restricted manner. By FMF with appropriate antibody reagents, these cells were shown to be Thy-1+, CD4-, CD5-, and CD8+ and to express CD3 and the alpha beta-TCR. These results are consistent with the view that Ts cells use Ag recognition structures similar to those reported for Th cells and CTL. A soluble factor (TsF) extracted from the cloned Ts cells also suppressed in vitro antibody responses in an Ag-specific and H-2Kd-restricted manner, i.e., restricted to MHC class I molecules. The suppressive activity of this TsF could be abrogated by addition of mAb H28-710 that reacts with a determinant on the alpha-chain of TCR. Moreover, the TsF bound to and could be recovered from an immunosorbent consisting of the anti-alpha-TCR mAb H28-710 coupled to Sepharose 4B. In contrast, the TsF was not bound by immunosorbents consisting of mAb to the beta-chain of TCR (H57-597) or to V beta 8 (F23.1). It was, therefore, concluded that the TsF of clone 23.32 is serologically related to the alpha-chain of the TCR; however, it is not identical to TCR, because it lacks the determinants expressed on the TCR beta-chain that are recognized by the two anti-beta mAbs used in this study.     

Single amino acid replacements in an antigenic peptide are sufficient to alter the TCR V beta repertoire of the responding CD8+ cytotoxic lymphocyte population.

Cytotoxic CD8+ T lymphocytes are activated upon the engagement of their Ag-specific receptors by MHC class I molecules loaded with peptides 8-11 amino acids long. T cell responses triggered by certain antigenic peptides are restricted to a limited number of TCR V beta elements. The precise role of the peptide in causing this restricted TCR V beta expansion in vivo remains unclear. To address this issue, we immunized C57BL/6 mice with the immunodominant peptide of the vesicular stomatitis virus (VSV) and several peptide variants carrying single substitutions at TCR-contact residues. We observed the expansion of a limited set of TCR V beta elements responding to each peptide variant. To focus our analysis solely on the TCR beta-chain, we created a transgenic mouse expressing exclusively the TCR alpha-chain from a VSV peptide-specific CD8+ T cell clone. These mice showed an even more restricted TCR V beta usage consequent to peptide immunization. However, in both C57BL/6 and TCR alpha transgenic mice, single amino acid replacements in TCR-contact residues of the VSV peptide could alter the TCR V beta usage of the responding CD8+ T lymphocytes. These results provide in vivo evidence for an interaction between the antigenic peptide and the germline-encoded complementarity-determining region-beta loops that can influence the selection of the responding TCR repertoire. Furthermore, only replacements at residues near the C terminus of the peptide were able to alter the TCR V beta usage, which is consistent with the notion that the TCR beta-chain interacts in vivo preferentially with this region of the MHC/peptide complex.     

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.