Molecular genetic analysis of 178 I-Abm12-reactive T cells

We have studied the genetic diversity of the TCR repertoire to the murine alloantigen I-Abm12 by generating a panel of 178 C57BL/10-derived I-Abm12-reactive T cell hybridomas. The expression of V alpha and V beta gene families was examined in this panel and the frequency of expression of V beta, but not ofV alpha, gene families differed significantly from that observed in a companion panel of random C57BL/10-derived hybridomas. The V beta 5 gene family was expressed significantly less frequently while the V beta 14, V beta 15, and V beta 16 genes were expressed significantly more frequently in the panel of I-Abm12-reactive than in the panel of random hybridomas. The junctional regions (VJ alpha and VDJ beta) of TCR V alpha and V beta genes from selected I-Abm12-specific hybridomas were amplified using the polymerase chain reaction, and directly sequenced. Surprisingly, no conserved J alpha, D beta, J beta, or N region-encoded sequences among these selected I-Abm12-reactive TCRs were identified. Thus, the T cell response to an I-A alloantigen that differs by only three amino acid residues from the I-A molecule of the responding strain is genetically complex but nonrandom. We have estimated that the repertoire to this alloantigen is comprised of at least 37 different TCRs.     

Expression of a transgenic T cell receptor beta chain enhances collagen- induced arthritis

SWR/J transgenic (tg) mice were generated expressing the TCR beta chain derived from an anticollagen type II (CII) arthritogenic T cell clone. The SWR/J strain was selected because it is resistant to collagen-induced arthritis (CIA) and lacks the V beta gene segment used by the T cell clone. Expression of the tg beta chain on all thymocytes and peripheral lymph node T cells led to a more efficient anti-CII immune response, but did not confer CIA susceptibility to SWR/J mice. Nevertheless, this tg beta chain enhanced predisposition to CIA as (DBA/1 x SWR) F1 beta tg mice were more susceptible than normal F1 littermates. Our results demonstrate that the expression of the tg beta chain contributes to CIA susceptibility, but by itself it is not sufficient to overcome CIA resistance in the SWR/J strain.     

Collagen-induced arthritis in T cell receptor V beta congenic B10.Q mice

B10.Q (H-2q) mice congenic for the truncated T cell receptor (TCR) V beta a and V beta c haplotypes were derived to examine the influence of TCR V beta genomic deletions in murine collagen-induced arthritis (CIA). Previous studies using gene complementation and segregation analyses suggested that in SWR (H-2q) mice, possession of the V beta a gene deletion results in CIA resistance. However, other studies have suggested alternative hypotheses. Thus, analysis of TCR V beta congenic mice allows for direct examination of V beta genotypes in CIA control. After immunization with bovine type II collagen, B10.Q-V beta a mice showed no difference in arthritis susceptibility, onset, or severity when compared with prototype B10.Q mice. In contrast, B10.Q-V beta c mice, which lack the V beta 6, 15, 17, and 19 families in addition to the V beta a deletion, were highly resistant to CIA. In vivo depletion of V beta 6+ T cells in B10.Q-V beta a mice significantly delayed arthritis onset suggesting that, among those V beta genes present in V beta a but absent in V beta c, V beta 6+ T cells contribute to arthritogenesis. Our findings show that, in B10.Q-V beta congenic mice, while the V beta a genotype does not prevent CIA, the highly truncated V beta c genotype renders B10.Q mice resistant to CIA. Thus, deletions within the V beta TCR genome can indeed influence CIA and suggests that the TCR repertoire displays only marginal flexibility in response to arthritogenic stimuli.     

H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity.

We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2Kd selects CTLs that bear TCRs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.     

T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire

We report here the first extensive study of a T cell repertoire for a class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) response. We have found that the T cell receptors (TCRs) carried by 28 H-2Kd-restricted CTL clones specific for a single Plasmodium berghei circumsporozoite nonapeptide are highly diverse in terms of V alpha, J alpha, and J beta segments and aminoacid composition of the junctional regions. However, despite this extensive diversity, a high proportion of the TCRs contain the same V beta segment. These results are in contrast to most previously reported T cell responses towards class II MHC-peptide complexes, where the TCR repertoires appeared to be much more limited. In our study, the finding of a dominant V beta in the midst of otherwise highly diverse TCRs suggests the importance of the V beta segment in shaping the T cell repertoire specific for a given MHC-peptide complex. As an additional finding, we observed that nearly all clones have rearranged both TCR alpha loci. Moreover, as many as one-third of the CTL clones that we analyzed apparently display two productive alpha rearrangements. This argues against a regulated model of sequential recombination at the alpha locus and consequently raises the question of whether allelic exclusion of the TCR alpha chain is achieved at all.     

Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain

CD4-CD8- (double negative [DN]) alpha/beta T cells are a largely uncharacterized subpopulation of unknown function. To investigate whether these cells are selected to recognize particular antigens or antigen-presenting molecules, DN alpha/beta T cells were purified from the peripheral blood of five normal donors and their T cell receptor (TCR) alpha and beta chains were examined. Random cloning of TCR alpha chains by single-sided polymerase chain reaction (PCR) amplification identified an invariant rearrangement between V alpha 24 and J alpha Q, with no N region diversity, which was expressed preferentially by DN alpha/beta T cells from all donors. Random cloning also identified a precise V alpha 7.2-J alpha (IGRJa14) rearrangement, with two variable amino acids encoded in the V-J junction, which was enriched in the DN alpha/beta T cell preparations from some, but not all, donors. Analysis of TCR beta chains by quantitative PCR amplification demonstrated that the expression of four V beta gene families, V beta 2, 8, 11, and 13, was markedly increased in these DN alpha/beta T cell preparations. The expression of particular TCRs by DN alpha/beta T cells from multiple donors indicates that these cells, or at least a subpopulation of cells with this phenotype, recognize a limited spectrum of antigens and suggests that they may use nonpolymorphic antigen-presenting molecules.     

Junctional sequences of fetal T cell receptor beta chains have few N regions

T cell receptors (TCRs) and immunoglobulins (Igs) derive a large fraction of their repertoire from diversity generated at the junctions of the V, D, and J coding segments. This diversity is derived both from the random deletion of nucleotides from the ends of coding regions and from the subsequent addition of nontemplated N region nucleotides. While the vast majority of TCRs and Igs from adult mice have N regions, less than 5% of both TCR-gamma/delta and Ig from fetal and neonatal mice have N regions. This study analyzed the ontogeny of junctional diversity of TCR-alpha/beta. Genomic DNA or C beta-primed cDNA was prepared from thymocytes of mice at varying stages in ontogeny, and the rearranged V beta 8 or V beta 5 sequences were amplified by polymerase chain reactions. Sequencing of the V beta-D beta-J beta junctions showed few N regions early in ontogeny, although the fraction of sequences with N regions exceeded that previously reported for Ig and for TCR-gamma/delta. N regions were found in 13% of V beta junctional sequences from day 18-19 fetal thymocytes, 33% of sequences from newborn thymocytes, 76% of sequences from day 4 postnatal thymocytes, and 88% of sequences from 5-wk-old thymocytes. In addition, nonrandom usage of the D beta and J beta segments was observed in both fetal and adult TCR sequences. While the usage of each of the six J beta 2 segments was different, the same pattern of usage was seen regardless of whether D beta 1 or D beta 2 was used, suggesting that a factor controlling the rate of usage of each J segment is intrinsic to the J gene itself. Since TCRs derive so much of their diversity from N regions, the relative paucity of N regions in fetal alpha/beta T cells would create a fetal TCR-alpha/beta repertoire that would be quite different from, and smaller than, the adult repertoire. The lack of N regions might be predicted to limit the range of affinities of TCR-MHC + peptide interactions, which may have important consequences for positive and negative selection of fetal and newborn T cells.     

Preferential V beta gene usage and lack of junctional sequence conservation among human T cell receptors specific for a tetanus toxin-derived peptide: evidence for a dominant role of a germline-encoded V region in antigen/major histocompatibility complex recognition.

To investigate the structural and genetic basis of the T cell response to defined peptide/major histocompatibility (MHC) class II complexes in humans, we established a large panel of T cell clones (61) from donors of different HLA-DR haplotypes and reactive with a tetanus toxin-derived peptide (tt830-844) recognized in association with most DR molecules (universal peptide). By using a bacterial enterotoxin-based proliferation assay and cDNA sequencing, we found preferential use of a particular V beta region gene segment, V beta 2.1, in three of the individuals studied (64%, n = 58), irrespective of whether the peptide was presented by the DR6wcI, DR4w4, or DRw11.1 and DRw11.2 alleles, demonstrating that shared MHC class II antigens are not required for shared V beta gene use by T cell receptors (TCRs) specific for this peptide. V alpha gene use was more heterogeneous, with at least seven different V alpha segments derived from five distinct families encoding alpha chains able to pair with V beta 2.1 chains to form a tt830-844/DR-specific binding site. Several cases were found of clones restricted to different DR alleles that expressed identical V beta and (or very closely related) V alpha gene segments and that differed only in their junctional sequences. Thus, changes in the putative complementary determining region 3 (CDR3) of the TCR may, in certain cases, alter MHC specificity and maintain peptide reactivity. Finally, in contrast to what has been observed in other defined peptide/MHC systems, a striking heterogeneity was found in the junctional regions of both alpha and beta chains, even for TCRs with identical V alpha and/or V beta gene segments and the same restriction. Among 14 anti-tt830-844 clones using the V beta 2.1 gene segment, 14 unique V beta-D-J beta junctions were found, with no evident conservation in length and/or amino acid composition. One interpretation for this apparent lack of coselection of specific junctional sequences in the context of a common V element, V beta 2.1, is that this V region plays a dominant role in the recognition of the tt830-844/DR complex.     

Structure-function relationships among highly diverse T cells that recognize a determinant from influenza virus hemagglutinin.

We have analyzed the structural and genetic basis for T cell recognition of the complex formed between antigen and class II products of the major histocompatibility complex by performing sequence analysis of T cell receptors (TCRs) induced in response to the helper T cell site 1 of the influenza virus hemagglutinin. The results demonstrate, first, that structurally highly diverse TCRs can be utilized in recognition of the same antigen/I-Ed complex: 12 of 13 TCRs utilize unique V alpha/V beta gene segment combinations, suggesting that approximately 70 different V alpha/V beta combinations are available to BALB/c mice in response to this determinant. Second, comparison of these sequences with the ability of each hybridoma to recognize a panel of peptide analogues suggests that alpha and beta chains of these TCRs frequently determine specificity for the NH2-terminal and the COOH terminal portions, respectively, of the site 1 determinant.     

The V beta repertoire of mouse gut homodimeric alpha CD8+ intraepithelial T cell receptor alpha/beta + lymphocytes reveals a major extrathymic pathway of T cell differentiation

Gut intraepithelial lymphocytes (IEL) contain two independent T cell receptor alpha/beta + T cell populations, with different V beta repertoires. In DBA/2 mice (Mlsa, IE+), the CD4+ and heterodimeric alpha/beta CD8+ thymodependent T cell pool shows the same deletion of V beta 6, 8.1, and 11+ cells as found in peripheral lymphoid organs. In contrast, such deletions are not observed in the pool of IEL bearing homodimeric alpha CD8+ chains, in which these V beta families are frequently observed in high amounts. The size of this gut homodimeric alpha CD8+ IEL pool and its different V beta repertoire selection demonstrate the existence of a major extrathymic pathway of T cell differentiation with a gut-restricted localization. The large amount of the thymo-independent, homodimeric alpha CD8+ IEL found in the small bowel may contribute to a first line of defense against exogenous superantigens.     

Transient rearrangements of the T cell antigen receptor alpha locus in early thymocytes

The dull Ly-1 double-negative (Ly-1dull, Lyt-2-, L3T4-) subpopulation appears to be the major precursor group of T lymphocytes in the thymus. In examining the status of the alpha, beta, and gamma chain genes for T cell receptors (TCR) in this population of cells and hybridomas made from them, we find that all of these loci appear to begin DNA rearrangements in a nearly simultaneous fashion. In the case of the gamma genes, these involve V gamma----J gamma C gamma gene rearrangements; with the beta chain genes, both D beta----J beta C beta rearrangement and V beta----D beta J beta C beta rearrangements are evident; and in the case of the alpha locus, assayed in part by pulsed-field gel electrophoresis, they take the form of a novel series of rearrangements occurring 80 kb or more 5' to the C alpha gene. These alpha locus rearrangements are well away from any of the J alpha gene segments found in cDNA clones to date and are deleted in most mature thymocytes and functional T cell lines. Therefore they appear to represent a distinct class of rearrangement that occurs before V alpha----J alpha joining. These distinctions between the character of the TCR gene rearrangements in these cells represent useful markers in further distinguishing different stages of T cell differentiation within this compartment of early T cells. In addition, the unexpected discovery of clonal rearrangements so far away from any of the expressed J alpha gene segments, and at a stage where there is little or no stable C alpha RNA present, has interesting implications for the hierarchy of TCR gene expression.     

Presentation of antigen by mixed isotype class II molecules in normal H-2d mice.

A panel of DBA/2 T cell hybridomas specific for the sperm whale myoglobin epitope 110-121 was found to recognize antigen presented by the mixed isotype class II molecule E alpha dA beta d. The response was blocked by monoclonal antibodies specific for E alpha and A beta d chains; in addition, the hybridomas responded to antigen presented by L cells expressing E alpha A beta d molecules, and made no response with L cells expressing I-Ad or I-Ed molecules. Two more groups of hybridomas isolated from DBA/2 and B10.D2 mice immunized with myoglobin also recognized peptide 110-121 presented by E alpha d A beta d. Thus, although it is expressed at biochemically undetectable levels on spleen cells, the E alpha d A beta d molecule is an important presenting element in normal H-2d mice making a conventional immune response to a protein antigen. These results suggest that high levels of class II expression are not a prerequisite for T cell activation.     

T cell receptor gene usage in the response to lambda repressor cI protein. An apparent bias in the usage of a V alpha gene element

The T cell response to the lambda repressor cI protein is directed to the same region of the protein (residues 12-26) in both BALB/c and A/J mice. A panel of T cell hybridomas specific for P12-26 in the context of either I-Ek or I-Ad have been isolated To further understand the molecular interaction between the TCR and the Ia-P12-26 complex, the primary structures of the TCR of five T cell hybridomas have been determined. Southern and Northern analyses indicate that two members of the V alpha 3 gene family are used by 13 out of 14 I-Ek-restricted T cells. Four different V beta genes are used by these T cell hybridomas, while the majority (8 out of 13) express V beta 1 in combination with the J beta 2.1 element. No clear correlation can be seen in this system between gene usage and MHC restriction. In addition, the fine specificity of I-Ek-restricted T cells to a single amino acid substitution [Phe22/His22]P12-26 is not attributed to the usage of particular V alpha and V beta elements. The V alpha 3 family gene is also used by a few I-Ad-restricted T cells. Interestingly, these I-Ad T cells share a reactivity pattern more similar to that of I-Ek-restricted T cells than other I-Ad-restricted T cells. The nonrandom selection V alpha 3 is also demonstrated by the fact that V alpha 3 is used by P12-26-specific, but not by cytochrome c- or staphylococcal nucleus-specific, I-Ek-restricted T cells. This suggests that although antigen specificity may not be accounted for by either chain of the TCR, the members of V alpha 3 genes may be selected by the antigen (P12-26).     

CDR3 length in antigen-specific immune receptors

In both immunoglobulins (Ig) and T cell receptors (TCR), the rearrangement of V, D, and J region sequence elements during lymphocyte maturation creates an enormous degree of diversity in an area referred to as the complementarity determining region 3 (CDR3) loop. Variations in the particular V, D, and J elements used, precise points of recombination, and random nucleotide addition all lead to extensive length and sequence heterogeneity. CDR3 loops are often critical for antigen binding in Igs and appear to provide the principal peptide binding residues in TCRs. To better understand the physical and selective constraints on these sequences, we have compiled information on CDR3 size variation for Ig H, L (kappa and lambda) and TCR alpha, beta, gamma, and delta. Ig H and TCR delta CDR3s are the most variable in size and are significantly longer than L and gamma chains, respectively. In contrast, TCR alpha and beta chain distributions are highly constrained, with nearly identical average CDR3 lengths, and their length distributions are not altered by thymic selection. Perhaps most significantly, these CDR3 length profiles suggest that gamma/delta TCRs are more similar to Igs than to alpha/beta TCRs in their putative ligand binding region, and thus gamma/delta and alpha/beta T cells may have fundamentally different recognition properties.     

The MHC molecule I-E is necessary but not sufficient for the clonal deletion of V beta 11-bearing T cells

We have generated an mAb, RR3-15, that recognizes murine TCRs containing the V beta 11 domain. Using this antibody to stain peripheral T cells, we have demonstrated that V beta 11-bearing T cells are largely absent from strains of mice that express the class II MHC molecule, I-E. Studies with F1 mice demonstrate that this effect is dominant, consistent with tolerance. The clonal deletion of V beta 11-bearing T cells appears to occur intrathymically, as immature but not mature V beta 11+ T cells are present in the thymus of I-E-bearing mice. Examination of B6 x DBA/2 recombinant inbred strains demonstrates that the expression of I-E molecules is necessary for the clonal deletion of V beta 11-bearing T cells, but that other non-MHC genes control the clonal deletion process, as well. Paradoxically, only a small fraction of V beta 11+ T cell hybridomas are I-E reactive.     

Analysis of T cell receptor repertoire of muscle-infiltrating T lymphocytes in polymyositis. Restricted V alpha/beta rearrangements may indicate antigen-driven selection.

Polymyositis is an inflammatory myopathy characterized by mononuclear cell infiltration of muscle tissue. Myocytotoxic T lymphocytes have been recognized in the infiltrates, but the muscle antigen, target of the immune attack, has not been identified. Molecular characterization of the variable regions of T cell receptors (TCRs) on the infiltrating lymphocytes can be expected to provide insights into the pathogenic process. The V alpha/beta TCR repertoire was investigated by RNA-PCR in muscle biopsies from 15 polymyositis patients and 16 controls (6 Duchenne muscular dystrophy and 10 with no inflammatory or dystrophic myopathy). A variety of rearranged variable TCR genes was found in polymyositis, V alpha 1, V alpha 5, V beta 1, and V beta 15 being the most common (present in 60-100% of patients). In Duchenne muscular dystrophy patients TCR V alpha or beta rearrangements were found although no restriction was observed; no rearrangements were found in muscles from the other controls. Sequence analysis revealed the presence of the J beta 2.1 region in 90% of the V beta 15 clones studied, no random N additions in the diversity region, and a common motif within the CDR3 region. These results suggest that selection of muscle-infiltrating T lymphocytes is antigen driven in polymyositis.     

Possible role of V beta T cell receptor genes in susceptibility to collagen-induced arthritis in mice

Arthritis was induced by immunization of type II collagen in adjuvant in mice from H-2q-bearing crosses between SWR (H-2q/q) and B10 (H-2b/b mice), two strains known to be resistant to collagen-induced arthritis (CIA). The resistance of B10 is known to be due to its MHC haplotype, but it was postulated that the resistance of SWR mice which expresses the susceptible MHC haplotype could be due to the deletion of close to 50% of the V beta genes of the T cell receptor (TCR) in them. 17% of the F1 hybrids, 33% of the SWR backcrosses, 68% of the B10 backcrosses, and 52% of the F2 hybrids developed arthritis on follow-up to 5 mo after primary immunization with collagen. There was no significant difference in anti-type II collagen antibody titers between the arthritic and nonarthritic mice in each of these crosses. The segregation of the TCR genes with arthritis was determined in the F2 population by typing with F23.1 mAb that reacts with T cells using V beta 8 subfamily genes in their TCRs. SWR mice are F23.1- as V beta 8 genes are deleted in them. All six of arthritic mice homozygous for H-2q, and thus with an H-2 haplotype similar to SWR mice, expressed the F23.1 marker. These studies indicate that for complete susceptibility to collagen-induced arthritis, not only is a susceptible MHC haplotype (H-2q) important, but possibly also the presence of a subset of T cells using certain specific V beta genes in their TCRs. Other background genes may, however, modulate the severity of arthritis.     

An invariant V alpha 24-J alpha Q/V beta 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells

The T cell receptor (TCR)-alpha/beta CD4-8- (double negative, DN) T cell subset is characterized by an oligoclonal repertoire and a restricted V gene usage. By immunizing mice with a DN T cell clone we generated two monoclonal antibodies (mAbs) against V alpha 24 and V beta 11, which have been reported to be preferentially expressed in DN T cells. Using these antibodies, we could investigate the expression and pairing of these V alpha and V beta gene products among different T cell subsets. V alpha 24 is rarely expressed among CD4+ and especially CD8+ T cells. In these cases it is rearranged to different J alpha segments, carries N nucleotides, and pairs with different V beta. Remarkably, V alpha 24 is frequently expressed among DN T cells and is always present as an invariant rearrangement with J alpha Q, without N region diversity. This invariant V alpha 24 chain is always paired to V beta 11. This unique V alpha 24-J alpha Q/V beta 11 TCR was found in expanded DN clones from all the individuals tested. These findings suggest that the frequent occurrence of cells carrying this invariant TCR is due to peripheral expansion of rare clones after recognition of a nonpolymorphic ligand.