The influence of self-MHC and non-MHC antigens on the selection of an antigen-specific T cell receptor repertoire

We have examined the influence of self-Ag on TCR expression and specificity in the immune response to the Ag pigeon cytochrome c. Previous work has shown that most Ek-restricted cytochrome c-specific T cells from B10 background mice express TCR alpha beta-heterodimers encoded by V beta 3 and V alpha 11 genes, but that T cells expressing V beta 3 proteins are eliminated due to self-tolerance in Mls-2a mouse strains. Thus, EK-restricted cytochrome c-specific T cells from Mls-2a mice fail to express any V beta 3. In the current study the influence of self-MHC and non-MHC Ag on TCR usage in the immune response to cytochrome c was further examined. First, it was demonstrated that the absence of V beta 3 expression in Mls-2a mice does not alter Ir gene function. Specifically, Mls-2a/Eb haplotype V beta 3- [C3H.SW x B10.A(5R)]F1 mice were high responders to cytochrome c despite the fact that previous structure function analyses have shown a very close correlation between Eb-restricted cytochrome c recognition and V beta 3 expression. This demonstration of the plasticity of TCR expression suggests that relatively few Ir gene defects result from tolerance induced by self-Ag. We also examined differences in V alpha 11 expression among cytochrome c-specific T cells from various H-2k haplotype mouse strains. In particular, the low level of expression of V alpha 11 in cytochrome c-specific T cells from C57BR (H-2k) mice was shown not to be due to self-tolerance. Rather, evidence for limited strain polymorphism of V alpha 11 genes, plus the fact that cytochrome c-specific T cells from F1 hybrids between H-2k, Mls-2b identical C57BR and B10.BR mice express high levels of V alpha 11, suggested the possibility that the variable V alpha 11 usage in the cytochrome c-specific responses of these two strains reflected differences in positive selection during ontogeny by non-MHC non-Mls self-Ag.     

Interactions between the amino-terminal domains of MHC class II molecules have a profound effect on serologic and T cell recognition: an analysis using recombinant HLA-DR/H-2E molecules.

A series of transfected L cell lines were generated expressing the products of wild-type or recombinant HLA-DR1/H-2Ek beta-chain-encoding genes paired to DR alpha or E alpha. The recombinant genes were created by reciprocal exchange of the gene segments encoding the amino (NH2)-terminal and carboxy (COOH)-terminal halves of the beta 1 domain and the beta 2 domain. The majority of the serologic determinants, predicted from the genetic composition of the class II dimers, were expressed indicating that no gross conformational changes were induced by the creation of the interspecies recombinant molecules. Subtle conformational variation was detected by the anti-H-2Eb,k,s mAb Y17. Epitope expression was dependent on the presence of the E alpha-chain and NH2-terminal sequence from the beta 1 domain of H-2Ek. Substitution of DR1 sequence in either region led to loss of recognition by Y17. This pattern of reactivity maps the Y17 epitope either to the E alpha-chain or to an exposed sequence on the fourth strand of the beta sheet of the beta 1 domain. If the Y17 epitope is located on the E alpha-chain this raises the interesting possibility that the conformation of this chain, which is invariant by sequence, may vary according to the beta-chain with which it is coexpressed. The ability of the recombinant class II dimers to present Ag to the pigeon cytochrome c-specific, H-2Ek-restricted T cell hybridoma 2B4 was assessed. Transfected L cells expressing E beta k paired to E alpha or DR alpha presented Ag with equal efficiency, and the beta 2 domain of H-2Ek could be substituted with the equivalent region from DR1 without any loss of response. Wild-type DR1 failed to function as a restriction element, however, substitution of the COOH-terminal portion of the beta 1 domain with the equivalent sequence from H-2Ek was sufficient to produce a partial recovery of Ag recognition. Cells expressing a recombinant beta 1 domain comprising the COOH-terminal sequence from H-2Ek and the NH2-terminal sequence from DR1 presented Ag when paired to DR alpha but failed to do so when paired to E alpha. This indicates that a subtle conformational disturbance caused by mismatching of the NH2-terminal region of the beta-chain and the alpha-chain can have pronounced effects on T cell recognition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Arsonate-specific murine T cell clones. IV. Properties of I-E- and I-A-restricted clones

The T cell antigen L-tyrosine-p-azobenzenearsonate is unique in being a simple determinant that can be presented in the context of both I-A and I-E. I-E-restricted T cell clones derived from B10.A(5R) mice were found to fall into three groups: Type I clones recognized antigen only in the context of syngeneic apcs, Type II clones recognized antigen with the same highly specific major histocompatibility complex restriction but in addition proliferated in response to allogeneic stimuli; Type III clones were "degenerate" in their major histocompatibility complex-restricted recognition of antigen and proliferated when antigen-presenting cells bearing Eb beta Ek alpha (syngeneic), Ek beta Ek alpha, or Ed beta Ed alpha were used. These observations allow some conclusions to be drawn about sites on the I-E molecule that may be functionally significant in the presentation of this antigen. By using the B cell hybridoma LK35.2 as target cells, some of these T cell clones act as cytotoxic cells in the Class II-restricted manner predicted from the results of proliferative assays. Class II-restricted cytotoxicity can therefore be controlled by both I-A and I-E mouse Ir gene loci.     

Structural analysis of anti-DR1 allorecognition by using DR1/H-2Ek hybrid molecules. Influence of the beta 2-domain correlates with CD4 dependence.

A segmental analysis of the key regions of HLA-DR1 that control T cell allorecognition was performed by using a series of transfected cell lines expressing the products of recombinant DRB/H-2Eb genes, paired with either DR alpha or H-2E alpha. Four of eight human T cell clones tolerated substitution of the H-2E alpha chain, but only one clone showed any response to the DR alpha/H-2E beta k dimer. Both the membrane-proximal and the membrane-distal domains of the beta-chain played an important part in stimulating these clones. The response of four of eight clones was markedly inhibited by substitution of the H-2E beta 2 for the DR beta 2 domain. This inhibition showed a complete correlation with the sensitivity of the clones to inhibition by anti-CD4 mAb. Taken together, these results suggest that the interaction site for CD4 may include residues on the beta 2-domain. Introduction of H-2Ek sequence into either half of the beta 1-domain led to a complete loss of response by all but two of the clones. This is consistent with these clones having dual specificity for exposed DR1-specific polymorphisms and for DR1-bound peptides. The pattern of response of one of the clones suggested that indirect conformational effects on the alpha 1-domain may also contribute to the influence of the amino-terminal half of the beta 1-domain on T cell recognition. In the presence of H-2E alpha, this clone responded more strongly when the amino-terminal half of the beta 1-domain was of H-2Ek rather than DR1 sequence. This implies that species matching of the floor of the beta 1-domain with the alpha-chain is more important than the presence of the alpha-chain of the parental species.     

Ek beta mutant antigen-presenting cell lines expressing altered Ak alpha molecules

Antigen-presenting cells (APC) expressing mutant Ek beta and Ak alpha proteins were isolated after chemical mutagenesis of TA3 cells and negative immunoselection for altered Ek beta molecules. Mutant clones were analyzed for biosynthesis, assembly, and cell surface expression of altered Ia molecules, and were assayed for antigen-presenting function by using a variety of T cell clones. Three types of mutants were detected: type 1, which had lost expression of the Ek beta chain and produced altered Ak alpha chains; type 2, which also expressed altered Ak alpha chains, and which expressed Ek beta proteins that had lost reactivity to the 17.3.3 and 74D monoclonal antibodies (mAb), but retained reactivity to other anti-Ek beta mAb; and type 3, which had lost expression of both Ek beta and Ak beta: Ak alpha surface molecules. Thus, all of the mutant clones that produced modified Ak alpha proteins also displayed either total loss or serologic modification of the Ek beta molecule. Ek beta:E alpha-reactive T cell clones were not stimulated when type 1 or type 3 cells were used as APC, but all such T cells were fully reactive with type 2 mutant APC. Most Ak beta:Ak alpha-reactive T cell clones could respond to type 1 and 2 APC, and none were responsive to type 3 APC. However, two autoreactive Ak beta:Ak alpha-specific T cell hybridomas were stimulated only very weakly by type 1 and type 2 cells expressing modified Ak alpha proteins. These results demonstrate that Ia mutations can have highly selective effects on antigen presentation to T cells as well as on mAb binding, and thus suggest that individual Ia molecules may be composed of many different functional subsites.     

Stimulation of normal inducer T cell clones with antigen presented by purified Ia molecules in planar lipid membranes: specific induction of a long-lived state of proliferative nonresponsiveness

Culture of normal inducer T cell clones with antigen and purified Ek beta:Ek alpha incorporated into planar lipid membranes resulted in specific T cell activation as determined by cell volume increase and IL 3 production. However, in contrast to results obtained with T cell hybridomas, antigen presentation by planar membranes did not induce measurable IL 2 production, and proliferative responses were not detected. Rather, recognition of only Ek beta:Ek alpha and antigen resulted in the specific induction of a long-lived state of proliferative nonresponsiveness to subsequent stimulation by conventional APC and antigen. Induction of nonresponsiveness required protein synthesis, and was not simply due to the absence of IL 2. The antigen-nonresponsive cells could respond to either PMA plus ionomycin or IL 2, and they expressed normal levels of surface antigen-receptor molecules. These results demonstrate that recognition by normal T cell clones of antigen and Ia molecules in the absence of other accessory cell molecules and signals results in a prolonged state of proliferative nonresponsiveness, possibly similar to a state of T cell tolerance in vivo.     

Haplotype-specific suppression of T cell response to lactate dehydrogenase B in (responder x nonresponder)F1 mice

Mouse strains that express the Ek (Ek beta E-1k alpha) molecule are nonresponders (NR) to the enzyme lactate dehydrogenase B (LDHB) in terms of T cell proliferation. Nonresponsiveness is caused by T suppressor (Ts) cells recognizing LDHB in the context of Ek molecules on the antigen-presenting cells. The data presented here demonstrate that the Ek-restricted Ts cells function in (R x NR)F1 mice in a remarkable haplotype-specific fashion: they selectively interfere with the Ak (ANR)-restricted response, and do not affect the response channeled through the A molecules of the responder parent. This haplotype-specificity of suppression provides an explanation of the dominance of responsiveness in (R x NR)F1 mice.     

High frequency and nonrandom distribution of alloreactivity in T cell clones selected for recognition of foreign antigen in association with self class II molecules

Previous studies have demonstrated that a single T cell clone can respond to both a foreign antigen in the context of self major histocompatibility complex (MHC)-encoded molecules (self plus X) and to an allogeneic class I or class II molecule in the absence of antigen (non-self). We have used limiting dilution of T cells obtained from the draining lymph nodes of antigen-primed B10.A mice to establish a large number of T cell clones that recognize either GAT, pigeon cytochrome c, or sheep insulin in association with syngeneic antigen-presenting cells. Sixty-two antigen-specific T cell clones were assayed for their ability to proliferate in response to a panel of nine different allogeneic haplotypes. Of these, 38 (61%) responded to at least one allogeneic haplotype, and 15 of the 38 (39%) responded to more than one allogeneic stimulator. In addition, the patterns of alloreactivity varied with the immunizing antigen. The GAT-specific T cells had at least one responder to every haplotype tested, although H-2u-responsive T cell clones were the most common. In contrast, no pigeon cytochrome c-specific T cells responded to stimulators of the H-2u haplotype, but rather predominantly responded to H-2t4/H-2s and H-2i5/H-2b. Finally, sheep insulin-reactive T cell clones preferentially responded to H-2u stimulators, although stimulation by antigen-presenting cells of the H-2p and H-2q haplotypes was also common. A chi 2 analysis of the data demonstrated that the dependence of the pattern of alloreactivity observed upon the antigen used for immunization was statistically significant (p less than 0.01). The high frequency of alloreactivity found in antigen-specific T cell clones is discussed, as well as the implications that the antigen-dependent skewing of the distribution of alloreactivity have for a one-receptor model vs a two-receptor model of T cell recognition.     

Restricted V-segment usage in T-cell receptors from cytotoxic T lymphocytes specific for a major epitope of lymphocytic choriomeningitis virus.

Cytotoxic T lymphocytes (CTL) play an important role in recovery from a number of viral infections. They are also implicated in virus-induced immunopathology as best demonstrated in lymphocytic choriomeningitis virus (LCMV) infection of adult immunocompetent mice. In the present study, the structure of the T-cell receptor (TCR) in LCMV-specific CTL in C57BL/6 (B6) mice was investigated. Spleen T cells obtained from LCMV-infected mice were cultured in vitro with virus-infected stimulator cells and then stained with anti-TCR V beta antibodies. A skewing of V beta usage was noticeable in T cells enriched for their reactivity to LCMV, suggesting that particular V segments are important for the recognition of LCMV T-cell epitopes in B6 mice. To gain more detailed information on the structure of the TCR specific for LCMV epitopes, we studied CTL clones. It has been shown that approximately 90% of LCMV-reactive CTL clones generated in H-2b mice are specific for a short peptide fragment of the LCMV glycoprotein, residues 278 to 286, recognized in the context of the class I major histocompatibility complex molecule, Db. Four CTL clones possessing the specificity were randomly selected from a collection of clones, and their TCR genes were isolated by cDNA cloning or by the anchored polymerase chain reaction. All four clones were found to use V alpha gene segments belonging to the V alpha 4 subfamily. By RNA blot analysis, two more clones with the same specificity were also shown to express the V alpha 4 mRNA. In contrast, three different V beta gene segments were used among the four clones examined. J beta 2.1 was used by three of the clones. Although amino acid sequences in the V(D)J junctional regions were dissimilar, aspartic acid was found in the V alpha J alpha and/or V beta D beta J beta junctions of all four of these clones, suggesting that this residue is involved in binding the LCMV fragment. Restricted usage of V alpha and possibly J beta segments in the CTL response to a major T-cell epitope of LCMV raises the possibility that immunopathology in LCMV infection can be treated with antibodies directed against such TCR segments. Thus, similar analysis of the TCR in other virus infections is warranted and may lead to therapeutic strategies for immunopathology due to virus infections.     

Asymmetric selection of T cell antigen receptor alpha- and beta-chains in HLA-B27 alloreactivity.

Endogenous peptides constitutively bind to class I MHC Ag and are thought to be integral parts of allospecific T cell epitopes. However, allospecific TCR can recognize structural features of the alloantigen as foreign. To define some crucial parameters determining HLA-B27 allorecognition, the structure of TCR alpha- and beta-chains from HLA-B27-specific CTL was analyzed. A strategy, based on V alpha and V beta family-specific oligonucleotides, was used for specific amplification and direct sequencing of TCR-alpha and -beta cDNA. We observed nonrandom usage of V beta segments and recurrent structural motifs within beta-chain junctional regions. In contrast, no structural restrictions were apparent among alpha-chains, even from CTL clones of related fine specificity. These results indicate an asymmetric contribution of TCR alpha- and beta-chains to HLA-B27 allospecificity among the CTL clones analyzed. They suggest recognition of multiple peptides and involvement of beta-chain junctional regions in recognizing shared motifs among some of these peptides.     

Restricted V-(D)-J junctional regions in the T cell response to lambda-repressor. Identification of residues critical for antigen recognition.

The T cell response to lambda-repressor is directed to a 15 amino acid peptide (P12-26) of the protein in A/J mice. Previous studies have demonstrated a preferential use of V alpha 2 and V beta 1 amongst the T cell hybridomas specific for P12-26 in the context of I-Ek. By using the polymerase chain reaction, the sequences of a panel of the T cells using V alpha 2 and V beta 1 were determined. A highly conserved alpha-chain V-J junctional sequence was found in six of the eight T cell hybrids. This consensus alpha-chain VJ sequence may be combined with different members of V alpha 2, indicating a more restricted selection on the junctional region than on the V element in these T cells. In contrast, greater diversities were found on the V-D-J region of beta-chains despite the same V beta 1 and J beta 2.1 were used. However, a highly conserved glutamic acid residue was found at the same position of beta-chains where a similar conservation was identified in cytochrome c-specific T cells. The correlation of the TCR sequence with the fine specificities of these T cells suggests that a single amino acid deletion in the V alpha-J alpha region may reduce the P12-26 response and abolish the recognition of an altered peptide [Phe22] P12-26. In addition, three amino acid difference in the V-D-J region of the beta-chain also determine the P12-26 reactivity. Thus the V(D)J junctional regions of both alpha- and beta-chains may be critical for the recognition of the peptide Ag presented by the specific MHC molecule.     

Restricted use of T cell receptor V genes in murine autoimmune encephalomyelitis raises possibilities for antibody therapy

Experimental allergic encephalomyelitis (EAE) is a paralytic autoimmune disease induced in susceptible animals by active immunization with myelin basic protein (MBP) or by passive transfer of MBP-specific T helper (TH) lymphocytes. We have analyzed the T cell receptor genes of 33 clonally distinct TH cells specific for a nonapeptide of MBP inducing EAE in B10.PL (H-2u) mice. All 33 TH cells used two alpha variable gene segments (V alpha 2.3, 61%; V alpha 4.2, 39%), the same alpha joining gene segment (J alpha 39), and two V beta and J beta gene segments (V beta 8.2-J beta 2.6, 79%; V beta 13-J beta 2.2, 21%). The anti-V beta 8 monoclonal antibody F23.1 was found to block completely recognition of the nonapeptide by V beta 8 TH cells in vitro and to reduce significantly the susceptibility of B10.PL mice to peptide-induced EAE.     

Predominant use of a T-cell receptor V beta gene family in simian immunodeficiency virus Gag-specific cytotoxic T lymphocytes in a rhesus monkey.

To explore the structural basis for AIDS virus recognition by CD8+ lymphocytes, we sought to determine whether there is a diverse or restricted usage of T-cell receptors (TCR) by simian immunodeficiency virus of macaques (SIVmac) Gag-specific cytotoxic T lymphocytes (CTL) in the rhesus monkey. Six Gag-specific CTL clones were independently generated from an SIVmac-infected rhesus monkey. All six CTL clones recognized a single SIVmac Gag peptide in association with a single major histocompatibility complex class I gene product, Mamu-A*01. TCR alpha-chain sequences from these six CTL clones employed four different V alpha families and five different J alpha gene segments. In contrast, five of the six CTL clones expressed V beta genes that were members of the same family, a human V beta 23 homolog. Furthermore, only one J beta gene was expressed by four of the six CTL clones. These results indicate that TCR of SIVmac Gag-specific CTL from a rhesus monkey can exhibit a restricted usage of V beta gene families and J beta genes.     

T cell receptor gene segment utilization by HLA-DR1-alloreactive T cell clones.

Transplantation of histoincompatible tissues leads to allograft rejection, which involves recognition of allogeneic MHC molecules by Ag-specific receptors expressed on T cells. The interaction of these molecules is highly specific yet poorly understood. We have investigated the relationship between TCR gene utilization and allo-MHC restriction patterns by using a one-way polymerase chain reaction to amplify the alpha- and beta-chain mRNA from a panel of 10 HLA-DR1-alloreactive T lymphocyte clones. Two previously unreported V alpha and five J alpha gene sequences were obtained. Although a few V alpha, V beta, and J alpha genes were utilized more than once, no correlation between TCR gene usage and DR1 alloreactivity was identified. At the sequence level, the presumed TCR alpha- and beta-chain CDR1 and CDR2 regions displayed limited diversity, whereas the CDR3 or junctional sequences were highly variable. Although most TCR probably interact with subtly different surface features of the DR1 alloantigen, we predict that TCR with similar CDR1 and CDR2 sequences would contact essentially identical regions of the DR1 molecule. The lack of sequence conservation in the junctional regions suggests that different endogenous peptides also may be recognized. Thus, alloreactive T cells may recognize not only allogeneic MHC molecules but perhaps also bound endogenous peptides.     

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.     

Polymorphic HLA-DR7 beta 1 chain residues that are involved in T cell allorecognition

The contributions to allorecognition of polymorphic amino acids in the HLA-DR7 beta 1 chain were analyzed by using mutant DR7 beta 1 chains with single amino acid substitutions at position 4, 11, 13, 25, 30, 37, 57, 60, 67, 70, 71, 74, or 78. Transfectants expressing mutant DR7 molecules were used as stimulators for six DR7-alloreactive T cell clones. The majority of the substitutions had profound effects on the ability of the DR7 molecule to stimulate one or more T cell clones. Nine of the 13 substitutions completely abrogated recognition by at least one clone. The finding that each of the substitutions in the beta-strands in the floor of the peptide binding groove affected T cell allorecognition supports the model of allorecognition in which the complex of a self-peptide bound to a class II molecule is recognized by the TCR. Interestingly, the substitution at position 4, which is predicted to be located outside the peptide binding groove, decreased the ability of the DR7 molecule to stimulate some clones. Each of the DR7-alloreactive T cell clones had a unique reactivity pattern in response to the different mutant molecules, indicating that the TCR of each clone recognized the DR7 molecule differently. Surprisingly, many of the mutant DR7 molecules induced proliferation by one or more clones that was greater than 125% of the proliferation induced by the wild-type DR7 molecule. These data indicate that multiple polymorphic residues, predicted in the class II model to be located in both the beta-strands and alpha-helix of the DR7 beta 1 chain, contribute to allorecognition of the DR7 molecule.     

Structure-function analysis of the Abm12 beta mutation using site-directed mutagenesis and DNA-mediated gene transfer

The B6.C-H-2bm12 (bm12) mouse possesses a naturally occurring mutation in its class II MHC A beta gene. The three amino acid substitutions at positions 67, 70, and 71 that comprise this mutation lead to changes in both Ia expression and immune recognition of the resultant A beta A alpha molecule. The experiments reported here utilize a combination of oligonucleotide-mediated site-directed mutagenesis and DNA-mediated gene transfer to explore the roles played by each of the three mutant residues in these various phenotypic changes. A beta genes comprising all permutations of the residues distinguishing Ab beta from Abm12 beta were created and were individually co-transfected with Ab beta into mouse L cells. Sublines expressing high levels of membrane Ia were selected by preparative flow cytometry and were studied for reactivity with a panel of monoclonal anti-Ia antibodies, or for their ability to act as antigen-presenting cells (APC) for the stimulation of T cell hybridomas. During the generation of these transfectant lines, it was noted that expression of a high level of Abm12 beta Ab alpha was more difficult to achieve than a similar level of Ab beta Ab alpha. Northern blot analysis of specific A beta and A alpha mRNA levels in these various lines indicated that more class II mRNA, and presumably more A beta and A alpha chains, were required to achieve expression of Abm12 beta Ab alpha equal to that of Ab beta Ab alpha, suggesting that the previously noted reduction of Ia expression on cells from bm12 mice reflects a decreased ability of Abm12 beta Ab alpha chains to pair, or to reach the membrane. Staining of the panel of transfectants with monoclonal antibodies revealed that antibodies which did not distinguish Ab beta Ab alpha from Abm12 beta Ab alpha also reacted equally well with all molecules involving in vitro mutant A beta chains. Monoclonal antibodies reactive with Ab beta Ab alpha but not Abm12 beta Ab alpha were specific for an epitope primarily determined by the presence or absence of Arg 70 in Ab beta. In striking contrast, all three mutant positions were found to play crucial roles in T cell recognition, because all substitutions led to significant or complete loss of antigen-presenting function with all but one of the T hybridomas tested.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA-mediated transfer of major histocompatibility class II I-Ab and I-Abm12 genes into B lymphoma cells: molecular and functional analysis of introduced antigens

A20.2J B lymphoma cells have been co-transfected with the A alpha b, A beta b or with the A alpha b, A beta bm12 and neomycin resistance genes. The transfected cell lines constitutively express the I-Ab or I-Abm12 class II molecules at a level comparable with that of the endogenous I-Ad antigen. The I-Ab antigens expressed on three independently transfected B cell clones (A20.Ab.1, A20.Ab.2, and A20.Ab.3) are serologically and functionally indistinguishable from the I-Ab molecules expressed by control H-2bxd B hybridoma cells (LB cells). These transfected cell lines were potent I region-restricted antigen-presenting cells to a large panel of antigen-specific, autoreactive and alloreactive T cell hybridomas, as well as normal T cell clones. There were not significant differences in the efficiency of antigen presentation by the Ia molecules encoded by the transfected, as compared with the endogenous, I-A genes. The expression of a functional I-Ab antigen on the surface of cells transfected with A beta bm12 and A alpha b genes is consistent with previous work that implicated the A beta-chain alone in the bm 12 mutation. Furthermore, because the transfected A20.Ab and A20.Abm12 cells display the serologic and functional properties of normal spleen cells from the wild-type and mutant mouse strains, respectively, it is clear that class II genes do not undergo unexpected and unpredictable alterations after transfection in this system. This system permits us to investigate the structural requirements for interactions between class II major histocompatibility complex antigens, a foreign antigen, and the T cell receptor by in vitro site-directed mutagenesis coupled with DNA-mediated gene transfer.     

Receptor diversity of insulin-specific T cell lines from C57BL (H-2b) mice

To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.     

Conserved CDR3 regions in T-cell receptor (TCR) CD8(+) T cells that recognize the Tax11-19/HLA-A*0201 complex in a subject infected with human T-cell leukemia virus type 1: relationship of T-cell fine specificity and major histocompatibility complex/peptide/TCR crystal structure

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.