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Results for “Complementarity Determining Regions”

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PubMed1994

An analysis of sequence variation in the beta chain framework and complementarity determining regions of an allo-reactive T cell receptor.

Current models of T cell receptor (TCR) structure are generally based on the homology observed between the TCR and the immunoglobulins. Furthermore, these models have predicted the locations of framework and complementarity determining regions within the alpha- and beta-chain variable regions. In order to test the validity of these models, we have generated a series of mutations within the V beta domain of an allo-reactive TCR and determined their effect on antigen recognition.

Amino Acid Sequence↗

PubMed2005

CD1d-independent developmental acquisition of prompt IL-4 gene inducibility in thymus CD161(NK1)-CD44lowCD4+CD8- T cells is associated with complementarity determining region 3-diverse and biased Vbeta2/Vbeta7/Vbeta8/Valpha3.2 T cell receptor usage.

Among Ag-inexperienced naive T cells, the CD1d-restricted NKT cell that uses invariant TCR-alpha-chain is the most widely studied cell capable of prompt IL-4 inducibility. We show in this study that thymus CD161-CD44lowCD4+CD8- T cells promptly produce IL-4 upon TCR stimulation, a response that displays biased Vbeta(2/7/8) and Valpha3.2 TCR usage. The association of Vbeta family bias and IL-4 inducibility in thymus CD161-CD44lowCD4+CD8- T cells is found for B6, B10, BALB/c, CBA, B10.A(4R), and ICR mouse strains. Despite reduced IL-4 inducibility, there is a similarly biased Vbeta(2/7/8) TCR usage by IL-4 inducibility+ spleen CD161-CD44lowCD4+CD8- T cells. Removal of alpha-galacotosylceramide/CD1d-binding cells from CD161-CD44lowCD4+CD8- thymocytes does not significantly affect their IL-4 inducibility. The development of thymus CD161-CD44lowCD4+CD8- T cells endowed with IL-4 inducibility and their associated use of Vbeta(2/7/8) are beta2-microglobulin-, CD1d-, and p59fyn-independent. Thymus CD161-CD44lowCD4+CD8- T cells produce low and no IFN-gamma inducibility in response to TCR stimulation and to IL-12 + IL-18, respectively, and they express diverse complementarity determining region 3 sequences for both TCR-alpha- and -beta-chains. Taken together, these results demonstrate the existence of a NKT cell distinct, TCR-repertoire diverse naive CD4+ T cell subset capable of prompt IL-4 inducibility. This subset has the potential to participate in immune response to a relatively large number of Ags. The more prevalent nature of this unique T cell subset in the thymus than the periphery implies roles it might play in intrathymic T cell development and may provide a framework upon which mechanisms of developmentally regulated IL-4 gene inducibility can be studied.

Amino Acid Sequence↗

PubMed1994

Complementarity determining region 1 (CDR1)- and CDR3-analogous regions in CTLA-4 and CD28 determine the binding to B7-1.

T cell surface receptors CD28 and CTLA-4 are homologous members of the immunoglobulin superfamily (IgSF), each comprising a single V-like extracellular domain. CD28 and CTLA-4 bind to the B7-1 and B7-2 counter-receptors on antigen presenting cells (APCs), thereby triggering a costimulatory pathway important for optimal T cell activation in vitro and in vivo. Soluble forms of CD28 and CTLA-4 in which the V-like extracellular domains were fused to Ig constant domains (CD28Ig and CTLA4Ig), have been used to study their interactions with B7-1 and B7-2, with CTLA4Ig binding B7-1 more strongly than CD28Ig (approximately 20-fold higher avidity). We have now, by site-specific and homologue mutagenesis, identified regions in CTLA4Ig important for strong binding to B7-1. A hexapeptide motif (MYPPPY) in the complementarity determining region 3 (CDR3)-like region is fully conserved in all CD28 and CTLA-4 family members. Alanine scanning mutagenesis through the motif in CTLA4Ig and at selected residues in CD28Ig reduced or abolished binding to B7-1. Chimeric molecules HS4, HS4-A, and HS4-B were constructed in which CDR3-like regions of CTLA-4, COOH-terminally extended to include nonconserved residues, were grafted onto CD28Ig. These homologue mutants showed stronger binding to B7-1 than did CD28Ig. Grafting of the CDR1-like region of CTLA-4, which is not conserved in CD28 and is predicted to be spatially adjacent to CDR3, into HS4 and HS4-A, resulted in chimeric molecules (HS7 and HS8) which bound B7-1 even better. Inclusion of the CDR2-like domain of CTLA-4 into HS7 and HS8 did not further increase binding. Thus, the MYPPPY motifs of CTLA4Ig and CD28Ig are important for their binding to B7-1, but the increased strength of this binding by CTLA4Ig is mediated by nonconserved residues in the CDR1- and CDR3-analogous regions.

Abatacept↗

PubMed1996

Comparison of the immunoglobulin heavy-chain complementarity determining region-3 structure among the DNA sequences and the mu- and gamma-transcripts in human B-lineage cells.

To study the recombinational significance of the (immunoglobulin heavy) IgH chain gene in human B-cell development, we compared the complementarity determining region (CDR)-3 sequences of the DNA and the mu-transcripts from human normal pre-B cells and mature B cells, and the gamma-transcripts from bone marrow cells. The CDR-3 sequences were longer in the DNA than in the mu- and gamma-transcripts, and this was independent of whether or not the rearrangement was productive. The DLR family genes were less frequently used in the mu- and gamma-transcripts. When translated into amino acids, all CDR-3 sequences from the mu- and gamma-transcripts were productive, although 26.2% of the DNA sequences had stop codons in the D element and/or frameshifts of the JH gene segments. The CDR-3 of the productive DNA sequences in pre-B cells frequently (26.6%) contained at least three continuous hydrophobic amino acids, which were mainly coded by the DLR and DXP family genes at the third reading frame. However, such motifs were rate in the mu-transcripts of pre-B (7.7%) and mature B cells (3.9%), and in the gamma-transcripts of bone marrow cells (1.1%) as well as in the DNA of mature B cells (10.4%). These findings suggested that the length and/or hydrophobicity of the IgH CDR-3 might play a role in the selection mechanisms of B-cell development.

Adult↗

PubMed1996

Complementarity determining region residues aspartic acid at H55, serine at H95 and tyrosines at H97 and L96 play important roles in the B72.3 antibody-TAG72 antigen interaction.

Structural analysis derived from the crystallographic study of the chimeric B72.3 antibody illustrated some major atomic interactions between complementarity determining region (CDR) residues. For example, hydrogen bonds are formed between H35/H95, L50/H97, H53/H55 and H96/L96 respectively. These CDR residues may play important roles in the B72.3-TAG72 (antibody-antigen) interaction either by direct interaction with the TAG72 antigen or by maintaining a CDR loop conformation through atomic interactions between CDR residues. In order to confirm these assumptions, we altered these CDR residues by site-directed mutagenesis and determined binding affinities of these mutant chimeric antibodies for the TAG72 antigen in a solid-phase radioimmunoassay. We found that H55, H95, H97 and L96 are important CDR residues for the B72.3-TAG72 interaction. Single amino acid substitutions of aspartic acid and serine by alanine at H55 of CDR2 and at H95 of CDR3 respectively and of tyrosine by phenylalanine at H97 and L96 of CDR3, significantly reduced the binding affinity for the TAG72 antigen by 20-, 8-, 16- and 45-fold respectively. Therefore, this study reveals some of the requirements for maintaining the integrity of the B72.3 antibody combining sites.

Amino Acid Sequence↗

PubMed1996

Human T cell repertoire generation in the absence of MHC class II expression results in a circulating CD4+CD8- population with altered physicochemical properties of complementarity-determining region 3.

In this study, we have investigated the impact of deficient MHC class II expression on the use of TCRBV6 and TCRBJ gene elements, and on the pattern of amino acid incorporation exhibited in the N1-D-N2 segments of the third complementarity-determining region (CDR3) of these TCRBV6 rearrangements. To this end, we have analyzed circulating T cells from three, nonrelated MHC class II-deficient (bare lymphocyte syndrome (BLS)) patients and three MHC class II-expressing family members. The patients and healthy controls exhibited similar, nonrandom usage profiles of TCRBV6 and TCRBJ gene elements in both the CD4+CD8- and the CD4-CD8+ subsets of peripheral blood T cells. No statistically significant differences between patients and controls were detected in the length of CDR3, or in the amount of non-germline modification at the sites of recombination. However, detailed analysis of the TCRBV6 rearrangements derived from the CD4+CD8- subsets from the BLS patients revealed patterns of amino acid incorporation into the N1-D-N2 region of CDR3 that resulted in altered charge and hydropathicity properties of the presumed Ag binding site. In this way, we have been able to demonstrate that human T cell repertoire development in the absence of MHC class II expression results in a circulating CD4+CD8- T cell population bearing TCRs with altered CDR3 profiles. Such altered profiles are likely to be a direct reflection of the lack of MHC class II-mediated selection processes in these BLS patients.

Amino Acid Sequence↗

PubMed1999

Molecular analysis of the complementarity determining region 3 of the human T cell receptor beta chain. Establishment of a reference panel of CDR3 lengths from phytohaemagglutinin activated lymphocytes.

The T cell receptor (TCR) repertoire of a lymphocyte population may be characterized by the distribution of lengths of the hypervariable fragment known as the complementarity determining region 3 (CDR3). Immunological activity leading to clonal predominance will result in an over-representation of given CDR3 lengths and a distortion of the CDR3 length distribution. CDR3 length distribution may be studied by the in vitro amplification of TCRB cDNA followed by gel electrophoresis of the resulting product. We have established a simple, robust method for the evaluation of CDR3 length distribution in human lymphocyte samples. The CDR3 length distribution in phytohaemagglutinin (PHA) activated lymphocytes from a large number of healthy donors was established as a reference panel for each of 22 human TCR beta variable (BV) families. We propose that an abnormal CDR3 length distribution be defined as one in which one or more CDR3 lengths exceed the upper confidence limit (5% significance, one-sided test) given by this PHA reference population. Using this criterion in titration experiments, we were able to identify a clone when it constituted 2% of the cells analyzed. Over-dilution of cellular material or cDNA may produce falsely abnormal CDR3 length distributions. A nested technique using two separate amplification steps was found to yield results comparable in quality to the single amplification technique. When few cells are available, the nested method gives more material for CDR3 length analyses. However, it does not reduce the likelihood of a falsely abnormal distribution being recorded when the cellular material is too scarce.

Adult↗

PubMed1998

Structural requirements for a specificity switch and for maintenance of affinity using mutational analysis of a phage-displayed anti-arsonate antibody of Fab heavy chain first complementarity-determining region.

We previously showed that a single mutation at heavy (H) position 35 of Abs specific for p-azophenylarsonate (Ars) resulted in acquisition of binding to the structurally related hapten p-azophenylsulfonate (Sulf). To explore the sequence and structural diversity of the H chain first complementarity-determining region (HCDR1) in modulating affinity and specificity, positions 30-36 in Ab 36-65 were randomly mutated and expressed as Fab in a bacteriophage display vector. Ab 36-65 is germline encoded, lacking somatic mutations. Following affinity selection on Sulf resins, 55 mutant Fab were isolated, revealing seven unique HCDR1 sequences containing different amino acids at position H:35. All Fab bound Sulf, but not Ars. Site-directed mutagenesis in a variety of HCDR1 sequence contexts indicates that H:35 is critical for hapten specificity, independent of the sequence of the remainder of HCDR1. At H:35, Asn is required for Ars specificity, consistent with the x-ray crystal structure of the somatically mutated anti-Ars Ab 36-71, while Sulf binding occurs with at least seven different H:35 residues. All Sulf-binding clones selected following phage display contained H:Gly33, observed previously for Ars-binding Abs that use the same germline V(H) sequence. Site-directed mutagenesis at H:33 indicates that Gly plays an essential structural role in HCDR1 for both Sulf- and Ars-specific Abs.

Animals↗

PubMed1994

Conserved amino acid residues in the complementarity-determining region 1 of the TCR beta-chain are involved in the recognition of conventional Ag and Mls-1 superantigen.

Superantigens activate T cells by interacting primarily with the V beta region of the TCR. This report describes a series of studies performed to elucidate the role of the conserved amino acid motif (Asp-His-Asn) in the complementarity-determining region 1 (CDR1) of the TCR V beta chains that recognize murine endogenous superantigen Mls-1. By using site-directed mutagenesis of the Mls-1-reactive TCR V beta 6 gene, it is shown that the alterations of the conserved CDR1 motif disrupt Mls-1 superantigen and conventional Ag recognition in vitro. The loss of V beta 6 (mutant)+ TCR reactivity to Mls-1 superantigen is apparently dependent on the partner alpha-chain in the V beta 6/V alpha 3 TCR pairing shows some reactivity to Mls-1, whereas other TCR pairings do not. The examination of the developmental fate of the mutated form of the V beta 6 chain in Mls-1+ mice by using retroviral vector-mediated gene transfer confirms the critical role played by the CDR1 residues in Mls-1 recognition in vivo. Collectively, the results indicate that the CDR1 of the TCR V beta 6 chain is involved in interacting with peptide/MHC as well as in Mls-1 recognition. The observation that the conserved residues in selective TCR V beta chains are imparting a significant contribution to Ag recognition suggests a molecular basis for the intrinsic bias of some V beta chains for MHC molecules.

Amino Acid Sequence↗

PubMed1995

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen.

To gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-p-azophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among the monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars that the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment.

Amino Acids↗

PubMed1996

Proline-rich tandem repeats of antibody complementarity-determining regions bind and neutralize human immunodeficiency virus type 1 particles.

The proline-rich tandem repeat domain of human mucin MUC1 forms an extended structure containing large repeating loops that are crested by a turn. We show that the repeating-loop structure of MUC1 can be replaced by an antibody complementarity-determining region loop of a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing antibody to create a chimeric, multivalent, mucin-like, anti-HIV-1 compound. We used 8 residues of an antibody molecule to replace 8 of 20 residues of the MUC1 tandem-repeat sequence. The antiviral peptide discussed here contains three copies of a 20-residue tandem repeat, (IYYDYEEDPAPGSTAPPAHG)3, for a total of 60 residues. We demonstrate that the mucin-antibody chimera retains the binding specificity of the parent antibody (monoclonal antibody F58), GPGR of the HIV-1 gp120 V3 neutralizing epitope, and the ability to neutralize virus particles. In inhibition enzyme-linked immunosorbent assay, the mucin-antibody chimeric peptide could inhibit 71 to 84% of binding to a V3 loop peptide by monoclonal antibodies known to be specific for GPGR in the V3 loop. The mucin-antibody chimeric peptide could also inhibit monoclonal antibody binding to native gp120 captured from virus particles. In addition, the chimeric peptide neutralized the homologous HIV-IIIB virus in a standard neutralization assay. The methods of antiviral peptide design and construction presented here are general and theoretically limited only by the size of the antibody repertoire. This approach could be used to synthesize peptides for a variety of therapeutic applications.

Amino Acid Sequence↗

PubMed1995

Impact of the high tyrosine fraction in complementarity determining regions: measured and predicted effects of radioiodination on IgG immunoreactivity.

Although iodine-131 is the most widely used radionuclide for radioimmunotherapy, direct radiolabeling methods yield decreased immunoreactivity of the antibody as a function of increased iodine incorporation. We have studied the amino acid sequences of a therapeutic IgG (HuM195), and in particular its complementarity determining regions (CDR), in order to correlate the iodination of tyrosine residues in the antigen binding site with changes in immunoreactivity. The CDR contained an overabundance of tyrosines relative to an expected random distribution of amino acids. In contrast, lysine residues that can be used for ligand attachment were evenly distributed throughout the IgG. HuM195 was first trace labeled with 111In and then labeled with stable 127I at various specific activities. The immunoreactivity of each product was determined using the 111In tracer. The immunoreactivity measured after varying levels of iodination fit a theoretical curve that was generated based on the assumption that a single iodine incorporation anywhere on a tyrosine residue in a CDR destroys the immunoreactivity of the antibody. Similar theoretical curves for antibody fragments (Fab, Fv) suggest an even faster decrease in immunoreactivity with increasing iodination. A review of the sequences of other therapeutic IgG shows that a similar overabundance of tyrosine residues is found in the CDRs. Using enzyme digestion, the distribution of iodine on different parts of the antibody was also studied. The iodinated residues were distributed non uniformly throughout the IgG, with the heavy chain variable region tyrosines having a higher propensity for iodine incorporation than tyrosines in the other regions of the IgG. The common abundance of tyrosine in the CDR of IgG and its correlation with loss of function have important implications for therapeutic use of high specific activity radioiodinated monoclonal antibodies or fragments.

Amino Acid Sequence↗

PubMed1991

Design and synthesis of a mimetic from an antibody complementarity-determining region.

A technique for producing non-peptide compounds (mimetics) of designed specificities was developed that permitted the synthesis of a conformationally restricted molecule that mimicked the binding and functional properties of monoclonal antibody (MAb) 87.92.6, which recognizes the reovirus type 3 cellular receptor. Binding of either MAb 87.92.6, peptide analogs, or 87.1-mimetic to the cellular receptor inhibited cellular proliferation. The mimetic was a synthetic beta-loop structure that mimics the second complementarity-determining region of the MAb. These studies may lead to strategies for the synthetic design of antibody complementarity regions, ligands, and other pharmacologically active agents that are water soluble, resistant to proteolysis, and nonimmunogenic.

Antibodies, Monoclonal↗

PubMed1996

Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site.

We determined the extent to which additional binding energy could be achieved by diversifying the complementarity determining regions (CDRs) located in the center of the antibody combining site of C6.5, a human single-chain Fv (scFv) isolated from a non-immune phage library which binds the tumor antigen c-erbB-2. CDR3 of the light (V(L)) and heavy (V(H)) chain variable region of C6.5 were sequentially mutated, the mutant scFv displayed on phage, and higher affinity mutants selected on antigen. Mutation of V(L) CDR3 yielded a scFv (C6ML3-9) with a 16-fold lower Kd (1.0 x 10(-9) M) than C6.5. Due to its length of 20 amino acids, four V(H) CDR3 libraries of C6ML3-9 were constructed. The greatest increase in affinity from a single library was ninefold (Kd = 1.1 x 10(-10) M). Combination of mutations isolated from separate V(H) CDR3 libraries yielded additional ninefold decreases in Kd, resulting in a scFv with a 1230-fold increase in affinity from wild-type C6.5 (Kd = 1.3 x 10(-11) M). The increase in affinity, and its absolute value, are comparable to the largest values observed for antibody affinity maturation in vivo or in vitro and indicate that mutation of V(L) and V(H) CDR3 may be a particularly efficient means to increase antibody affinity. This result, combined with the location of amino acid conservation and substitution, suggests an overall strategy for in vitro antibody affinity maturation. In addition, the affinities and binding kinetics of the single-chain Fv provide reagents with potential tumor targeting abilities not previously available.

Alanine↗

PubMed1998

T cells with similar T-cell receptor beta-chain complementarity-determining region 3 motifs infiltrate inflammatory lesions of synthetic peptides inducing rat autoimmune myocarditis.

Experimental autoimmune myocarditis (EAM) resembles the giant cell myocarditis seen in humans, and recurrent forms lead to dilated cardiomyopathy. EAM has been shown to be a T cell-mediated autoimmune myocarditis. We have previously shown that cDNA encoding Vbeta complementarity-determining region (CDR) 3 from heart- and pericardial space-infiltrating T cells in EAM induced by rod cardiac myosin contains more restricted sequences than that from normal spleen T cells. Recently, it has become apparent that several epitopes of EAM exist in rod cardiac myosin; therefore, T cells infiltrating into lesions may recognize certain epitopes in EAM induced by rod cardiac myosin. In this study, we examined heart- and pericardial space-infiltrating T-cell clonotypes in EAM induced by synthetic peptides of cardiac myosin. EAM was produced by immunization with synthetic peptides corresponding to N-terminally acetylated amino acids 1539 to 1555 of rat cardiac myosin alpha heavy chain. Five of 12 rats receiving synthetic peptides developed macroscopic signs of myocarditis. To examine T-cell receptor (TCR) Vbeta expression and CDR3 of the TCR beta chain of lesion-infiltrating T cells in EAM, total RNA was isolated from heart, pericardial effusion, spleen, lymph node, and peripheral blood. TCR Vbeta expression of the T cells infiltrating the lesions revealed a predominance of Vbeta4. On the basis of single-strand conformation polymorphism analysis for CDR3 of the TCR Vbeta4 chain, heart- and pericardial space-infiltrating T cells were considered to be oligoclonal, whereas spleen, lymph node, and peripheral blood in a rat with EAM and spleen in a native rat were considered to be polyclonal. In the same rat, clonotypes of heart-infiltrating T cells were almost the same as those of pericardial space-infiltrating T cells. Furthermore, on sequence analysis for CDR3 of the TCR Vbeta4 chain, the amino acid motifs were similar among T cells infiltrating into lesions of different EAM rats. In the present study, TCR beta chains of heart- and pericardial space-infiltrating T cells in EAM induced by synthesized peptide consisting of 17 amino acids were examined. Vbeta4+ T cells with similar Vbeta CDR3 motifs that infiltrate the heart and pericardial space may recognize the same epitope.

Amino Acid Sequence↗

PubMed1994

B precursor acute lymphoblastic leukemia third complementarity-determining regions predominantly represent an unbiased recombination repertoire: leukemic transformation frequently occurs in fetal life.

To determine whether the ALL (acute lymphoblastic leukemia) CDR3 (third complementarity-determining region) repertoire represents the recombination repertoire, or shows evidence of selectional processes inherent to normal B cell differentiation or malignant transformation, we analyzed 68 ALL CDR3 regions and included 127 previously published sequences in the analyses. We found no evidence of selection prior to malignant transformation as recombination was random with 1/3 "in frame" and 2/3 "out of frame" joinings and usage of all three D reading frames was observed. D and JH gene segments were predominantly unmutated which allowed a detailed analysis of gene usage and rearrangement characteristics. JH4 and JH6 usage (both 32.2%) was significantly different (p = 0.005) from that observed in peripheral B lymphocytes. D gene family usage roughly represented D gene family size with the exception of the DXP and DA/K family which were over- and underrepresented (p = < or = 0.05), respectively. D-D fusions were found in 26.2% of CDR3 regions. If less stringent criteria were applied DIR homology was found in 40/65 sequences, suggesting the frequent involvement of DIR gene segments in human CDR3 formation. The rearranged D genes were evenly distributed over the D locus, suggesting that D recombination is a predominantly random process, independent of physical location at the locus. Also, there was no correlation between JH gene usge and physical location of the rearranged D gene segment, which excludes a major contribution of the DJH replacement recombination mechanism. In 36.1% of CDR3 regions N-nucleotides at the DJH junction were absent. This frequency is higher than observed for peripheral B lymphocytes. It is suggested that for a number of ALL the initial transformational event took place in early fetal life. We conclude that ALL CDR3 sequences show no evidence of selection prior to malignant transformation, nor of extensive changes subsequent to malignant transformation.

Age Factors↗

PubMed1990

Amino acid substitutions in the first complementarity-determining region of a murine T-cell receptor alpha chain affect antigen-major histocompatibility complex recognition.

The T-cell antigen receptor mediates recognition of foreign antigens physically associated with major histocompatibility complex (MHC) proteins. The tertiary structure of the T-cell receptor is thought to resemble that of immunoglobulin Fab fragments and to possess corresponding complementarity-determining regions (CDRs) that contact antigen-MHC. To test such a model for the T-cell receptor, we have generated T-cell hybridomas that express a wild-type or mutant form of the T-cell receptor present on the p-azobenzenearsonate-specific T-cell clone D5. Mutation of 2 amino acids (Tyr26 to serine, Gly28 to valine) in the predicted CDR1 of the D5 T-cell receptor alpha chain caused a markedly diminished response to antigen without affecting the response to anti-CD3 and anti-T-cell receptor antibodies. These results constitute the first test of the prediction that CDR1 in the T-cell receptor alpha chain is important for antigen-MHC recognition, thus providing strong evidence for the structural model of the T-cell antigen receptor based upon immunoglobulin.

Amino Acid Sequence↗

PubMed1998

Ig heavy chain third complementarity determining regions (H CDR3s) after stem cell transplantation do not resemble the developing human fetal H CDR3s in size distribution and Ig gene utilization.

Previous studies have suggested that the B-cell repertoire after stem cell transplantation resembles the developing repertoire in the fetus. Fetal and adult repertoires differ strikingly at the molecular level in Ig heavy chain third complementarity determining region (H CDR3) size distribution and Ig gene utilization. Previously, the posttransplant repertoire has not been studied fully in this regard. In this study, we analyzed H CDR3s posttransplant using CDR3 fingerprinting, single-strand conformation polymorphism (SSCP), and random sequencing. Eleven adult patients who received either autologous (n = 6) or allogeneic adult sibling (n = 5) hematopoietic stem cell transplants were studied. IgM H CDR3 repertoires demonstrated limited clonal diversity within the first 6 to 10 weeks posttransplant. By 3 to 4 months, the IgM H CDR3 repertoires were as diverse as those in healthy adults. Reconstitution of the IgM diversity correlated with the expansion of the multimember VH3 family. By contrast, the contribution of the single-member VH6 family was limited in most patients up to 6 to 9 months. No evidence was seen for greater contribution of VH6 posttransplant. IgG repertoires remained clonally restricted at all times. In all patients, H CDR3 sizes fell within adult limits. Direct nucleotide sequencing of H CDR3s showed adult-type N-nucleotide insertions and Ig gene utilization. These results indicate that the emerging repertoire posttransplant does not resemble the developing fetal repertoire at the molecular level.

Adult↗