Analysis of the CDR3 Length Repertoire and the Diversity of TCRα Chain in Human Peripheral Blood T Lymphocytes

Analysis of complementarity determining region 3 （CDR3） length of T lymphocyte receptors （TCRs） by immunoscope spectratyping technique has been used successfully to investigate the diversity of TCR in autoimmune diseases and infection diseases. In this study, we investigated the patterns of CDR3 length distribution for all 32 TCR AV gene families in human peripheral blood lymphocytes of four normal volunteers by the immunoscope spectratyping technique. It was found that PCR products exhibited an obscure band on 1.5% agarose gel electrophoresis. Each TCR AV family exhibited more than 8 bands on 6% sequencing gel electrophoresis. The CDR3 spectratyping of all TCR AV families showed a standard Gaussian distribution with different CDR3 length, and the expression frequency of CDR3 was similar among the gene families. Most of CDR3 in TCR AV family recombine in frame. However, some of the CDR3 showed out-of frame gene rearrangement. Additionally, we found that in some of TCR AV families there were 18 amino acid discrepancies between the longest CDR3 and shortest CDR3. These results may be helpful to further study the recombination mechanism of human TCR genes, the TCR CDR3 gene repertoire, and the repertoire drift in health people and disease state. Cellular ＆ Molecular Immunology.     

The expressed TCRβ CDR3 repertoire is dominated by conserved DNA sequences in channel catfish

We analyzed by high-throughput sequencing T cell receptor beta CDR3 repertoires expressed by αβ T cells in outbred channel catfish before and after an immunizing infection with the parasitic protozoan Ichthyophthirius multifiliis. We compared CDR3 repertoires in caudal fin before infection and at three weeks after infection, and in skin, PBL, spleen and head kidney at seven and twenty-one weeks after infection. Public clonotypes with the same CDR3 amino acid sequence were expressed by αβ T cells that underwent clonal expansion following development of immunity. These clonally expanded αβ T cells were primarily located in spleen and skin, which is a site of infection. Although multiple DNA sequences were expected to code for each public clonotype, each public clonotype was predominately coded by an identical CDR3 DNA sequence in combination with the same J gene in all fish. The processes underlying this shared use of CDR3 DNA sequences are not clear.     

T cell receptor CDR3 loop length repertoire is determined primarily by features of the V(D)J recombination reaction

The third complementarity-determining region (CDR) of the TCR alpha and beta chains forms loops that engage amino acid residues of peptides complexed with MHC. This interaction is central to the specific discrimination of antigenic-peptide-MHC complexes by the TCR. The TCRbeta chain CDR3 loop is encoded by the Dbeta gene segment and flanking portions of the Vbeta and Jbeta gene segments. The joining of these gene segments is imprecise, leading to significant variability in the TCRbeta chain CDR3 loop length and amino acid composition. In marked contrast to other pairing antigen-receptor chains, the TCR beta and alpha chain CDR3 loop size distributions are relatively narrow and closely matched. Thus, pairing of TCR alpha and beta chains with relatively similar CDR3 loop sizes may be important for generating a functional repertoire of alpha beta TCR. Here we show that the TCRbeta chain CDR3 loop size distribution is minimally impacted by TCRbeta chain or alpha beta TCR selection during thymocyte development. Rather, this distribution is determined primarily at the level of variable-region gene assembly, and is critically dependent on unique features of the V(D)J recombination reaction that ensure Dbeta gene segment utilization.     

Composition and variation analysis of the TCR β-chain CDR3 repertoire in systemic lupus erythematosus using high-throughput sequencing

The ability of T lymphocytes to mount an immune response against a diverse array of pathogens is primarily conveyed by the amino acid (aa) sequence of the hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (TCR). In this study, we used a combination of multiplex-PCR, Illumina sequencing and IMGT/HighV-QUEST for a standardized analysis of the characteristics and polymorphisms of the T-cell receptor BV complementarity-determining region 3 (TCR BV CDR3) gene in peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy donors (NC). We found the distributions of CDR3, VD indel, and DJ indel lengths to be comparable between the SLE and NC groups. The degree of clonal expansion in the SLE group was significantly greater than in the NC group, and the expression levels of 10 TRβV segments and 6 TRβJ segments were also significantly different in the SLE group. Regarding public T cell responses, 3CDR3 DNA sequences and 4 aa sequences were shared by all SLE patients and may serve as biomarkers for SLE disease risk, diagnosis and/or prognosis.     

In vitro positive selection of alpha beta TCR transgenic thymocytes by a conditionally immortalized cortical epithelial clone

Development of mature CD4 and CD8 single-positive T cells requires a process known as positive selection, which depends on the specific recognition of self-peptide-MHC complexes on thymic stromal cells by immature CD4+CD8+ thymocytes. We have used an in vitro reaggregate system to study the positive selection of thymocytes by conditionally immortalized thymic epithelial clones. Thymocytes from mice transgenic for the F5 alpha beta TCR, specific for a peptide from the influenza nucleoprotein in the context of H-2Db, are positively selected in the H- 2b MHC background, but fail to mature in mice expressing the H-2q haplotype. Development of embryonic day 15 F5 H-2q transgenic thymocytes was followed in reaggregate cultures supplemented with H-2b- expressing epithelial clones. A conditionally immortalized cortical epithelial clone, derived from H-2Kb-tsA58 transgenic mice, was found to be as efficient as freshly isolated thymic stromal cells in positively selecting CD8 transgenic thymocytes. In contrast, an H-2b- expressing kidney epithelial clone did not augment positive selection above background levels, implying that the effect of the thymic epithelial clone was not merely the presentation of selecting MHC molecules. Mature transgenic thymocytes generated in reaggregate cultures were able to differentiate into functionally competent cytotoxic T cells. This model provides an important in vitro system for the detailed study of the specific molecular interactions leading to positive selection of developing thymocytes.      

Dominant TCR V alpha usage by virus and tumor-reactive T cells with wide affinity ranges for their specific antigens

We have studied the TCR features and functional responses of three sets of human cytolytic T cell (CTL) clones, recognizing antigenic peptides presented by HLA-A2 and derived from the Epstein-Barr virus proteins BMLF1 and BRLF1 and from the melanoma protein Melan-A/MART-1. Within each set, a majority of clones used a recurrent V alpha region, even though they expressed highly diverse TCR beta chains and V(D)J junctional sequences. Functional assays and peptide/MHC multimer binding studies indicated that this restricted V alpha usage was not associated with the affinity/avidity of the CTL clones. The V alpha dominance, which may be a frequent feature of antigen-specific T cells, likely reflects a restricted geometry of TCR/peptide/MHC complexes, primarily determined by V alpha CDR.     

CML病人TCR Vβ21寡克隆T细胞及其CDR3序列特点

目的 分析慢性粒细胞白血病（ＣＭＬ）病人的克隆性增殖Ｔ细胞及其ＣＤＲ序列的特点。方法 利用ＲＴ－ＰＣＲ扩增３例ＣＭＬ中血单个核细胞中２４个ＴＣＲ Ｖγ基因的ＣＤＲ３。了解ＴＣＦ ＶγＴ细胞的分布。阳必平物进一步经基因扫描分析，了解其克隆性。寡克隆的ＰＣＲ产物再进行序列分析。结果 ３例病人仅存在３～９个ＴＣＲ Ｖβ亚家族Ｔ细胞，部分Ｖγ亚家族Ｔ细胞呈我隆重恶性Ｔ细胞呈我隆性增殖，Ｖγ２１的我隆Ｔ细胞     

Skewing of TCR V genes to CD4+ and CD8+ subsets of T lymphocytes is not determined by amino acid composition of CDR3

TCR V genes show differing expression patterns, termed skewing, in CD4+ and CD8+ subsets of T lymphocytes. To determine which elements of the TCR V regions contribute to these observed TCR V gene skewing patterns, we have performed an in-depth analysis, taking advantage of RT-PCR and DNA sequencing, which was focused on the multi-member TCRBV6 gene family. These studies allowed us to evaluate the contributions of the various elements, that constitute the TCR beta chain variable region, to the observed TCR V gene skewing patterns. The results of these analyses revealed that within the TCRBV6 family individual members exhibited differing skewing patterns, i.e. TCRB6S7 was significantly skewed towards the CD4+ T cell subset, whereas TCRBV6S5 was significantly skewed towards the CD8+ subset. Scrutiny of the usage of TCRBV6 family members in combination with TCRBJ gene usage and amino acid composition of CDR3 did not reveal obvious structural characteristics which would explain the differing skewing patterns between TCRBV6S7 and TCRBV6S5. Further examination of these TCR V regions showed that the CDR1 and 2 regions within these TCRBV elements were composed of different amino acids. These observations suggests that these components contribute to the observed TCR V gene skewing patterns.      

Restricted alpha/beta receptor gene usage of idiotype-specific major histocompatibility complex-restricted T cells: selection for CDR3-related sequences.

We have sequenced the T cell receptor (TcR) V alpha and V beta genes of seven independent BALB/c CD4+ T cell clones specific for the immunoglobulin lambda 2 light chain produced by the MOPC 315 myeloma (lambda 2(315)). All the clones recognize a peptide of residues 91-101 of lambda 2(315) and are restricted by the major histocompatibility complex (MHC) molecule I-E(d). The results indicate that in BALB/c mice, this anti-idiotypic response uses a very limited number of TcR. The four clones which cross-react between Phe94 and Tyr94 peptide analogues use very similar receptors (V alpha 3, J alpha 1, V beta 6, J beta 1.1). The V alpha 3 gene used by all of these clones is identical and has not been previously described. Although the four clones differ in nucleotide sequence in the V/J borders, two had identical receptors at the amino acid level. One of the cross-reactive clones exhibits a heteroclitic response to the Tyr94 peptide variant resulting from a single amino acid exchange in the V/J junction of the alpha chain. The three remaining clones which recognize only the Phe94 and not the Tyr94 peptide have somewhat more diverse TcR, however, two of these three clones use V beta 6. One of these non-crossreacting clones is alloreactive, the specificity of which can be attributed to differences in the N-D-J sequences. Taken together these data indicate that this T cell response to an immunoglobulin idiotope is very restricted in terms of the TcR used. These data in conjunction with recently published results indicate that, although there can be strong preference for individual V alpha or V beta gene segments, certain V alpha/V beta combinations are preferentially selected for interacting with a given peptide/MHC combination, and that the CDR3-related regions are crucial for antigen fine specificity and alloreactivity.     

Non-random employment of V{beta}6 and J{beta} gene elements and conserved amino acid usage profiles in CDR3 regions of human fetal and adult TCR {beta} chain rearrangements

We have studied the usage of V_ß36, D_ß, andJ_ß elements, and the composition of the CDR3 regionsof human fetal TCR ß chain rearrangements In a 17week old fetal thymus and In fetal liver, bone marrow, spleen,and cord blood at 11 and 13 weeks of gestation. These fetalsequences were compared with TCR ß chain rearrangementsobtained from post-partum thymus, adult spleen, and adult peripheralblood mononuclear cells. Both fetal and adult TCR V_ß6rearrangements exhibited a non-random usage of V_ßand J_ß elements. Up to 90% of the sequences obtainedat 11 weeks of gestation used J_ß2 elements, most notablyJ_ß2.1. In the 13 and 17 week old fetal and in theadult tissues, J_ß1 elements were used In 30% of therearrangements while, within the J_ß2 rearrangements,J_ß2.1 and J_ß2.7 were used most frequently.Both fetal and adult TCR ß chain CDR3 regions showednon-random usage of amlno acids. However, the early fetal repertoirewas further limited due to the relative absence of N-reglonsin up to 60% of the 11 and 13 week old TCR ß chainrearrangements, resulting In smaller antigen binding sites.In fetal and adult TCR ß chain rearrangements thedistribution patterns of the length of N-regions and the usageprofiles of J_ß elements were similar in hematopoletlcand peripheral organs, suggesting no apparent preference forparticular TCR ß chain rearrangements. On the whole,the data showed that both the available fetal and adult TCRV_ß6 repertoires are seemingly smaller than expectedon the basis of random usage of V_ß and J_ßelements and amlno acid composition of the CDR3 regions.     

An example of immunodominance: engagement of synonymous TCR by invariant CDR3 beta

The structural basis of the T cell response against immunodominant tetanus toxin (TT)-derived peptides was investigated using TT-specific T cell clones raised from a DRB1*0301 homozygous donor. Three peptides forming T cell epitopes were identified, including one, TT(1272-1284), that stimulated four different TT-specific T cell clones. TCR sequence analysis revealed that these synonymous TCR shared only arginine at the third position of the CDR3 beta loop. This prominent residue may form a salt bridge with a corresponding aspartate at the relative position 8 (P8) of the antigenic peptide TT(1272-1284) as suggested from amino acid replacement analysis. A similar scenario was observed for a second TT epitope, TT(279-296), and its corresponding TCR. These examples show that immunodominance may result from a single strong amino acid interaction between TCR CDR3 beta loops here in contact with the C-terminus of the antigenic peptide. Such a dominant interaction could compensate for weaker contacts between other residues of the TCR and the antigenic peptide, and would allow the recognition of a single peptide-MHC complex by a broader synonymous TCR repertoire and could thus contribute to its immunodominance.     

Excision products of TCR V{alpha} recombination contain in-frame rearrangements: evidence for continued V(D)J recombination in TCR+ thymocytes

Cortical thymocytes that express a TCR on their surface continueto express mRNA for the recombination activating genes RAG-1and RAG-2. As the expression of these genes appears to be necessaryand sufficient for V(D)J recombination to occur, this findingsuggests that productive TCR gene rearrangement may not by itselfterminate TCR recombination. To further study this question,we have utilized a polymerase chain reaction-based strategyto isolate and sequence excision products of secondary TCR generearrangement events. Our data shows that TCR excision productsfrom TCR⁺ thymocytes contain in-frame V–J joints and thussupport the concept that V(D)J recombinatorial activity maycontinue even in thymocytes which have undergone productiveTCR gene rearrangement.     

VH CDR3-dependent positive selection of murine VH12-expressing B cells in the neonate

Five to fifteen percent of peritoneal B1 (CD5⁺) cells from unmanipulated mice produce antibodies that bind bromelain-treated mouse red blood cells and the hapten phosphatidylcholine (PtC). The majority of these B cells express either of two V_H/V? gene combinations, V_H12/_V?4 or V_HH/V?9. Both the V_H11 and V_H12 genes are rearranged to J_H1 and encode third complementarity determining regions (CDR3) of restricted length and sequence. These and other observations argue strongly that PtC-specific Bl cells are antigen selected. To determine when selection of PtC-specific Bl cells begins in mice we have used the polymerase chain reaction to amplify V_H12-D-J_h1 rearrangements from livers of fetal and neonatal mice, and determined the CDR3 encoding sequences of individual clones. We find an unusually low ratio of productive (P) to non-productive (NP) rearrangements (0.4–1.0) at both developmental stages. P rearrangements in day 1 neonates are biased in D gene use and in the sequence and length of their deduced V_H CDR3. These biases are similar to those of PtC-specific B1 cells in the adult peritoneum. D gene use and CDR3 length and sequence are significantly less biased among V_H12 P rearrangements 2 to 3 days earlier in the day 18 fetal liver. We suggest that this rapid change in repertoire is due to positive ligand selection that is dependent on the sequence of V_H CDR3. We suggest further that the majority of V_H12-expressing cells are not ligand selected and consequently undergo programmed cell death. The evidence of restriction in day 1 neonatal livers and the low P/NP ratio in the fetus suggests that selection of V_H12-expressing cells begins before birth.     

Analysis of T cell repertoire and function in mice transgenic for the human V{beta}3 TCR

We have constructed mice containing the human V_ß3TCR gene from the influenza virus haemagglutinin specific humanCD4⁺ T cell clone HA1.7. Similar cell yields were obtained fromtransgenic and non-transgenic lymphoid tissue, with normal levelsof T cells and with no unusual bias of the CD4 or CD8 subpopulations.Immunostaining and FACS analysis of transgenic thymocytes, spleen,and mesenteric lymph nodes revealed that the majority of T cellsexpressed the human V_ß3 TCR on the cell surface. Smallnumbers of cells expressing murine TCR_ßchain werealso detected. Polymerase chain reaction analysis revealed thatan extensive V TCR repertoire was used in the human V_ß3transgenic mice. Lymphocytes from the spleen and bmesentericlymph nodes of transgenic mice were assessed for functionalactivity in vitro. Isolated cells were stimulated with mitogenor superantigen, as well as directly through the TCR-CD3 complex,and their ability to proliferate and secrete lymphokines analysed.Cells from transgenic mice responded well after stimulationwith phytohaemagglutinin, concanavalin A, anti-CD3 antibody,anti-CD3 antibody with phorbol ester, and Staphylococcus aureusenterotoxin B, and also showed alloreactivity in a mixed lymphocytereaction. Minimal levels of response were detected after stimulationwith murine TCRß antibody. Together, these data suggestthat a human TCRß chain is able to associate witha murine TCR chain, to form a fully functional surface TCR-CD3complex.     

采用荧光定量PCR溶解曲线法监测人TCR alpha链CDR3谱系漂移技术的初步探讨

目的:初步探讨荧光定量PCR溶解曲线分析技术监测人外周血T细胞TCR alpha链CDR3谱系漂移(单/寡/多克隆增生).方法:提取4例正常人、2例淋巴瘤型白血病患者PBMC中的总RNA,逆转录成cDNA,以32个人TCR alpha 链胚系可变区基因家族 (TRAV)设计上游引物,共同的TCR alpha 胚系链恒定区基因家族(TRAC)设计下游引物,荧光定量PCR(FQ-PCR)扩增32个TRAV基因各家族CDR3谱系,溶解曲线法分析各家族CDR3谱系的单/寡/多克隆增生.结果:正常人外周血T细胞TCR alpha链32个家族CDR3表达频率不一致,各家族PCR产物的溶解曲线谱型图(melting curve spectratyping)呈现熔点不同的CDR3多态性,为多克隆增生的高斯分布,2例淋巴瘤型白血病患者外周血T细胞TCR alpha链32个家族CDR3表达频率不一致,部分家族呈缺失状态,患者各家族PCR产物的溶解曲线谱型图上,多数家族为多克隆增生的高斯分布,但每个患者均出现数量不等的单克隆和寡克隆增生家族.结论:荧光定量PCR溶解曲线分析TCR alpha链CDR3谱系漂移技术方法稳定简便,能较好地监测正常人和临床样本外周血T细胞TCR alpha链CDR3谱系漂移(单/寡/多克隆增生).     

TCR v(beta) repertoire restriction and lack of CDR3 conservation implicate TCR-superantigen interactions in promoting the clonal evolution of murine thymic lymphomas

Thymic lymphoma development is a multistage process in which genetic and epigenetic events cooperate in the emergence of a malignant clone. The notion that signaling via TCR-ligand interactions plays a role in promoting the expansion of developing neoplastic clones is a matter of debate. To investigate this issue, we determined the TCR V(beta) repertoire of thymic lymphomas induced in AKR/J mice by either endogenous retroviruses or the carcinogen, N-methyl-N-nitrosourea (MNU). Both spontaneous and MNU-induced lymphomas displayed restricted V(beta) repertoires. However, whereas V(beta)6, V(beta)8 and V(beta)9 were expressed by a greater than expected frequency of MNU-induced lymphomas, V(beta)8, V(beta)7, V(beta)13 and V(beta)14 were over-represented on spontaneous lymphomas. The dissimilar TCR V(beta) profiles indicate that different endogenous ligands promote neoplastic clonal expansion in untreated and MNU-treated mice. Although the nature of these ligands is not clear, the lack of conservation in TCR beta chain CDR3 regions among lymphomas that express the same V(beta) segment suggests that endogenous superantigens (SAG), as opposed to conventional peptide ligands, are likely to be involved in the selection process. The biased representation of lymphomas expressing V(beta)6-, V(beta)7- and V(beta)9-containing TCRs that recognize endogenous SAG is consistent with this hypothesis. The finding that Bcl-2 is expressed at high levels in spontaneous and MNU-induced lymphomas suggests that preneoplastic thymocytes may be resistant to SAG-induced clonal deletion. A working model is presented in which preneoplastic clones expressing TCRs that recognize endogenous SAG are selectively expanded as a consequence of sustained TCR-mediated signaling.     

Enhanced positive selection of a transgenic TCR by a restriction element that does not permit negative selection

Very little is known about the conformational properties ofthe MHC molecules that are able to signal positive selectionof a given TCR. To try to understand these parameters and todetermine whether these requirements are shared with interactionsduring negative selection and antigen recognition, we have studiedselection and antigen recognition of a transgenic TCR (specificfor lymphocytic choriomeningitls virus glycoprotein and H-2D^b)in the context of two D^b mutants, H-2^bm13 and H-2^bm14. The datashowed that the transgenic TCR was not positively selected bythe H-2^bm14 haplotype but, interestingly, enhanced positiveselection was seen in H-2^bm13 mice. The transgenic TCR couldnot be negatively selected In H-2^bm13animals persistently infectedwith the virus (neonatal virus carrier mice), nor could thetransgenic TCR be activated by H-2^bm13 infected cells in vivoor in vitro. These experiments show that although a TCR maybe selected by a mutant MHC molecule, the corresponding viralantigen cannot be recognized in the context of the mutant MHCmolecule, as Judged by both negative selection and T cell reactivityin vivo and in vitro. The ‘enhanced’ positive selectionoccurring in the context of D^bm13 suggests that a differentconformation of the MHC molecule is able to select the sameTCR and also that various TCR-ligand avidities may permit positiveselection.