Objective To investigate the distribution and clonality of the T-cell receptor (TCR) Vβ repertoire in chronic graft versus host disease (cGVHD).Methods The complementarity determining region 3 (CDR3) of the TCRβ gen...Objective To investigate the distribution and clonality of the T-cell receptor (TCR) Vβ repertoire in chronic graft versus host disease (cGVHD).Methods The complementarity determining region 3 (CDR3) of the TCRβ gene with 24 variable regions was amplified in peripheral blood mononuclear cells drawn from one cGVHD patient after allogenic bone marrow transplantation (allo-BMT) 35, 39, 43 or 45 months respectively, using RT-PCR, to observe the expression of TCR Vβ repertoire T cells. The PCR products were further analyzed by genescan to evaluate clonality of T cells. Ressults Fourteen or 16 TCR Vβ subfamily T ceils were detected in each sample of cGVHD case. Oligoclonal T cells were identified in TCR Vβ 6, 16, 17, 19 and 21 subfamilies. The stable clonal T cells in all samples were identified in Vβ6, Vβ17 and Vβ21 subfamilies.Conclusion Skewing distribution and stable clonal expansion of T cells can be found in cGVHD cases and it may be related to the initiation of cGVHD.展开更多
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 d...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.展开更多
Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes （RAG） and RAG-mediated V（D）J recombination. This process is named re...Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes （RAG） and RAG-mediated V（D）J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor （TCR） gene recombination. Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles （sjTRECs） were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences （RSSs） in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 （CDR3） size of the TCRVβ chain was examined by the TCR GeneScan technique. Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining （NHEJ） pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ2 5＇ and Dβ 2 3＇ sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation. Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V（D）J recombination and as a ＂special＂ model of the coexistence of TCR gene rearrangements and ＂negative＂ receptor revision.展开更多
Background In general,it is very important to understand the state of T cell immune response against tumor cells in leukemia patients and it is especially critical to assess the T cell repertoire of untreated patients...Background In general,it is very important to understand the state of T cell immune response against tumor cells in leukemia patients and it is especially critical to assess the T cell repertoire of untreated patients. As we know,few studies have dealt with the distribution of oligoclonal T cells in leukemia,so we investigated the distribution and clonality of TCR Vβ repertoire of T cells in patients with chronic myelogenous leukemia (CML) in chronic phase. Methods The complementarity determining region 3 (CDR3) of TCR Vβ24 subfamily genes were amplified in peripheral blood mononuclear cells from 27 cases with CML using reverse transcription-polymerase chain reaction (RT-PCR). In order to observe the distribution of TCR Vβ repertoire,the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vβ T cells. The PCR products of the oligoclonal T cells from three cases were analyzed by direct sequencing to define the sequence of CDR3.Results The expression pattern of TCR Vβ repertoire in different individuals are different. Vβ2-21 subfamilies could be detected in CML cases. The frequent usage Vβ repertoire in CML was Vβ1,Vβ2 orVβ13. Most of the PCR products from 27 patients displayed polyclonality,while a part of the PCR products from 21 out of 27 samples displayed clonal expansion pattern. The clonal expanded T cells in CML could be found in Vβ16 subfamilies. The frequent usage of Vβ genes in clonal expansion was Vβ3,Vβ13 or Vβ21. Multiple Vβ clonal expansion was a general phenomenon in the same patient. The CDR3 sequence of Vβ21 oligoclonal T cells from 3 cases showed some difference in splice regions and in the usage of J segments.Conclusions These results indicated that clonal expanded T cells could be found in patients with CML and were tendentious in Vβ3,Vβ13 and Vβ21 subfamilies that may be related to the specific immune response for leukemia cell associated antigen.展开更多
文摘Objective To investigate the distribution and clonality of the T-cell receptor (TCR) Vβ repertoire in chronic graft versus host disease (cGVHD).Methods The complementarity determining region 3 (CDR3) of the TCRβ gene with 24 variable regions was amplified in peripheral blood mononuclear cells drawn from one cGVHD patient after allogenic bone marrow transplantation (allo-BMT) 35, 39, 43 or 45 months respectively, using RT-PCR, to observe the expression of TCR Vβ repertoire T cells. The PCR products were further analyzed by genescan to evaluate clonality of T cells. Ressults Fourteen or 16 TCR Vβ subfamily T ceils were detected in each sample of cGVHD case. Oligoclonal T cells were identified in TCR Vβ 6, 16, 17, 19 and 21 subfamilies. The stable clonal T cells in all samples were identified in Vβ6, Vβ17 and Vβ21 subfamilies.Conclusion Skewing distribution and stable clonal expansion of T cells can be found in cGVHD cases and it may be related to the initiation of cGVHD.
文摘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.
基金grants from Major State Basic Research Development 973 Program of China(No.2001CB510008 and 2003CB514113)NSFC & Research Grant Coancil of Hong Kong Joint Research Fund(No.30418003).
文摘Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes （RAG） and RAG-mediated V（D）J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor （TCR） gene recombination. Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles （sjTRECs） were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences （RSSs） in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 （CDR3） size of the TCRVβ chain was examined by the TCR GeneScan technique. Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining （NHEJ） pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ2 5＇ and Dβ 2 3＇ sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation. Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V（D）J recombination and as a ＂special＂ model of the coexistence of TCR gene rearrangements and ＂negative＂ receptor revision.
文摘Background In general,it is very important to understand the state of T cell immune response against tumor cells in leukemia patients and it is especially critical to assess the T cell repertoire of untreated patients. As we know,few studies have dealt with the distribution of oligoclonal T cells in leukemia,so we investigated the distribution and clonality of TCR Vβ repertoire of T cells in patients with chronic myelogenous leukemia (CML) in chronic phase. Methods The complementarity determining region 3 (CDR3) of TCR Vβ24 subfamily genes were amplified in peripheral blood mononuclear cells from 27 cases with CML using reverse transcription-polymerase chain reaction (RT-PCR). In order to observe the distribution of TCR Vβ repertoire,the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vβ T cells. The PCR products of the oligoclonal T cells from three cases were analyzed by direct sequencing to define the sequence of CDR3.Results The expression pattern of TCR Vβ repertoire in different individuals are different. Vβ2-21 subfamilies could be detected in CML cases. The frequent usage Vβ repertoire in CML was Vβ1,Vβ2 orVβ13. Most of the PCR products from 27 patients displayed polyclonality,while a part of the PCR products from 21 out of 27 samples displayed clonal expansion pattern. The clonal expanded T cells in CML could be found in Vβ16 subfamilies. The frequent usage of Vβ genes in clonal expansion was Vβ3,Vβ13 or Vβ21. Multiple Vβ clonal expansion was a general phenomenon in the same patient. The CDR3 sequence of Vβ21 oligoclonal T cells from 3 cases showed some difference in splice regions and in the usage of J segments.Conclusions These results indicated that clonal expanded T cells could be found in patients with CML and were tendentious in Vβ3,Vβ13 and Vβ21 subfamilies that may be related to the specific immune response for leukemia cell associated antigen.
