ANALYSIS OF T CELL CLONALITY BY CDR3 SIZE OF T-CELL ANTIGEN RECEPTOR Vβ REPERTOIRE IN HCL AND c-ALL

Objective: To analyze the distribution and clonality of TCR Vβ subfamily T cells in hairy cell leukemia (HCL) and common-acute lymphoblastic leukemia (c-ALL). Methods: Peripheral blood mononuclear cell samples from 3 cases of HCL and 1 case of c-ALL were investigated for analysis of complementarity determining region 3 (CDR3) size of T cell receptor Vβ repertoire using reverse transcriptase-polymerase chain reaction (RT-PCR). The products were further analyzed by genescan to identify T cell clonality. Results: Some Vβ subfamily PCR products from 4 patients contained monopeak (monoclone) or a dominant peak (oligoclone). In contrast, multipeak (polyclone) distributions were found in all Vβ subfamily PCR products from normal control cases. Conclusion: T cell clonal expansion may be found in HCL and c-ALL cases that may indicate a host response directed against leukemia related antigen. In addition, it may be useful to detect the minimal residual disease.

Stable clonal expansion of the T-cell receptor Vβ6, Vβ17 and Vβ19 T cells in a cGVHD case using genescan analysis

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.

T cell receptor Vβ repertoire usage and clonal expansion of T cells in chronic myelogenous leukemia

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.

Changes in the T-cell receptor Vβ gene repertoire after allogeneic hematopoietic stem cell transplantation

Background We distinguished graft-versus-host disease (GVHD) from graft-versus-leukemia (GVL) effects and to investigate the distribution of T-cell receptor (TCR) Vβ gene repertoire in individuals with leukemia before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods Peripheral blood mononuclear cells (PBMC) were obtained from 10 normal individuals, 8 donors and 11 patients with leukemia before and after transplantation. Polymerase chain reaction (PCR) amplification of complementarity-determining region 3 (CDR3) of 24 TCR Vβ genes was used to examine serial samples of PBMC. The PCR products were further analyzed by genescan to evaluate clonality of T cells.Results The 24 TCR Vβ gene repertoire displayed highly diverse and polyclonal spectratypes in all normal individuals and 4 of 8 donors. Anoth er 4 donors expressed part of the 24 TCR Vβ subfamily and 1 donor had oligoclonality. The expressions of the 24 TCR Vβ subfamilies were skewed and restric ted in 11 leukemia patients before and after transplantation. Some absences of 24 TCR Vβ subfamily expression were quite similar between the recipients pro-transplantation and related donors. The number of subfamilies expressed increased over time post-transplantation, but the restricted expressions of the subfamily could last 6-30 months after transplantation. All patients with GVHD and some without GVHD exhibited T cell clonal expansion. The expansive T cell clone was distributed in Vβ 2-3, 16-17, 18-19, 21 and Vβ 23 in patients with GVHD and in Vβ 7, 9, 16 and 19 in patients without GVHD. One patient with syngeneic-HSCT (syn-HSCT) had Vβ 15 and 16 T cell expansion after transplantation. One patient displayed Vβ 18 T cell expansion after donor lymphocyte infusion (DLI).Conclusions Normal individuals express the entire 24 TCR Vβ ge ne repertoire and have polyclonal distribution. However, the TCR Vβ gene repertoire is only partially expressed in some donors. The TCR Vβ gene repertoire is restrictedly expressed in a skew fashion in patients with leukemia before and after transplantation. The number of TCR Vβ gene subfamilies increases over time post- transplantation. GVHD and GVL effects may induce the proliferation of T cell clones.Clinical GVL response may be distinguished from GVHD alloreactivity through the host MHC antigen.