Qingsong Yin

Reduced levels of recent thymic emigrants in acute myeloid leukemia patients.

BackgroundT cell immunodeficiency is a common feature in cancer patients, which may relate to initiation and development of tumor. Our previous study showed skewed expression of T cell receptor beta variable region (TRBV) subfamilies and clonal expansion of T cells in leukemia patients. In the present study, in order to further characterize the T cell immunity in acute myeloid leukemia (AML) patients, the level of recent thymic emigrants (RTE) was analyzed.Materials and methodsQuantitative analysis of signal joint T cell recombination excision circles (δRec-ψJα sjTRECs) was performed in peripheral blood mononuclear cells (PBMCs) by real-time PCR (TaqMan), and the analysis of 23 TRBV-BD1 sjTRECs was performed by semi-nested PCR. Eighty-eight cases with AML were selected for this study; ten AML cases in complete remission (AML-CR) and 38 healthy individuals served as controls.ResultsThe levels of δRec-ψJα sjTRECs in PBMCs and CD3+ T cells were significantly decreased in AML patients, compared with healthy individuals and in patients in completive remission. Also the frequency of 23 TRBV-BD1 sjTRECs, and the number of detectable TRBV subfamily sjTRECs were significantly lower in AML patients than in healthy individuals. Moreover, the sjTRECs numbers and the frequency of TRBV-BD1 sjTRECs showed a progressive linear decline with age in AML patients.ConclusionsThe decreased numbers of universal (δRec-ψJα) and family-specific (TRBV-BD1) sjTRECs indicate that the severe T cell immunodeficiency in AML patients is associated with reduced levels of recent thymic emigrants. In patients achieving complete remission both sjTREC counts return to normal values indicating the recovery of thymic function. Better understanding of the mechanisms underlying persistent immunodeficiency in leukemia patients may lead to novel treatment strategies to enhance immune competence.

Generation of diffuse large B cell lymphoma-associated antigen-specific Vα6/Vβ13+T cells by TCR gene transfer

BackgroundOur previous study had amplified antigen-specific full-length TCR α and β genes of clonally expanded T cells in the peripheral blood (PB) of patients with diffuse large B-cell lymphoma (DLBCL). The transfer of T cell receptor (TCR) genes endows T cells with new antigen specificity. Therefore, the aim of this study is to generate diffuse large B cell lymphoma (DLBCL)-specific T cells by T cell receptor (TCR) gene transfer.Materials and methodsTwo different eukaryotic expression plasmids harboring TCR Vα6 and TCR Vβ13 genes specific for DLBCL-associated antigens were constructed and subsequently transferred into human T cells using Nucleofector™ technique. The expression of targeted genes in TCR gene-modified cells was detected by real-time PCR, and western blot using TCR Vβ antibody. The specific cytotoxicity of TCR gene-transferred T cells in vitro was estimated using a lactate dehydrogenase (LDH) release assay.ResultsTwo different eukaryotic expression plasmids harboring TCR Vα6 and TCR Vβ13 genes specific for DLBCL-associated antigens were constructed and subsequently transferred into T cells from healthy donors. Specific anti-DLBCL cytotoxic T lymphocytes (CTL) could be induced by transduction of specific TCR gene to modify healthy T cells. The transgene cassette of TCR Vβ13-IRES-TCR Vα6 was superior to the other in the function of TCR-redirected T cells.ConclusionsSpecific anti-DLBCL cytotoxic T lymphocyte (CTL) could be inducted by transduction of specific TCR gene to modify healthy T cells.

Decreased level of recent thymic emigrants in CD4+ and CD8+T cells from CML patients.

BackgroundT-cell immunodeficiency is a common feature in cancer patients, which may relate to initiation and development of tumor. Based on our previous finding, to further characterize the immune status, T cell proliferative history was analyzed in CD4+ and CD8+ T cells from chronic myeloid leukemia (CML) patients.MethodsQuantitative analysis of δRec-ψJα signal joint T cell receptor excision circles (sjTRECs) was performed in PBMCs, CD3+, CD4+ and CD8+T cells by real-time PCR, and the analysis of 23 TRBV-D1 sjTRECs was performed by semi-nested PCR. Forty eight CML cases in chronic phase (CML-CP) were selected for this study and 17 healthy individuals served as controls.ResultsThe levels of δRec-ψJα sjTRECs in PBMCs, CD3+, CD4+, and CD8+ T cells were significantly decreased in CML patients, compared with control groups. Moreover, the numbers of detectable TRBV subfamily sjTRECs, as well as the frequency of particular TRBV-BD 1 sjTRECs in patients with CML were significantly lower than those from healthy individuals.ConclusionsWe observed decreased levels of recent thymic emigrants in CD4+ and CD8+ T cells that may underlay the persistent immunodeficiency in CML patients.

Characterization of conserved CDR3 sequence of TCR α- and β-chain genes in peripheral blood T-cells from patients with diffuse large B-cell lymphoma

Abstract T-cell immunodeficiency is a common feature in cancer patients, but clonally expanded T cells which were considered to have the specific anti-tumor cytotoxicity can be identified in most cancer patients, including those with hematological malignancies. We previously reported that there were skewed usages of T-cell receptor (TCR) Vα and TCR Vβ subfamily in clonally expanded T cells in the peripheral blood (PB) of patients with diffuse large B-cell lymphoma (DLBCL). In order to investigate the characteristics of the complementarity determining region 3 (CDR3) of TCR α- and β-chain genes in clonally expanded T cells, the sequences of TCR-CDR3 region of clonally expanded TCR Vα and TCR Vβ subfamily T cells were analyzed. Conserved amino acid motifs of TCR-CDR3 region were identified in different clonally expanded T cells. RSxYNTDKLI, SxGG, GGS, and DSxY motifs were found in the TCR Vα subfamily T-cell clones, while SxGTG, SRG, and GTxD motifs existed in the TCR Vβ subfamily T-cell clones. Moreover, the TCR harboring conserved motif in CDR3 region of some T-cell clones had high homology in the three-dimensional spatial structures. These findings revealed the mono/oligoclonally expanded T cells were derived from the stimulation of some antigens in the PB of patients with DLBCL, and may be recognizing an identical antigenic epitope.

Clonal expanded TRA and TRB subfamily T cells in peripheral blood from patients with diffuse large B-cell lymphoma

Abstract T cell immunodeficiency is a common feature in cancer patients and may be a contributing factor to the initiation and development of the tumor. In order to characterize the immune status in a group of patients with diffuse large B-cell lymphoma (DLBL), the repertoires of T cell receptor alpha and beta variable regions (TRAV and TRBV) were analyzed. The CDR3 of 29 TRAV and 24 TRBV subfamily genes were analyzed in peripheral blood mononuclear cells from six cases with DLBL using RT-PCR and GeneScan technique. Six normal individuals served as controls. Marked restriction of TRBV repertoire was observed in peripheral blood mononuclear cells (PBMCs) from DLBL. Clonal expanded T cells were found frequently in PBMCs from all DLBL patients; the oligoclonality was most frequent in TRAV6, TRAV8, TRAV12, TRAV21, TRAV22 and TRAV25 subfamilies. Similarly, clonally expanded TRBV subfamily T cells were found in all DLBL patients. The oligoclonality was most frequent in TRBV3, TRBV13 and TRBV15 subfamilies, which could be detected in four out of six cases. In conclusion, the frequent and restricted clonal expansion αβ+ T cells were thought to be the specific immune response to lymphoma-associated antigen.

Alteration of the gene expression profile of T-cell receptor αβ-modified T-cells with diffuse large B-cell lymphoma specificity

Abstract Antigen-specific, T-cell receptor (TCR)-modified cytotoxic T lymphocytes (CTLs) that target tumors are an attractive strategy for specific adoptive immunotherapy. Little is known about whether there are any alterations in the gene expression profile after TCR gene transduction in T cells. We constructed TCR gene-redirected CTLs with specificity for diffuse large B-cell lymphoma (DLBCL)-associated antigens to elucidate the gene expression profiles of TCR gene-redirected T-cells, and we further analyzed the gene expression profile pattern of these redirected T-cells by Affymetrix microarrays. The resulting data were analyzed using Bioconductor software, a two-fold cut-off expression change was applied together with anti-correlation of the profile ratios to render the microarray analysis set. The fold change of all genes was calculated by comparing the three TCR gene-modified T-cells and a negative control counterpart. The gene pathways were analyzed using Bioconductor and Kyoto Encyclopedia of Genes and Genomes. Identical genes whose fold change was greater than or equal to 2.0 in all three TCR gene-redirected T-cell groups in comparison with the negative control were identified as the differentially expressed genes. The differentially expressed genes were comprised of 33 up-regulated genes and 1 down-regulated gene including JUNB, FOS, TNF, INF-γ, DUSP2, IL-1B, CXCL1, CXCL2, CXCL9, CCL2, CCL4, and CCL8. These genes are mainly involved in the TCR signaling, mitogen-activated protein kinase signaling, and cytokine–cytokine receptor interaction pathways. In conclusion, we characterized the gene expression profile of DLBCL-specific TCR gene-redirected T-cells. The changes corresponded to an up-regulation in the differentiation and proliferation of the T-cells. These data may help to explain some of the characteristics of the redirected T-cells.