[The Genome-Wide Changes in Expression Profile of CML T Cells After Up-regulation of TCRζ Chain Expression].

OBJECTIVE: To analyze the changes in the gene expression profile of T cells in CML patients after TCRζ up-regulation expression, and to explore the molecular mechanism of T cell reactivation after transgenic up-regulation of TCRζ. METHODS: The peripheral blood mononuclear cells(PBMCs) from 3 newly untreated chronic-stage CML patients were collected, and the CD3+ T cells were obtained by MACS method. The TCRζ-IRES2-EGFP (experimental group) and pIRES2-EGFP (control group) plasmids were transfected into T cells by nuclear transfection technique. The gene expression profiles of CML T cells up-regulated TCRζ chain and control cells were detected by Affymetrix GeneChip Human Gene 2.0 ST Array. The differentially expressed genes were analyzed by GO functional annotation analysis and KEGG pathway enrichment analysis. RESULTS: A total of 2248 differentially-expressed genes were obtained, including 553 up-regulated genes and 1695 down-regulated genes in experimental group as compared with those in control group (P<0.05) . The GO and KEGG enrichment analyses showed that differentially expressed genes involved in the biological processes related to T cell immune function, such as TCR signaling pathway, T cell proliferation and activation. Some of core genes involved in promoting the TCR signaling pathway, T cell proliferation, activation and apoptosis pathways were significantly up-regulated, while some core genes involved in inhibiting T cell activation were significantly down-regulated. CONCLUSION: The molecular mechanism of the significantly improved T cell activation and proliferation ability in CML patients after TCRζ up-regulation may be related to the differential transcripts mediated signaling pathways of T cell activation, proliferation and apoptosis.

[Expression and Clinical Significance of PD-L1 and MicroRNA-138-5p in Patients with Acute Myeloid Leukemia].

OBJECTIVE: To investigate the expression and clinical significance of PD-L1 and microRNA-138-5p in the peripheral blood mononuclear cells of patients with acute myeloid leukemia. METHODS: The SYBR GreenⅠreal-time PCR was used to detect the expression levels of PD-L1 mRNA and miR-138 in 20 cases of primary AML, 9 cases of relapsed/refractory AML and 8 cases of complete remission. The samples of peripheral blood of 20 healthy peoples were used as controls. RESULTS: The expression levels of PD-L1 in both the primary AML and the relapsed/refractory AML groups were significantly higher than those in the healthy control group (P<0.01), and the expression level of PD-L1 in the relapsed/refractory AML group was significantly higher than that in the primary AML group (P<0.01). The expression level of miR-138 in both the primary AML and the relapsed/refractory AML groups were significantly lower than that in the healthy control group (P<0.01). The 8 sampes out of 20 cases of primary AML patients achieved complete remission (CR) were collected and detected. The results showed that the expression level of miR-138 in the complete remission group was higher than that in the primary AML group (P<0.05), but the expression level of PD-L1 mRNA in the CR group was not significantly different from that in the primary AML group (P>0.05). and there was a negative correlation between PD-L1 mRNA and miR-138 in primary AML patients (P<0.05). CONCLUSION: The expression of PD-L1 increases and the expression of miR-138 down-regulates in PBMNCs of AML patients, furthermore, both correlate each other.

[Establishment of stable subline of K562 cells expressing human leucocyte antigen a1101].

The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A(*)1101 protein, which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte (CTL) effects against chronic myeloid leukemia (CML). The HLA-A(*)1101 protein encoding gene was amplified from peripheral blood mononuclear cell (PBMNC) of CML patient by RT-PCR; the 2A peptide linker (D-V-E-X-N-P-G-P) gene was linked to the 3'terminal of the HLA-A(*)1101 gene by recombinant PCR, then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene, and the eukaryotic recombinant expression vector containing HLA-A(*)1101-T2A-EGFP transcription box was constructed; the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells. The expression of GFP was monitored by fluorescence microscopy, finally stably transfected sublines of K562 cells containing HLA-A(*)1101 gene, and of K562 containing pEGFP-N3 vector were obtained by G418 selection; the transcriptional or translational expression of HLA-A(*)1101 gene was detected with RT-PCR and flow cytometry respectively. The results indicated that the eukaryotic expression vector HLA-A(*)1101-T2A-EGFP plasmid was successfully constructed; after G418 selection for 2 months, two sublines of K562 cells (HLA-A(*)1101(+)K562, pEGFP-N3(+)K562) expressing GFP were constructed. The expression of HLA-A*A1101 gene could be determined in HLA-A(*)1101(+)K562 cell line by RT-PCR, while the pEGFP-N3(+)K562 cells could not express HLA-A*A1101 gene. HLA-A(*)1101 protein and GFP double positive HLA-A(*)1101(+)K562 cells were up to 88.5%, which was obviously higher than pEGFP-N3(+)K562 cells (0.698%) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A(*)1101(+)K562 cells has been established and a subline of K562 cell expressing HLA-A(*)1101 protein on its cell membrane was successfully constructed, which provides the tool cells for further studying the specific cellular immunity against-CML.

[TCRζ gene expression in TCR gene-modified T cells specific for DLBCL-associated antigen].

AIM: To analyze the expression level of TCRζ chain gene in the DLBCL-associated antigen-specific T cells before and after being activated by coculture with Toledo cells (DLBCL cell line). METHODS: Real-time PCR with SYBR GreenI technique was used for detecting TCRζ chain expression in activated and unactivated DLBCL-associated antigen-specific T cells. ß2 microglobulin gene (ß2M) was used as an endogenous reference. Relative mRNA expression level of TCRζ gene was analyzed by the formula of both 2(-δCt); ×100% and 2(-δδCt);. RESULTS: Compared with (1.74±0.28)% of the relative mRNA expression level of TCRζ gene in TCR gene-untransduced T cells, the expression level of TCRζ gene was (1.78±0.22)% in unactivated TCR gene-transduced T cells and showed no obvious increase. While the expression of TCRζ gene arrived at (11.54±1.98)% in the activated TCR gene-modified T cells, which was significantly higher than that in unactivated TCR gene-modified and TCR gene-untransduced T cells (P<0.05), and was increased (6.59±0.80) and (6.48±0.36) times, respectively. CONCLUSION: The expression of TCRζ chain was up-regulated, when TCR gene-modified T cells were activated by the stimulation of specific antigens.