Jing Lai

Oligoclonal expansion of TCR Vδ T cells may be a potential immune biomarker for clinical outcome of acute myeloid leukemia

BackgroundRecent data have shown that γδ T cells can act as mediators for immune defense against tumors. Our previous study has demonstrated that persisting clonally expanded TRDV4 T cells might be relatively beneficial for the outcome of patients with T cell acute lymphoblastic leukemia after hematopoietic stem cell transplantation (HSCT). However, little is known about the distribution and clonality of the TRDV repertoire in T cell receptor (TCR) of γδ T cells and their effects on the clinical outcome of patients with acute myeloid leukemia (AML). The aim of this study was to assess whether the oligoclonal expansion of TCR Vδ T cells could be used as an immune biomarker for AML outcome.Findingsγδ T cells were sorted from the peripheral blood of 30 patients with untreated AML and 12 healthy donors. The complementarity-determining region 3 (CDR3) sizes of eight TCR Vδ subfamily genes (TRDV1 to TRDV8) were analyzed in sorted γδ T cells using RT-PCR and GeneScan. The most frequently expressed TRDV subfamilies in the AML patients were TRDV8 (86.67 %) and TRDV2 (83.33 %), and the frequencies for TRDV1, TRDV3, TRDV4, and TRDV6 were significantly lower than those in healthy individuals. The most frequent clonally expanded TRDV subfamilies in the AML patients included TRDV8 (56.67 %) and TRDV4 (40 %). The clonal expansion frequencies of the TRDV2 and TRDV4 T cells were significantly higher than those in healthy individuals, whereas a significantly lower TRDV1 clonal expansion frequency was observed in those with AML. Moreover, the oligoclones of TRDV4 and TRDV8 were independent protective factors for complete remission. Furthermore, the oligoclonal expansion frequencies of TRDV5 and TRDV6 in patients with relapse were significantly higher than those in non-recurrent cases.ConclusionsTo the best of our knowledge, we characterized for the first time a significant alteration in the distribution and clonality of the TRDV subfamily members in γδ T cells sorted from AML patients. Clonally expanded TRDV4 and TRDV8 T cells might contribute to the immune response directed against AML, while oligoclonal TRDV5 and TRDV6 might occur in patients who undergo relapse. While the function of such γδ T cell clones requires further investigation, TRDV γδ T cell clones might be potential immune biomarkers for AML outcome.

Lower expression of PD-1 and PD-L1 in peripheral blood from patients with chronic ITP

Background: T-cell dysregulation is a major event involved in immune thrombocytopenic purpura (ITP). Increasing data have indicated that abnormal expression of T-cell immunosuppressive receptors, such as programmed death (PD) 1 and cytotoxic T lymphocyte antigen-4 (CTLA-4), may be related to autoimmune disease pathogenesis. Methods: We analyzed the expression levels of PD-1, its ligand PD-L1, and CTLA-4 in peripheral blood mononuclear cells from 18 patients with chronic ITP by real-time polymerase chain reaction, and samples from 20 healthy individuals served as control. Results: The results demonstrated significantly lower expression of PD-1 (median: 0.0015) and PD-L1 (median: 0.0572) in chronic ITP patients compared with healthy individuals (PD-1: median: 0.0117, P < 0.0001; PD-L1: median: 0.5428, P < 0.0001), while there was no significant difference in the CTLA-4 expression level between the chronic ITP patients (median: 0.0818) and healthy individuals (median: 0.1667) (P = 0.219). Moreover, a positive correlation between the expression levels of PD-1 and PD-L1 (rs = 0.486, P = 0.041) and CTLA-4 and PD-1 (rs = 0.643, P = 0.004) in the chronic ITP patients was found. Conclusion: Consistently lower expression of T-cell immunosuppressive receptors is a common characteristic of chronic ITP, which may be associated with its pathogenesis.

Re-balance of memory T cell subsets in peripheral blood from patients with CML after TKI treatment

T cell immune surveillance is considered an important host protection process for inhibiting carcinogenesis. The full capacity of T cell immune surveillance is dependent on T cell homeostasis, particularly for central memory T (TCM) cells and stem cell memory T (TSCM) cells. In this study, distribution of T cell subsets in peripheral blood from 12 patients with chronic myeloid leukemia (CML) and 12 cases with CML in complete remission (CR) was analyzed using a multicolor flow cytometer, and 16 samples from healthy individuals (HIs) served as control. The proportion of CD8+ TSCM and CD4+ and CD8+ TCM cells were lower, while CD4+ effector memory T (TEM) cells and CD4+ and CD8+ terminal effector T (TEF) cells were higher in CML patients compared with HIs. Moreover, the proportion of CD8+CD28- T cells, which were found to have the immune suppressive function, increased in the naive T (TN) cell and TCM subsets in CML patients compared with HIs. Our study reveals that elimination of leukemia cells by treating with tyrosine kinase inhibitors (TKIs) restores the memory T cell distribution from a skewed pattern in CML patients who are under leukemia burden, indicating that leukemia-specific immune responses mediated by T cells might be induced and maintained in CML patients, however, these responsive T cells might gradually become exhausted due to the continued existence of leukemia cells and their environment; therefore, T cell activation using a different approach remains a key point for enhancing global T cell immunity in CML patients, even for those with CR status.

Higher PD-1 expression concurrent with exhausted CD8+ T cells in patients with de novo acute myeloid leukemia

Objective To investigate the association between the T cell inhibitory receptor programmed death 1 (PD-1) and T cell exhaustion status in T cells from patients with de novo acute myeloid leukemia (AML) and AML in complete remission (CR). Methods Surface expression of PD-1 and the exhaustion and immunosenescence markers CD244 and CD57 on CD3+, CD4+ and CD8+ T cells from peripheral blood samples from 20 newly diagnosed, untreated AML patients and 10 cases with AML in CR was analyzed by flow cytometry. Twenty-three healthy individuals served as control. Results A significantly higher percentage of PD-1+ cells were found for CD3+ T cells in the de novo AML group compared with healthy controls. In addition, an increased level of PD-1+CD8+ T cells, but not PD-1+CD4+, was found for CD3+ T cells in the de novo AML and AML-CR samples. A higher percentage of CD244+CD4+, CD244+CD8+, CD57+CD4+ and CD57+CD8+ T cells was found in CD3+ T cells in samples from those with de novo AML compared with those from healthy controls. Strong increased PD-1+CD244+ and PD-1+CD57+ co-expression was found for CD4+ and CD8+ T cells in the de novo AML group compared with healthy controls. Conclusions We characterized the major T cell defects, including co-expression of PD-1 and CD244, CD57-exhausted T cells in patients with de novo AML, and found a particular influence on CD8+ T cells, suggesting a poor anti-leukemia immune response in these patients.

The Effects of Antisense miRNA-20a Alone or in Combination with Imatinib on K562 Cell Proliferation

Objective: The effects of microRNA-20a (miR-20a) antisense oligonucleotides (ASODNs) on the proliferation and apoptosis of K562 cells were investigated, and the effects of these ASODNs in combination with imatinib on K562 cells were preliminarily observed. Methods: miR-20a ASODNs and scrambled oligonucleotides (SODNs) were chemically synthesized, and the later was used as the control. miR-20a ASODNs were transfected into K562 cells using Lipofectamine 2000 transfection reagent, and the expression of miR-20a was detected using real-time quantitative RT-PCR (qRT-PCR). The CCK8 assay was performed to detect the inhibition of the cell growth rate. The cells were stained by Hoechst 33258 to detect apoptotic cell morphology. Annexin V/PI double staining was used to detect the cell apoptosis rate using flow cytometry. The protein expression levels of E2F1, P21, and Bim in the K562 cell line were detected using western blotting. Results: The qRT-PCR results showed that the expression level of miR-20a in K562 cells transfected with miR-20a ASODNs was lower than those in the normal control, SODN and blank transfection groups (p < 0.05). miR-20a ASODNs significantly inhibited the growth of K562 cells as compared to the controls (p < 0.05). The Hoechst staining results showed morphological changes, suggesting apoptosis. The cell apoptosis rates in the ASODN group was (13.9 ± 1.5)%, which was significantly higher than that in the normal control group (1.84 ± 0.21)%, blank transfection group (3.21 ± 0.32)%, and SODN group (3.72 ± 0.44)% (p < 0.05). The protein expression of E2F1 and P21 in K562 cells transfected with miR-20a ASODNs were higher, while the level of Bim protein was significantly lower than that in the control groups. When miR-20a ASODNs were combined with imatinib, the growth of K562 cells was significantly inhibited as compared to the ASODN treatment alone, imatinib alone, and SODN+imatinib groups (p < 0.05). Conclusions: miR-20a ASODNs could induce apoptosis and inhibit the proliferation of K562 cells. In addition, imatinib combined with miR-20a ASODNs can increase the inhibitory effect on K562 cell proliferation.