Changes of Th1/Th2/Th17 in patients received non-myeloablative haploidentical hematopoietic stem cell transplantation detected by flow cytometric bead array / 中国实验血液学杂志

This study was purposed to investigate the changes of Th1/Th2/Th17 in patients received non-myeloblastic haploidentical hematopoietic stem cell transplantation (NAHSCT). The levels of IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ, as well as IL-17 level were determined by flow cytometric bead array (CBA) in samples from 18 patients underwent allo-peripheral NAHSCT at different time points before and after transplantation. The results showed that all cytokines changed obviously after transplantation, and their serum levels were higher than that before transplantation. The expression levels of IL-2, IL-4 and IL-17 changed early, and their obviously up-regulation was found after transplantation. The expression levels of IL-6, IL-10 and TNF-α changed significantly, and were high as compared with that before transplantation. The change of INF-γ serum level was observed late, its rising occurred at week 4 after transplantation. The expression of all cytokines kept increasing during 4 weeks after transplantation and peaked at week 4. It is concluded that the serum levels of all cytokines from the patients after NAHSCT increased significantly, in which the levels of IL-2, IL-4 and IL-17 increased early, but the level of INF-γ changed late. The detection of cytokines is helpful for deep understanding the pathophysiologic mechanism of transplant-related complications.

Relationship between WT1-specific T-cell subsets and graft-versus-host disease after nonmyeloablative allogeneic transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the relationship between WT1-induced T-cell subsets and graft-versus-host disease (GVHD) after nonmyeloablative allogeneic hematopoietic stem cell transplantation (NST).</p><p><b>METHODS</b>Peripheral blood mononucleated cells (PBMCs) from 19 patients who expressed WT1 and developed GVHD after NST were simulated by WT1126-134 peptide in vitro, and proportions of WT1-induced-T-cell subsets (Tc1, Tc2, Th1, Th2 cells) before and after transplant were detected by intracellular cytokine staining (ICCS) assay. WT1-specific CD8(+) CTLs of 14 patients with HLA-A*0201 were detected by HLA-A*0201/WT1 pentamer.</p><p><b>RESULTS</b>(1) 17 of 19 patients developed GVHD, among whom proportions of Tc1 and Th1 cells, achieved peak value in 16 patients at occurrence of GVHD (P = 0.039); (2) The peak proportions of Tc1 and Th1 cells in patients with aGVHD above grade II were higher than those with grade I, but being no statistical difference (P = 0.900 and P = 0.140, respectively); (3) The peak proportion of Th1 cells (P = 0.004), but not Tc1 cells (P = 0.060) in patients with extensive cGVHD was significantly higher than that in patients with limited one; (4) Proportions of Tc1, Th1 and WT1(+)CD8(+)CTL in patients without GVHD were similar to those in patients with Grade I aGVHD, but lower than those in aGVHD above grade II.</p><p><b>CONCLUSION</b>GVHD promotes the generation of WT1-induced GVL effect, and the intensity of the latter maybe correlated with the intensity of GVHD, especially cGVHD. Th1 cells play a more important role in the enhancement of WT1-induced GVL effect in extensive cGVHD patient than in limited cGVHD patients.</p>

Application of HLA-A*0201/WT1 pentamer combined with intracellular IFNgamma+ staining in detecting circulating WT1 specific T cells in leukemia / 中国实验血液学杂志

This study was purposed to investigate the value of combination of pentamer and intracellular IFNgamma staining in the qualitative and quantitative detection of circulating antigen-specific T cells. WT1 expressions in 14 HLA-A*0201+ patients and their matched donors were detected by RT-PCR, and circulating WT1 specific T cells were assayed by HLA-A*0201/WT1 pentamer combined with intracellular IFNgamma+ staining. The results showed that the low level of WT1 expression was found only in 2 cases out of 14 donors, but different levels of WT1 expression could be observed in all leukemic patients. The WT1+CD8+ CTL and WT1+IFNgamma+ cells did not detected in all 14 donors, but WT1+CD8+ CTL cells in 2 patients and WT1+IFNgamma+ cells in 3 patients could be detected before transplantation respectively, there was no significant difference between them, while the WT1+CD8+ CTL cells and WT1+IFNgamma+ cells both could be detected in all 14 patients after transplantation, the positive detection rate after transplantation was obviously higher than that before transplantation. The WT1+CD8+ and WT1+ IFNgamma+ cells could be detected within 30 days after transplantation, but the positive detection rate of WT1+IFNgamma+ cells was higher than that of WT1+CD8+ CTL cells (p=0.014). The median peak value of WT1+CD8+ CTL cells was 0.18% in 14 patients, and the median peak value of WT1+IFNgamma+ cells was 0.83% in 14 patients, the later was significantly higher than former. The median peak time of WT1+CD8+ CTL cells was 75 days after transplantation, while the WT1+IFNgamma+ cells was 105 days after transplantation, there was no significant difference between them. It is concluded that pentamer and intracellular IFNgamma staining may effectively detect circulating WT1 specific T cells in leukemic patients, and the combination of these two methods profit to the exact qualitation and quantitation of circulating antigen-specific T cells.

Changes of lymphocyte subsets in acute leukemia patients after HLA-mismatched nonmyeloablative hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was purposed to investigate the reconstitution of immune system in patients with acute lymphocyte leukemia (ALL) or acute myeloid leukemia (AML) after HLA-mismatched nonmyeloablative hematopoietic stem cell transplantation (NHSCT) and its relation with infection and GVHD. 6 ALL and 4 AML patients having HLA-mismatched related donors received the nonmyeloablative precondition regimen composed of fludarabine (Fln), ATG, Ara-C, CTX and total body irradiation (TBI) in dose 2 Gy. The GVHD was prevented and treated by CsA, anti-CD25 antibody and mycophenolic mofetil (MMF) before and after transplantation. The flow cytometry was used to detect the changes of total T cells, help/inducer T cells, suppressor/killer T cells, gamma/delta T cells, B cells, NK cells, NKT cells, regulatory T cells, activated T cells, naive T cells, memory T cells and ratio of CD4/CD8 in patients with remission resulting from chemotherapy before transplantation, and analyse the relation of immunofunctional cells to infection and GVHD after transplantation, compare the difference in recovery of immune system of ALL and AML patients. The results showed that the recovery of total lymphocytes and lymphocyte subsets displayed one's own regular pattern. As compared with patients without GVHD, the counts of lymphocyte subsets in patients with GVHD was higher, while the counts of gamma/delta T cells, regulatory T cells, NK cells, the counts of B cells, NK cells, naive cells and CD4/CD8 ratio as well as the counts of B cells, naive T cells and NK cells were lower at 1 month, 2 - 3 months and 6 - 8 months after transplantation respectively. The total T cells and subsets recovered slowly, but NK cells and NKT cells recovered rapidly in patients with infection at early period after transplantation, the B cells and naive B cells recovered rapidly at 3 months after transplantation. There was no difference in lymphocyte recovery between ALL and AML patients. It is concluded that the analysis of each lymphocyte subsets may indirectly show the recovery of thymus function in patients, the changes of NK cells, B cells and naive T cells have an important significance for identifying and forecasting the GVHD and infection.

Effects of IL-21 alone or in combination with IL-15/IL-2 on proliferation and anti-tumor activity of G-CSF-mobilized peripheral blood mononuclear cells in vitro / 中国实验血液学杂志

This study was purposed to investigate the proliferation and antitumor activity of rhG-CSF-mobilized peripheral blood mononuclear cells (G-PBMNCs) activated by interleukin 21 (IL-21) alone or in combination with interleukin 15 (IL-15)/interleukin 2 (IL-2) and to evaluate the feasibility and value of tumor immunotherapy with cytokine combinations. G-PBMNCs were activated by IL-21 alone or in combination with IL-15/IL-2 in vitro, and the proliferation of the activated G-PBMNCs was analyzed by CCK-8 assay. The cytotoxicity of the activated G-PBMNC to the K562 cells was studied by the test principle which is based on target cell labeling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNA-labeling with propidium iodide (PI) for identification of target cells with compromised cell membranes. The phenotypes of the activated G-PBMNCs were assayed by flow cytometry. The results showed that the cytotoxicity of IL-21 group had no difference from which of IL-2 group. When G-PBMNCs were exposed to the combinations of IL21+IL15/IL21+IL15+IL2, the cytotoxicity was significantly enhanced at E:T ratio of 25:1, as compared with combination of IL21+IL2 (p<0.05). The cytotoxicity of the cytokines combinations was significantly higher than that in cytokine used alone at E:T ratio of 50:1 (p<0.05). The cryopreservative and resuscitative G-PBMNCs showed the same result with the fresh G-PBMNCs in cytotoxicity test. The proportions of CD3+ and CD8+ T cells were increased when G-PBMNCs were incubated with cytokines for 72 hours. CD4, CD3-56+ and CD3+56+ counts were significantly elevated when G-PBMNCs were exposed to IL21 + IL15 (p<0.05). It is concluded that IL-21 alone enhance the antitumor activity of G-PBMNCs, which further strengthens when IL-21 combinated with IL-15.

The correlated research of acute graft versus host disease after allogeneic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the relationship between the expression of FasL, Perforin and Granzyme B and the development of acute graft versus host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>The peripheral blood mRNA expression of granzyme B, perforin, fasL from 17 patients after allo-HSCT was detected by competitive quantitative RT-PCR and the relationship between FasL, Granzyme B and Perforin expressions and clinical symptom of aGVHD was analyzed.</p><p><b>RESULTS</b>The expression level of Granzyme B, Perforin and FasL was 4.6 +/- 0.2, 4.5 +/- 0.1, 1.4 +/- 0.1 before aGVHD occurrence respectively, and was 98.7 +/- 2.5, 91.8 +/- 3.4, 61.5 +/- 2.2, after the occurrence in 14 patients (P < or = 0.05). Over expressions of Granzyme B, Perforin, and FasL during acute GVHD were detected in 13 of 14, 12 of 14, and 12 of 14 patients respectively. The upregulated expressions occurred prior to clinical symptom of aGVHD.</p><p><b>CONCLUSION</b>The expressions of Granzyme B, Perforin, and FasL were significantly high in patients with acute aGVHD. Monitoring of the expressions, might predict the occurrence of clinical aGVHD and it severity and prognosis.</p>

Clinical study on anti-leukemia effect mediated by dentritic cells / 中国实验血液学杂志

Immunotherapy of tumor is extensively attentioned as an important part of combined therapy of tumor in recent years. Dendritic cell (DC) is the most powerful antigen presenting cell (APC) by now which not only activates auto-immunity to attack tumor cells, but also does help to enhance antitumor effect for allogenic bodies. To explore the feasibility and safety of clinical therapy application of peripheral blood derived DC cultured ex vivo, and analyze the influence of DC-inducing-immunotherapy upon long-term survival of ANLL patients accepted autologous bone marrow transplantation, peripheral blood mononuclear cells (PBMNC) of 13 ANLL patients after autologous bone marrow transplantation were collected by using CS3000Plus. DC immunotherapy was administered after cultivation of PBMNC ex vivo for 2 weeks, desease-free survival time was observed after therapy for long time follow-up. The results showed that no any severe adverse event associated with DC therapy was observed, the survival analysis of Kaplan-Meier suggested that five year survival rate was 75.52% in DC group while 45.71% in non-DC group. DC group surpassed non-DC group in accumulative survival rate. It is concluded that the ex vivo cultivation and clinical therapy application of DC derived from peripheral blood are feasible and safe, DC immunotherapy in patients with acute non-lymphocytic leukemia after autologous bone marrow transplantation prolongs desease-free survival time and enhances long-term survival rate.

Aberrant expression and deletion of FHIT gene in leukemias / 中国实验血液学杂志

FHIT (fragile histidine triad) gene at chromosome 3p14.2 usually expresses at a very low level in human tissue and cells. A high frequency of abnormalities in FHIT gene has been demonstrated in various cancers. FHIT is proposed as a putative tumor-suppressor gene. To evaluate the expression of the FHIT gene in various leukemias, bone marrow or peripheral blood samples from 98 leukemia patients were tested by RT-PCR: 38 from patients with AML-[M(2)(9), M(3)(12), M(4)(8), M(5)(9)], 16 with ALL, and 34 with CML-[CP(20), AP(4), BC(10)] of various FAB types, as well as 10 patients with other hematological malignancies. To detect a deletion in sequencing the FHIT gene, the representative aberrant PCR products were cloned and then sequenced. The results showed that 22/38 (58%) patients with AML, 9/16 (56%) patients with ALL and 19/34 (56%) patients with CML were detectable of aberrant FHIT mRNA transcripts or deletion of FHIT. In 6 (16%) AML patients, 3 (19%) ALL patients, and 5 (15%) CML patients, the wild-type product was absent. Some patient's samples - 6 (42%) AML, 6 (38%) ALL, and 14 (41%) CML revealed aberrant FHIT transcripts in addition to a normal-sized band. Samples from healthy donors (PB, n = 12; BM, n = 5) did not indicate any abnormal expression. Eleven isolated fragments from various patterns of FHIT gene expression were investigated using cDNA sequencing. Sequence analysis revealed deletion of exon 4-8, exon 5-8, and exon 5-6 in various leukemias, as well as the deletion of the full FHIT gene sequence. The fused transcripts included: exon 3 and exon 9, exon 3 and exon 7, exon 4 and exon 9, exon 5 and exon 7. Sequence analysis of aberrant fragments present in samples from an AML and a CML patients was detected for point mutations and insert mutations located in exons 2, 8 and 10, plus a variety of aberrant transcripts. Deletion or aberrant FHIT mRNA transcripts in 50/98 (51%) leukemia patients were found. All samples with aberrant FHIT lacked gene product. A Kaplan-Meier plot of survival in patients with AML in relation to FHIT expression revealed that aberrance or loss of FHIT gene significantly correlated with a low clinical remission rate and poor overall survival.

The Effects of Cyclosporin A on Leukemia Cells In Vitro / 中国实验血液学杂志

To explore the mechanism of CSA in the reversal of drug-resistance of leukemia cells, K562 and K562/ADM cells were used for in vitro study. Drug sensitivity of cells was evaluated by MTT assay and cell survival by trypan blue exclusion. Apoptosis was detected by terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL) and agarose gel electrophoresis. The results showed that 1.0 mg/L CSA alone have no effect on the survival of K562 and K562/ADM cell lines, but it could enhance the chemotherapy sensitivity of the 2 cell lines, and accelerate the apoptosis induced by ADM. These effects were stronger in K562/ADM cell line, when the concentration of CSA > 1.0 mg/L, CSA itself could kill leukemia cells and induce the apoptosis of the 2 cell lines. It is concluded that CSA can reverse leukemia cells' drug-resistance by enhance apoptosis of the leukemic cells, CSA itself can induce leukemia cells' apoptosis and kill leukemia cell too.