Detection, enrichment and expansion of T lymphocytes mediating alloresponse based on cytokine / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To detect, enrich and expand the cytokine secreting T lymphocytes after allogeneic PBMNCs stimulation.</p><p><b>METHODS</b>The novel cytokine secretion assay (CKSA) was applied to detect T lymphocytes secreting IFN-gamma at single cell level in human mixed lymphocytes reaction. IFN-gamma secreting T cells were enriched by means of magnetic sorting system and expanded with OKT(3), anti-CD(3)mAb and IL-2 combination. Antigen specificity of the expanded cells was confirmed using enzyme linked immunospot assay.</p><p><b>RESULTS</b>A sizable proportion of IFN-gamma secreting T lymphocytes could be detected [(1.12 +/-0.13)% compared with (0.23 +/-0.07)%] and be further enriched to (67.3 +/-10.5)%, or (93.8 +/-22.1) fold. T lymphocytes could be expanded up to 600-fold within 21-28 days and the specific IFN-gamma response of expanded cells was confirmed with stimulation of the relevant allogeneic PBMNC, which was significantly higher than the irrelevant PBMNC control.</p><p><b>CONCLUSION</b>It is feasible to detect significantly increased IFN-gamma secreting T lymphocytes after allogeneic PBMNCs stimulation based on the CKSA technique at single cell level and these cells can be efficiently enriched and expanded for further research.</p>

Evaluation of interferon-gamma producing-cells using enzyme linked immunospot assay in mice model of acute graft versus host disease / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate IFN-gamma producing-cells (IFN-gamma PCs) in allogeneic mixed lymphocyte reaction (MLR) and acute graft versus host disease (aGVHD) model of mice.</p><p><b>METHODS</b>Enzyme linked immunospot assay (ELISPOT) was applied to study IFN-gamma PCs in MHC mismatched mice spleen cell MLR and aGVHD model of mice.</p><p><b>RESULT</b>IFN-gamma PCs increased significantly in MLR after allogeneic mice spleen cell stimulation. In the experimental mice aGVHD model, IFN-gamma PCs were significantly higher in the severe aGVHD group than those in the moderate aGVHD. In the moderate aGVHD group, mice with GVHD prophylaxis regimen demonstrated significantly lower level of IFN-gamma PCs, compared with those without prophylaxis. IFN-gamma PCs were significantly correlated with the GVHD clinical scores in the group with moderate aGVHD and prophylaxis regimen.</p><p><b>CONCLUSION</b>ELISPOT is a fast, sensitive and specific approach to evaluate alloresponse in allogeneic mice MLR and IFN-gamma PCs are correlated closely with the severity of aGVHD and prophylaxis regimen in the MHC-mismatched mice model.</p>

Relationship between the IFN-gamma producing cells specific for recipient and acute graft versus host disease / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the relationship between the IFN-gamma producing cell specific for recipient (IFN-gamma-PCSR) in allogeneic stem cell transplantation (allo-HSCT) and acute graft versus host disease (aGVHD).</p><p><b>METHODS</b>In 37 consecutive HLA-identical sibling allo-HSCT pairs, peripheral blood mononuclear cells (PBMNC) from donors before allo-HSCT and recipients after allo-HSCT were taken as responder cells (RC), and PBMNC from recipients before allo-HSCT as allogeneic stimulator cells (allo-SC) in mixed lymphocyte reaction (MLR). IFN-gamma-PCSR in PBMNC were assayed using enzyme linked immunospot assay (ELISPOT).</p><p><b>RESULTS</b>Pretransplantation frequencies of IFN-gamma-PCSR in donor PBMNC were significantly higher in aGVHD group than in non-aGVHD group (P < 0.01) and IFN-gamma PCSRs (>or= 20/2 x 10(5)RC) were significantly associated with the occurrence of grade II-IV GVHD (P < 0.05). Compared with that before allo-HSCT, IFN-gamma PCSR in PBMNC of aGVHD patients was significantly increased (P < 0.05). When PBMNC from aGVHD patients reacted with donor PBMNC, the IFN-gamma PC was significantly lower than that with recipient PBMNC before allo-HSCT. Longitudinal analysis of IFN-gamma PCSR following allo-HSCT showed that compared with that in patients without aGVHD, the IFN-gamma PCSR were significantly higher in patients with that in aGVHD on day +14 (P < 0.01) and day +28 (P < 0.01), respectively. After immunosuppressive therapy for 7 days, IFN-gamma PC declined significantly (P < 0.05).</p><p><b>CONCLUSION</b>The recipient-specific IFN-gamma PC is closely correlated with the allo-response during allo-HSCT and may be helpful for the prediction, diagnosis and monitoring of aGVHD.</p>

Study on unrelated donor allogeneic bone marrow transplantation with Bu-CY2 conditioning regimen for myelodysplastic syndrome / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of Bu-CY(2) conditioning regimen on allogeneic bone marrow transplantation (BMT) with unrelated donor for myelodysplastic syndrome.</p><p><b>METHODS</b>Six patients received chemotherapy regimen of busulfan (Bu) and cyclophosphamide (CY) before allogeneic BMT (Bu 4 mg . kg(-1) . d(-1), -7 d - -4 d, CY 60 mg . kg(-1) . d(-1), -3 d - -2 d). Mycophenolate mofetil combined with cyclosporin A and methotrexate was used for prevention of acute graft-versus-host disease after transplantation. Lipo prostaglandin E(1)was used in prophylactic regimen for hepatic veno-occlusive disease.</p><p><b>RESULT</b>Neutrophil count began to be higher than 0.5 x 10(9)/Lat the 18th day after BMT. Platelet count began to be higher than 20 x 10(9)/Lat the 21st day after BMT. Disease-free survival in the six patients was 27 months.</p><p><b>CONCLUSION</b>Bu-CY(2) conditioning regimen on allogeneic bone marrow transplantation with unrelated donor is an effective therapy for patients with myelodysplastic syndrome.</p>

Tandem autotransplants of peripheral blood stem cells following sequential high-dose CHOEP chemotherapy for aggressive lymphoma / 中国实验血液学杂志

The purpose of this study was to evaluate the effecacy and safety of CHOEP mobilization regimen, and the effect and tolerance of sequential chemotherapy combined with tandem autotransplants of peripheral blood stem cells for aggressive lymphoma. The clinical data of 5 patients with recurrent, aggressive lymphoma treated with of sequential chemotherapy combined with tandem autotransplants were analyzed retrospectively. The patients included 1 HD and 4 NHL. Mobilization regimen was CHOEP combined with G-CSF 5 microg/(kg x d). The conditioning regimen for the tandem transplantation was high-dose CHOEP. The interval of the tandem autotransplantation was 9 (5 - 31) weeks. In tandem autotransplant, the cell number of MNC transfused was 3.05 (1.91 - 4.14) x 10(8)/kg and 3.55 (2.23 - 6.0) x 10(8)/kg; CD34(+) cells were 4.11 (2.59 - 4.94) x 10(6)/kg and 5.70 (2.77 - 10.6) x 10(6)/kg; CFU-GM was 2.96 (2.01 - 4.54) x 10(5)/kg and 2.44 (1.78 - 2.9) x 10(5)/kg respectively (P > 0.05). The results showed that all patients gained prompt and sustained hemotopoietic reconstitution. The interval of ANC >or= 0.5 x 10(9)/L was 10 (8 - 12) days and 10.5 (9 - 12) days; Pt >or= 2.0 x 10(9)/L was 11 (10 - 14) days and 12.5 (10 - 15) days respectively (P > 0.05). Four patients survived, three patients among them were alive in disease-free for median of 46 (9 - 88) months. The overall survival was 80%, and the disease-free survival was 60%. In conclusion, the method of sequential high-dose CHOEP chemotherapy combined with autotransplants of peripheral blood stem cells in tandem for aggressive lymphoma is probably safe and effective.

Detection of cytokine-secreting T lymphocytes in allogeneic reaction and a preliminary study on its clinical significance / 中国实验血液学杂志

In order to explore a new way to study allogeneic reactive T lymphocytes, detection of cytokine-secreting T lymphocytes after allogeneic peripheral blood mononuclear cells (PBMNCs) stimulation and investigation of its clinical significance were performed. A novel cytokine secretion assay (CKSA) was first applied to detect T lymphocytes secreting cytokine including IFN-gamma, IL-4 and IL-10 at single cell level in human mixed lymphocytes reaction. IFN-gamma-secreting T cells from PBMNCs were then evaluated in 2 patients with acute graft versus host disease (aGVHD) after allogeneic bone marrow transplantation. The results showed that compared with IL-4 and IL-10 (which were 0.12 +/- 0.03% and 0.10 +/- 0.03% respectively), a sizable proportion of IFN-gamma-secreting T lymphocytes could be detected (1.12 +/- 0.13)% after allogeneic PBMNCs stimulation. Preliminary results indicated that frequency of IFN-gamma-secreting T lymphocytes correlated with the onset and severity of clinical aGVHD. In conclusion, it is feasible to detect IFN-gamma secreting T lymphocytes after allogeneic PBMNCs stimulation and to apply the CKSA technique for clinical identification of aGVHD.

A comparison of clinical outcomes between HLA allele matched and 1 - 2 alleles mismatched unrelated allogeneic bone marrow transplantations / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare the clinical outcomes between HLA allele matched (HLA-M) and 1 approximately 2 alleles disparity mismatched (HLA-mis) unrelated allogeneic bone marrow transplantation (URD-BMT).</p><p><b>METHODS</b>Thirty-nine patients received HLA-M and 21 received HLA-mis URD-BMT for the treatment of acute leukemia, chronic myeloid leukemia in chronic phase (CP) and myelodysplastic syndromes (MDS) in our hospital between November 1998 and December 2002. Conditioning regimen was Bu 16 mg/kg plus CTX 120 mg/kg, and mycophenolate mofetil (MMF), CsA and MTX were given to prevent aGVHD.</p><p><b>RESULTS</b>Thirty-eight of the HLA-M group and 18 of the HLA-mis group were engrafted successfully. The median follow-up duration was 11 (2.5 - 52.0) months for HLA-M group and 9 (2 - 46) months for HLA-mis group. The 3-year probabilities of disease-free survival (DFS) for HLA-M and HLA-mis group were (79.2 +/- 7.1)% and (45.8 +/- 15.5)%, respectively (P < 0.05). Grade II - IV aGVHD occurred in 10 (26.3%) patients in HLA-M group and 6 (33.3%) in HLA-mis group, respectively (P > 0.05).</p><p><b>CONCLUSION</b>URD-BMT is an effective modality for the treatment of leukemia and MDS. The outcome after URD-BMT can be optimized by matching the HLA-A, B and DR alleles between the donor and recipient.</p>