Immunological consequences of interleukin 12 administration after autologous stem cell transplantation.

PURPOSE: The purpose is to determine the immunological effects of recombinant human interleukin (rhIL)-12 therapy after autologous stem cell transplantation. EXPERIMENTAL DESIGN: Twelve patients (8 non-Hodgkin's lymphoma, 2 Hodgkin's disease, and 2 plasma cell myeloma) were treated with rhIL-12 by bolus i.v. injection in doses of 30, 100, or 250 ng/kg starting at a median of 66 days posttransplant. Immunological assays were performed using serum and peripheral blood mononuclear cell (PBMC) samples obtained on study. RESULTS: Dose-dependent increases in the total lymphocyte count occurred during rhIL-12 therapy. The absolute number of peripheral blood CD4 T cells increased up to 16.3-fold, CD8 T cells up to 20.5-fold, B cells up to 11-fold, and natural killer (NK) cells up to 12.3-fold during rhIL-12 administration and returned to pretreatment baseline levels after discontinuation of rhIL-12. CD56(bright) NK cells expanded dramatically in the blood of a patient with baseline lymphopenia before rhIL-12 therapy. In vitro proliferation of patient PBMCs in response to IL-12 was indistinguishable from that of PBMCs obtained from healthy control sub-jects. Moreover, spontaneous in vitro proliferation of patient PBMCs increased significantly during rhIL-12 therapy. Increased levels of IFN-gamma and IL-18 were detected in the serum of patients treated in the 100 and 250 ng/kg dose cohorts during the first multiple dose cycle. CONCLUSIONS: Expansion of T, B, and NK cells occurs in vivo during rhIL-12 therapy after autologous stem cell transplantation for hematological malignancies. In contrast to their striking defect in IL-12-induced IFN-gamma production, posttransplant patient PBMCs exhibit normal proliferative responses to IL-12 in vitro. Additional investigation of rhIL-12 for posttransplantation immunotherapy is warranted.     

双次自体造血干细胞移植治疗恶性血液病的临床分析

目的：探讨双次自体造血干细胞移植对恶性血液病的临床疗效。方法：回顾性分析我院1995年－1999年间12例双次自体造血干细胞移植的资料,并与同期17例单次自体移植效果比较。结果：双次移植12例患者 移植后持续缓解6－68个月,中位数27个月,其中8例（66．7％）至今无病生存,1例于第二次移植后6个月出现复发,3例死于移植相关性疾病。单次自体移植17例患者移植后持续缓解1－36个月,中位数12个月,其中6例（35．3％）至今无病生存,8例肿瘤复发,3例死于移植相关性疾病。结论：双次自体造血干细胞移植治疗恶性血液病的疗效较好,其移植后18个月生存率有优于单次自体造血干细胞移植的趋势。     

Activated natural killer cells from patients with acute myeloid leukemia are cytotoxic against autologous leukemic blasts in NOD/SCID mice.

Natural killer (NK) cells are implicated in the surveillance of hematological malignancies. They participate in the immune response against residual acute myeloid leukemia (AML) after hematopoietic stem cell transplantation with partial HLA class I disparity. However, the role of NK cells in autologous leukemia-specific immunity remains poorly understood. We studied the function of NK cells in AML patients at diagnosis. Following isolation, CD56+CD3- cells exhibited a high proliferative potential in vitro in response to interleukin (IL)-2. The polyclonal population of activated AML-NK cells expressed normal levels of the activating receptor NKG2D and the major natural cytotoxicity receptor NKp46. AML-NK cells were highly effective with respect to interferon-gamma production, cytotoxicity against HLA class I-deficient K562 erythroleukemia cells in vitro and retardation of tumor growth in vivo in K562-bearing NOD/SCID mice. Importantly, when AML blasts were injected into NOD/SCID mice, a single dose of adoptively transferred autologous AML-NK cells significantly reduced the AML load by 8-77%. Recognition of AML blasts may be related to the observed upregulation of ligands for NKG2D and natural cytotoxicity receptors in vivo. We conclude that AML patient-derived NK cells are fully functional, in support of exploring the benefit of AML immunotherapy with IL-2-stimulated autologous NK cells.     

超高CD34+采集的动员方案序贯二次自体造血干细胞移植治疗难治性 霍奇金淋巴瘤

目的:探讨超高CD34+采集的动员方案后序贯二次自体造血干细胞移植治疗难治性霍奇金淋巴瘤的疗效和安全性。方法:对1例经过多疗程一线、二线、新药、免疫等均难治的霍奇金淋巴瘤患者,予以IA+C方案化疗+G-CSF动员干细胞后采集出超高水平CD34+细胞,之后行自体造血干细胞移植,移植后获得完全缓解,再予序贯第二次自体造血干细胞移植进行巩固治疗。结果:总计输注单个核细胞数13.67×108/kg,CD34+细胞48.68×106/kg,第一次自体造血干细胞移植术后第7天造血功能恢复,复查全身PET-CT提示获得完全缓解,第二次自体造血干细胞移植术后第8天造血功能恢复,两次自体造血干细胞移植相关并发症均在可控范围内。结论:超高CD34+细胞采集的IA+C方案化疗+G-CSF动员可以让患者有机会进行多次自体造血干细胞移植,是临床动员的创新方案。对于难治性霍奇金淋巴瘤,序贯二次自体造血干细胞移植可达到更深层次缓解,且安全性较高,延长患者无疾病生存期及总生存期,为难治性霍奇金淋巴瘤治疗提供更多临床依据。     

Immune Reconstitution of CD4+T Cells after Allogeneic Hematopoietic Stem Cell Transplantation and its Correlation with Invasive Fungal Infection in Patients with Hematological Malignancies

Objective: To explore the immune reconstitution of CD4+T cells after allogeneic hematopoietic stem celltransplantation (Allo-HSCT) and its relationship with invasive fungal infection (IFI) in patients with hematologicalmalignancies. Materials and Methods: Forty-seven patients with hematological malignancies undergoing Allo-HSCT in Binzhou Medical University Hospital from February, 2010 to October, 2014 were selected. At 1, 2and 3 months after transplantation, the immune subpopulations and concentration of cytokines were assessedrespectively using flow cytometry (FCM) and enzyme linked immunosorbent assay (ELISA). The incidenceof IFI after transplantation and its correlation with immune reconstitution of CD4+T cells were investigated.Results: The number of CD4+T cells and immune subpopulations increased progressively after transplantationas time went on, but the subpopulation cell count 3 months after transplantation was still significantly lowerthan in the control group (p<0.01). In comparison to the control group, the levels of interleukin-6 (IL-6) andIL-10 after transplantation rose evidently (p<0.01), while that of transforming growth factor-β (TGF-β) wasdecreased (p<0.01). There was no statistically significant difference level of interferon-γ (IFN-γ) (p>0.05). Theincidence of IFI was 19.2% (9/47), and multivariate logistic regression revealed that IFI might be related toTh17 cell count (p<0.05), instead of Th1, Th2 and Treg cell counts as well as IL-6, IL-10, TGF-β and IFN-γlevels (p>0.05). Conclusions: After Allo-HSCT, the immune reconstitution of CD4+T cells is delayed and Th17cell count decreases obviously, which may be related to occurrence of IFI.     

Differences in transplant-related complications between hematologic malignancies and solid tumors receiving high-dose chemotherapy and autologous peripheral blood stem cell transplantation

Multiple factors contribute to transplant-related complications after high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation, including conditioning regimens, number of infused stem cells and clinical characteristics of patient at transplant. We compared the transplant-related complications of 141 patients affected with hematological malignancies with those of 109 patients with solid tumors. The total number of peripheral blood stem cell transplantations performed was 339. High-dose chemotherapy mainly consisted of melphalan-, busulphan- or thiotepa-based regimens. Despite the equal number of infused CD34+ cells, patients with a hematological malignancy showed a slower absolute neutrophil count (days to neutrophils > 0.5 x 10(9)/L, 10.6 +/- 3.6 for hematological malignancies versus 9.1 +/- 1.2 for solid tumors, P < 0.0001) and platelet recovery (days to platelets > 20 x 10(9)/L, 16.4 +/- 9.8 for hematological malignancies versus 12.3 +/- 4.1 for solid tumors, P < 0.0001) than patients with a solid tumor. A significantly higher requirement of red blood cell (3.3 +/- 4.1 versus 2.0 +/- 1.9, P < 0.0029) and platelet units (7.5 +/- 10.4 versus 4.2 +/- 3.4, P < 0.0001) was observed for hematological malignancies than for solid tumors. Five graft failures were documented exclusively in patients with a hematological malignancy. Moreover, such patients displayed a longer duration of mucositis (P < 0.0028) and hospital stay (P < 0.0001), but no difference was observed in terms of febrile episodes. Transplant-related mortality was similar between the two groups. In conclusion, patients with a hematological malignancy overall have more complications than those with a solid tumor.     

单一剂量G-CSF用于异基因外周血造血干细胞动员和移植后造血恢复的临床研究

背景与目的:应用粒系集落刺激因子(granulocytecolony-stimulatingfactor,G-CSF)等动员剂从健康供者中动员和获取造血干细胞用于异基因外周血造血干细胞移植(allogeneicperipheralbloodstemcelltransplantation,allo-PBSCT)已在临床广泛开展,但在其作用机理、给药剂量和给药方案等方面还存在许多问题有待进一步研究。本研究旨在探讨单一剂量的G-CSF(250μg/d)用于allo-PBSCT的外周血干细胞(peripheralbloodstemcell,PBSC)动员以及移植后造血恢复的安全性和有效     

化疗联合单一剂量rhG-CSF用于自体外周血造血干细胞移植的临床研究

背景与目的:总结广东省干细胞多中心研究协作组自1999年6月至2001年12月间55例自体外周血造血干细胞移植治疗造血系统恶性疾病的资料,对化疗联合单一剂量rhG-CSF用于自体外周血造血干细胞移植前动员及移植后造血重建的效果进行研究和评价。方法:全部病例(急性髓细胞性白血病28例,急性淋巴细胞性白血病9例,非霍奇金淋巴瘤14例,其他4例)采用化疗+重组粒系集落刺激因子(rhG-CSF,格拉诺赛特)联合动员方案,其中白血病患者主要采用EA方案,恶性淋巴瘤患者主要采用以CTX为主的方案。rhG-CSF用量为250μg/d,WBC升至>4×109/L后,连续1～2天采集PBSC。移植后+3天开始使用rhG-CSF250μg/d,并观察造血重建情况。结果:动员所需的时间即自化疗开始至采集的平均时间为(18.08±3.63)天,rhG-CSF平均应用剂量为4.15μg·(kg·d)-1,应用时间平均7.12天。55例患者平均采集1.38次,采集到的MNC细胞数为(4.09±1.69)×109/kg,CD34+细胞平均值为8.5×106/kg,CFU-GM平均为(6.1±5.8)×105/kg。WBC恢复至>1.0×109/L及中性粒细胞绝对值>0.5×109/L的中位天数分别为10天和10.5天,全部移植患者均获满意的造血重建。结论:我们采用的EA和以CTX为主的化疗联合单一剂量rhG-CSF,是一种安全有效的动员自体外周血造血干细胞的方法,单一剂量rh     

Cellular therapy for intensification in oncology and hematology: manipulation of peripheral blood stem-cell products

Peripheral blood stem cells transplantation after high dose chemotherapy is increasingly used for the treatment of hematological malignancies and solid tumors. Autologous transplantation are now used as first line therapy. The use of hematopo?etic growth factors allowed to collect peripheral hematopoietic progenitors in quantity large enough for several autologous reinfusions. As for bone marrow, peripheral blood stem cells allow fast and long-term hematopoietic reconstitution. The fast regeneration is strictly correlated to the number of hematopoietic progenitors infused. Some questions are still opened anyway, notably about the tumoral contamination of the graft which has been clearly demonstrated. Even if residual tumor cells are clearly shown to participate to relapse, the interest of ex vivo purging is still matter of debate. Several techniques of positive selection are now available, selecting normal stem cells thanks to the CD34+ antigen. Negative selection is also available either using clinical purging or monoclonals antibodies against tumoral antigens. Endly, ex vivo expansion of hematopoietic stem cells is under investigation using medical progress in the field of growth factors. Therefore, improvement of mobilisation protocols, of technology for positive or negative selection, as well as the strategy for ex vivo expansion will be the tools for the development of the treatment of some hematological malignancies and solid tumors reducing the hematopoietic and extra hematopoietic toxicity.     

Is There a Place for Immunotherapy with Interleukin-2 to Prevent Relapse after Autologous Stem Cell Transplantation for Acute Leukemia?

Chemotherapy-resistant cells cause disease recurrence in a significant proportion of patients with acute leukemia treated with autologous stem cell transplantation due to the lack of immune-mediated effects which contribute significantly to the prevention of post-treatment disease recurrence. This conclusion is based on the observation that relapse after high dose chemotherapy supported by a stem cell transplant from a twin donor is 3-4 times higher than after transplant from an allogeneic donor. This anti-leukemic mechanism of transplanted donor cells has been termed graft-versus-leukemia (GVL) effect, and efforts are being directed toward utilizing such an immune-mechanism after autologous transplantation. Since interleukin-2 (IL-2) can induce remissions in selected patients with advanced leukemia, it has become a candidate cytokine to be used in attempts to introduce GVL after autologous stem cell transplantation. Here we review the available clinical data with IL-2 and critically evaluate whether IL-2 has a place as adjunct treatment to prevent relapse after autologous transplantation for acute leukemia.     

Transplantation of IL-2-mobilized autologous peripheral blood progenitor cells for adults with acute myelogenous leukemia in first remission.

In order to improve leukemia-free survival we evaluated the feasibility and efficacy of autologous transplantation of interleukin-2 (IL-2)-mobilized peripheral blood progenitor cells for adult patients with acute myelogenous leukemia in first remission. Forty-nine consecutive patients (median age 49, range 21-70) with acute myelogenous leukemia in first remission were enrolled on a study of high-dose cytarabine/mitoxantrone consolidation chemotherapy with post-recovery IL-2 used as a method of in vivo purging for the purpose of autologous peripheral blood progenitor cell transplantation. A median of 2.08 x 10(6) CD34+ peripheral blood progenitor cells/kg were infused 1 day after preparative conditioning with 11.25 Gy total body irradiation and cyclophosphamide (120 mg/kg). Forty-one patients received myeloablative chemoradiotherapy followed by the infusion of IL-2-mobilized autologous peripheral blood progenitor cells. The median times to both neutrophil and platelet recovery were 16 days (range, 2-43) and 23 days (8-318+ days), respectively. Twenty-seven patients remain alive with 24 in continued first complete remission. Median remission duration for all eligible patients is 8 months, and actuarial leukemia-free survival is 49+/-15%. The actuarial risk of relapse is 43+/-16%. Toxicity of autologous peripheral blood progenitor cell transplant included treatment-related death in three patients and serious organ toxicity in 12. Advanced age was a negative prognostic factor for leukemia-free survival. Results were compared to an age-matched historical control treated with autologous transplantation of chemotherapy-mobilized progenitor cells; no significant difference in favor of IL-2 mobilization could be demonstrated. Our results demonstrate that autologous transplantation of IL-2-mobilized peripheral blood progenitor cells is feasible in an unselected population of adult patients with acute myelogenous leukemia in first remission with minimal toxicity but no clear evidence of benefit in leukemia-free survival.     

Dendritic cell vaccines for leukemia patients

Dendritic cells are the most professional antigen-presenting cells to elicit T-cellular responses toward microbial agents and cancer cells. The graft-versus-leukemia effect observed after allogeneic stem cell transplantation strongly suggests that T lymphocytes play a major role in the rejection of leukemic cells. This graft-versus-leukemia effect might be enhanced through dendritic cell vaccination. The characterization of leukemia-specific antigens eliciting immune responses in the autologous host has prompted researchers and clinicians to broaden the spectrum of dendritic cell vaccines to hematological malignancies. Recently, the focus is on acute myeloid leukemia and chronic lymphocytic leukemia. This review summarizes data on the administration of autologous and allogeneic dendritic cells to leukemia patients as an interesting approach in cellular therapy of leukemias.     

Dendritic cell vaccines for leukemia patients

Dendritic cells are the most professional antigen-presenting cells to elicit T-cellular responses toward microbial agents and cancer cells. The graft-versus-leukemia effect observed after allogeneic stem cell transplantation strongly suggests that T lymphocytes play a major role in the rejection of leukemic cells. This graft-versus-leukemia effect might be enhanced through dendritic cell vaccination. The characterization of leukemia-specific antigens eliciting immune responses in the autologous host has prompted researchers and clinicians to broaden the spectrum of dendritic cell vaccines to hematological malignancies. Recently, the focus is on acute myeloid leukemia and chronic lymphocytic leukemia. This review summarizes data on the administration of autologous and allogeneic dendritic cells to leukemia patients as an interesting approach in cellular therapy of leukemias.     

Cellular immune responses in multiple myeloma patients with treatment-induced cytopenia early after high-dose chemotherapy and autologous peripheral blood stem cell transplantation

Proliferative T cell responses were compared for two patient groups with severe treatment-induced leukopenia (white blood cell counts < 0.5 x 10(9)/l): (i). multiple myeloma patients receiving high-dose melphalan and autologous peripheral blood stem cell transplantation; (ii). patients receiving conventional intensive chemotherapy for acute leukemia or myelodysplasia. Although the majority of circulating leukocytes were CD2(+)TCRalphabeta(+) in both groups, the myeloma patients showed significantly lower T cell proliferation in responses to several activation signals (anti-CD3, anti-CD3 + IL2, anti-CD3 + anti-CD28, anti-CD3 + anti-CD28+IL2. Our results suggest that myeloma patients with post-transplant cytopenia have a more severe cellular immune defect than patients with other hematological malignancies and severe cytopenia due to conventional intensive chemotherapy.     

Factors Affecting Engraftment Time in Autologous PeripheralStem Cell Transplantation

Background: Rapid hematological engraftment at autologous peripheral stem cell transplantation (APSCT)is a significant factor in reduction of early transplant-related complications and costs. For this reason, it isimportant to determine influences on hematological recovery. Methods: This study was designed to evaluatefactors affecting leukocyte and platelet engraftment times after high dose chemotherapy following APSCT.A total of 228 patients (131 males and 97 females) were enrolled. Results: There were statistically significantdifferences between patients with CD34+ cell doses ≥2.5×106/kg (n=180) and <2.5×106/kg (n=48), regardingleukocyte engraftment at 11 and 12 days, respectively (p<0.02), between G-CSF (n=167) and GM-CSF (n=61)posttransplant regarding median leukocyte engraftment times (p=0.005), and between with (n=75) or without(n=153) history of pretransplant radiotherapy for both leukocyte and platelet engraftment times (p<0.001).Conclusions: For leukocyte engraftment, a history of pretransplant radiotherapy, type of growth factor usedand number of CD34+ cells infused, and for platelet engraftment, a history of pretransplant radiotherapy werefound to be independent variables on multivariate analysis with the Cox regression method.     

Treatment of chronic myeloid leukemia with autologous transplantation using peripheral blood stem cells or bone marrow cultured in IL-2 followed by IL-2, GM-CSF,and IFN-alpha administration

Interleukin-2 (IL-2) is able to generate nonspecific cytotoxic effectors from hematopoietic precursors. We evaluated the feasibility and efficacy of chronic myeloid leukemia (CML) treatment with autologous hematopoietic stem cell transplantation (HSCT) using grafts cultured in IL-2 followed by immunotherapy with IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-α. Eight patients with CML were enrolled: five in an accelerated phase and three in a chronic phase. They received peripheral blood stem cells (PBSC) or bone marrow (BM) cultured in a medium containing IL-2 for 24 h. A median of 1.29 × 10⁶ CD34+ cells/kg were infused after conditioning with busulfan (12–16 mg/kg) in PBSC recipients. BM was infused without prior myeloablative therapy. The engraftment occurred with a median of 15 d. Engraftment failure developed in one patient. The transplantation was followed by a 1-mo regimen of IL-2 (0.5 × 10⁶ IU/m² daily) and GM-CSF, and 6 mo of IFN-α. One complete and one transient minor cytogenetic remission were observed. At 24 mo after transplantation, two patients had died of progressive disease and one of infection. Five patients had stable disease in the chronic phase. Autologous transplantation using IL-2-activated graft is feasible and the subsequent IL-2, GM-CSF, and IFN-α administration has acceptable toxicity. However, no benefits in comparison with conventional autologous transplantation for CML were identified in our study. Tissue Bank, University Hospital Brno, Czech Republic     

Activation of autologous or HLA-identical sibling cytotoxic T lymphocytes by blood derived dendritic cells pulsed with tumor cell extracts.

This study describes the effect of dendritic cells (DC) pulsed with tumor cell extracts on the induction and activation of autologous and HLA-identical sibling tumor cell specific cytotoxic T lymphocytes (CTL). The subjects of the study were patients with hematological malignancies (acute myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma), and stem cell transplant donors. Although lymphocytes primed with non-pulsed DC did not show definite cytotoxic activity against autologous or HLA-identical sibling patient tumor cells, both autologous and HLA-identical sibling CTL activated by DC pulsed with tumor cell extracts demonstrated markedly significant killing activity against tumor cells in the fashion of higher cytotoxicity with the increase of effector:target ratio (p<0.05). These findings reveal that DC pulsed with tumor cell extracts could be efficiently applicable in anti-tumor immunotherapy for patients with hematological malignancies.     

Correlation Between Survival and Number of Mobilized CD34+ Cells in Patients with Multiple Myeloma or Waldenström Macroglobulinemia

High-dose chemotherapy followed by autologous stem cell transplantation is the established treatment for symptomatic multiple myeloma (MM) or Waldenström macroglobulinemia (WM). We retrospectively analyzed the impact of mobilized CD34+ cell number on clinical outcomes in patients with MM or WM who underwent autologous stem cell transplantation in our hospital from 1997 to 2007. A total of 39 patients were identified. All patients received peripheral stem cell support after a conditioning regimen. We defined patients with collection of a large number (≥ 8?×?10⁶/kg) of CD34+ cells as super mobilizers (SM), and all others as normal mobilizers (NM). Although hematological engraftment was earlier in the SM group, overall survival did not differ significantly between groups (P?=?0.392). Likewise, no significant differences were seen in progression-free survival (P?=?0.201) or survival after relapse (P?=?0.330). In conclusion, our retrospective study could not find any correlation between survival and number of mobilized CD34+ cells, in contrast to previously reported results.     

Hematopoietic stem cell transplantation in childhood leukemias]

With the recent remarkable progress in cell processing technology, the number of patients or types of disease that are treated with hematopoietic stem cell transplantation (HSCT) has been rapidly increasing in both autologous or allogeneic settings. Transplantable cells can be harvested from blood or the umbilical cord as well as from bone marrow, and CD34+ cells can be effectively isolated as a pure stem cell source. However, the clinical benefit of HSCT in the treatment of hematological malignancies has remained unclear. As HSCT is principally a measure of stem cell rescue after high-dose chemotherapy with or without radiation therapy, it is difficult to clarify its contribution to the cure of the disease. Given these circumstances, pediatric patients with extremely high-risk features like Ph1 ALL or 11q23 translocation are usually selected for allogeneic HSCT even in the 1st CR. Additionally, HSCT could also reasonably be applied to those in 2nd CR or later. These considerations are based upon the idea that transplant-related mortality (TRM) is still too high in allogeneic settings. Allogeneic HSCT may be the first choice for treatment of such patients, if TRM becomes low and it is scientifically proved to be effective. On the other hand, autologous HSC grafts may be contaminated with cancer cells and lack the potency of allogeneic cell-mediated immune function. To improve the clinical results in this setting, a new type of anticancer agent with lower toxicity and a new strategy including immunotherapy should be established.