脐血移植GVHD与GVL效应的分子机制

从脐血T细胞、NK LAK细胞以及脐血细胞因子谱等方面探讨脐血移植中GVHD与GVL效应 ,并为脐血移植中减弱GVHD的发生 ,而使GVL效应能力正常提供初步的理论基础     

130脐血移植GVHD与GVL效应的分子机制

从脐血T细胞、NK/LAK细胞以及脐血细胞因子谱等方面探讨脐血移植中GVHD与GVL效应,并为脐血移植中减弱GVHD的发生,而使GVL效应能力正常提供初步的理论基础.     

脐血移植GVHD与GVL效应的分子机制

从脐血T细胞、NK／LAK细胞以及脐血细胞因子谱等方面探讨脐血移植中GVHD与GVL效应，并为脐血移植中减弱GVHD的发生，而使GVL效应能力正常提供初步的理论基础。     

NK细胞在异基因造血干细胞移植中对移植物抗白血病效应及移植物抗宿主病作用的影响

白血病复发和移植物抗宿主病（GVHD）是异基因造血干细胞移植（allo-HSCT）后影响病人生存的主要障碍。自然杀伤细胞（NK）独特的受体表达使其具有不同于一般免疫细胞的“同种反应性”效应,这种作用使NK细胞在增强移植物抗白血病效应（GVL）的同时抑制GVHD的发生,从而达到两者的分离。NK细胞在异基因造血干细胞移植中产生GVL及GVHD作用的机制以及影响因素的深入研究可能为探讨如何改善异基因造血干细胞移植预后提供有益的参考。     

NK细胞在异基因骨髓移植中作用的动物实验研究

本文探讨NK细胞在异基因小鼠骨髓移植GVHD和GVL反应中的作用。受体小鼠以60 Co照射 ,总剂量 8 0Gy;骨髓细胞经尾静脉注入受体小鼠中 ;GVHD反应指标包括 (1)弓背体位、大小便及脱毛等一般表现 ;(2 )嵌合体、体内混合淋巴细胞反应、 30d生存率为检测GVHD反应的定量指标 ;GVL作用指标包括外周血白细胞总数及白血病细胞比例 ,脾脏白血病细胞浸润比例 ,以及死亡率观察 ;病理学检查作为鉴别GVL实验中是否主要死于GVHD的指标。GVHD反应结果表明 ,异基因与后输NK细胞的异基因骨髓移植组均于 15d内死亡。而先输NK细胞组的GVHD反应明显减轻 ,30d死亡率为5 1%。同时输NK细胞组 30d内死于GVHD反应。移植后第 7天与 15d时 ,以先输NK细胞组的嵌合率高 ,第 30天时各组均未检出明显嵌合体存在。GVL作用实验方面 ,异基因、同时输NK细胞组及后输NK细胞的骨髓移植组虽然于 12d内表现出抗白血病作用 ,但在 30d内相继死于GVHD反应。先输NK细胞组于移植后 12d开始观察到较明显GVHD样反应 ,而外周血或脾细胞中L615细胞比例变化结果证实该移植途径具有较好的抗白血病作用。先输注供体NK细胞的异基因骨髓移植方式既能减轻GVHD反应 ,又能保留较好的GVL作用     

异基因造血干细胞移植后的移植物抗白血病作用的临床观察

移植物抗白血病作用（GVL）是异基因造血干细胞移植后,引起造血系统肿瘤持续缓解状态的一种免疫介导的反应。目前,对GVL的机理仍不清楚。临床上观察到,在GVL出现前,病人多经历了一个急或慢性的移植物抗宿主病（GVHD）过程。GVHD使受者正常的组织和细胞受到损伤,而同时产生     

脐血体外同时扩增T、NK及干祖细胞移植治疗BALB/c小鼠白血病

目的观察体外同时扩增T、NK及干祖细胞脐血用于移植治疗BALB/c小鼠白血病效果.方法在不同的细胞因子组合作用下定向扩增脐血干祖细胞及T、NK免疫细胞.扩增7天后,5 × 106个/只脐血细胞经尾静脉接种X射线照射的白血病小鼠,观察各组小鼠存活时间及检查存活证据及微小残留白血病细胞.结果1.细胞因子SCF,IL-3,IL-6,IL-7,IL-2组合能显著扩增脐血中的CD34 、CD3 T、NK细胞.2.尾静脉接种2×106个/只K562产生可移植性白血病BALB/c裸鼠.3.BALB/c白血病小鼠移植6周后,实验组共存活鼠12只.而未移植者在60天内死于肿瘤.4.流式细胞分析移植30天后小鼠外周血中人CD3 细胞从最低新鲜脐血移植组(0.48±0.40)%,最高SCF IL-3,6,7组中为(3.01±1.25)%.RT-PCR 检测发现,在12只扩增后移植存活6周小鼠中,检测不到bcr/abl 基因.在3只新鲜脐血移植存活6周的小鼠中,1只呈现阳性,说明移植取得了造血与免疫重建.所有脐血移植小鼠均未出现明显临床GVHD 症状.结论这种扩增后含一定水平T、NK免疫细胞的脐血能重建可移植性BALB/c 白血病小鼠的造血和免疫.     

人NK细胞的辐射敏感性观察

<正> NK细胞在移植免疫学中的作用和地位,越来越受到重视,它们参与了造血干细胞移植的排斥反应,与GVHD的关系十分密切。在免疫监护中起着重要的作用。因此,人们十分关心NK细胞对药物、激素及射线的辐射敏感性。一些材料表明,小鼠经过约1,000拉德(rad)全身照射,其活性未出现明显下降。有关人体外周血NK细胞辐射敏感性的研究报告不多。本文主要观察了人     

重组人IL-15促人脐血CD34+造血干细胞定向分化为NK细胞

目的 探讨IL－15促CD34^ 造血干细胞定向分化为NK细胞的作用。方法 采用MACS法分离脐血中CD34^ 细胞，分别用IL－15、SCF（造血干细胞因子）和IL－15 SCF体外培养，流式细胞仪测定CD3、CD16、CD56分子，MTT法检测NK细胞杀伤活性。结果 IL－15可以促进CD34^ 细胞分化为以CD56^ CD16^-为主的NK细胞，SCF具有较强的协同作用。结论 IL－15具有促使NK细胞定向分化的作用。     

自体外周血造血干细胞移植后序贯CIK细胞治疗急性髓系白血病

背景：细胞因子诱导的杀伤细胞作为一种有效的过继免疫治疗方法,成为治疗急性髓系白血病的一种新的手段,目前关于急性髓系白血病自体移植后序贯细胞因子诱导的杀伤细胞治疗的报道尚少,值得进一步研究。 目的：观察急性髓系白血病M2患者自体外周血造血干细胞移植后序贯细胞因子诱导的杀伤细胞治疗的临床疗效和不良反应。 方法：入选45例低、中危急性髓系白血病M2患者,其中19例在自体外周血造血干细胞移植后序贯了细胞因子诱导的杀伤细胞治疗,另26例未序贯细胞因子诱导的杀伤细胞治疗,比较两组患者的复发率、无病生存率、总生存率,观察细胞因子诱导的杀伤细胞治疗的安全性。 结果与结论：①序贯细胞因子诱导的杀伤细胞治疗组的复发率低于未序贯细胞因子诱导的杀伤细胞治疗组(21.05%,38.46%,P < 0.05);序贯细胞因子诱导的杀伤细胞治疗组患者的2年无病生存率和2年总生存率均高于未序贯细胞因子诱导的杀伤细胞治疗组,差异均有显著性意义(P < 0.05)。②19例接受细胞因子诱导的杀伤细胞治疗的患者均顺利完成治疗方案,治疗过程中除4例出现寒战、发热外,无其他不良反应。结果显示低、中危急性髓系白血病M2患者自体外周血造血干细胞移植后序贯细胞因子诱导的杀伤细胞治疗可降低原发病的复发率,提高患者的无病生存率及总生存率,且无明显不良反应,是一种安全、有效、可行的治疗方法。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

High expression of NKG2A/CD94 and low expression of granzyme B are associated with reduced cord blood NK cell activity

Human umbilical cord blood （CB） has recently been used as a source of stem cells in transplantation. NK cells derived from CB are the key effector cells involved in graft-versus-host disease （GVHD） and graft-versus-leukemia （GVL）. It was reported that the activity of CB NK cells was lower than that of adult peripheral blood （PB） NK cells. In this study, we analyzed the expression of some NK cell receptors and cytotoxicity-related molecules in CB and PB NK cells. The expressions of activating NK receptors, CD16, NKG2D and NKp46, did not show significant difference between CB and PB NK cells. But the expression of inhibitory receptor NKG2A/CD94 was significantly higher on CB NK cells. As to the effector function molecules, granzyme B was expressed significantly lower in CB NK cells, but the expressions of intracellular perforin, IFN-γ, TNF-α and cell surface FasL and TRAIL did not show difference between CB and PB NK cells. The results indicated that the high expression of NKG2A/CD94 and low expression of granzyme B may be related with the reduced activity of CB NK cells.     

The Role of Gamma Delta T Cells in Haematopoietic Stem Cell Transplantation

Although haematopoietic stem cell transplantation (HSCT) is a potential curative treatment for haematological malignancies, it is still a procedure associated with substantial morbidity and mortality due to toxicity, graft‐versus‐host disease (GVHD) and relapse. Recent attempts of developing safer transplantation modalities increasingly focuses on selective cell depletion and graft engineering with the aim of retaining beneficial immune donor cells for the graft‐versus‐leukaemia (GVL) effect. In this context, the adoptive and especially innate effector functions of γδ T cells together with clinical studies investigating the effect of γδ T cells in relation to HSCT are reviewed. In addition to phospho‐antigen recognition by the γδ T cell receptor (TCR), γδ T cells express receptors of the natural killer (NK) and natural cytotoxicity (NCR) families enabling them to recognize and kill leukaemia cells. Antigen recognition independent from the major histocompatibility complex (MHC) allows for the theoretical possibility of mediating GVL without an allogeneic response in terms of GVHD. Early studies on the impact of γδ T cells in HSCT have reported conflicting results. Recent studies, however, do suggest an overall favourable effect of high γδ T cell immune reconstitution after HSCT; patients with elevated numbers of γδ T cells had a significantly higher overall survival rate and a decreased rate of acute GVHD compared to patients with low or normal γδ T cell counts. Further research in terms of effector mechanisms, subtypes and tissue distribution during the course of HSCT is needed to assess the potentially beneficial effects of γδ T cells in this setting.     

Enrichment of IL-12–Producing Plasmacytoid Dendritic Cells in Donor Bone Marrow Grafts Enhances Graft-versus-Leukemia Activity in Allogeneic Hematopoietic Stem Cell Transplantation

A critical question in the field of allogeneic hematopoietic stem cell transplantation (HSCT) is how to enhance graft-versus-leukemia (GVL) activity while limiting graft-versus-host-disease (GVHD). We have previously reported that donor bone marrow (BM) precursors of plasmacytoid dendritic cells (pre-pDCs) can polarize donor T cells toward Th1 immunity and augment the GVL activity of donor T cells while attenuating their GVHD activity in a murine model of allogeneic HSCT. Clinical data on the role of donor pre-pDCs and conventional DCs (cDCs) on transplantation outcomes has been conflicting. To test the effect of increasing the proportion of pre-pDCs versus cDCs in a BM graft, we enriched CD11b⁻ pDCs by selectively depleting the CD11b⁺ myeloid DC (mDC) population from BM using FACS sorting in a murine model of allogeneic BM transplantation. Donor T cell expansion and GVL activity were greater in mice that received BM depleted of mDCs compared with mice that received undepleted BM. GVHD was not increased by depleting mDCs. To examine the mechanism through which mDC depletion enhances the GVL activity of donor T cells, we used BM and pDCs from IL-12p40KO mice, and found that the increased GVL activity of mDC-depleted BM was IL-12–dependent. This study indicates that a clinically translatable strategy of engineering the DC content of grafts can improve clinical outcomes in allogeneic HSCT through the regulation of donor T cell activation and GVL activity.     

IL‐12/IL‐18‐preactivated donor NK cells enhance GVL effects and mitigate GvHD after allogeneic hematopoietic stem cell transplantation

Adoptive transfer of donor NK cells has the potential of mediating graft‐versus‐leukemia (GVL) effect while suppressing acute graft‐versus‐host‐disease (aGVHD) during allogeneic hematopoietic stem cell transplantation (allo‐HSCT). However, these beneficial effects are limited by the transient function of adoptively transferred NK cells. Previous studies demonstrate that cytokine‐induced memory‐like NK cells that are preactivated by IL‐12, IL‐15, and IL‐18 have enhanced effector functions and long life span in vivo. Here, we investigated the effects of IL‐12/18‐preactivated and IL‐12/15/18‐preactivated donor NK cells on GVL and aGVHD in a murine model of allo‐HSCT. We found that both IL‐12/18‐ and IL‐12/15/18‐preactivated NK cells mediated stronger GVL effect than control NK cells mainly due to their elevated activation/cytotoxicity and sustained proliferative potential. Interestingly, we observed that although both IL‐12/18‐ and IL‐12/15/18‐preactivated NK cells significantly inhibited severe aGVHD, only the IL‐12/18‐preactivated NK cells maintained the beneficial effect of donor NK cells on mild aGVHD. The IL‐12/15/18‐preactivated NK cell infusion accelerated aGVHD in the fully‐mismatched mild aGVHD model. Our results demonstrated that IL‐12/18‐preactivated NK cells displayed sustained and enhanced GVL functions, and could mitigate aGVHD despite the severity of the disease. IL‐12/18‐preactivated donor NK cell infusion may be an effective and safe adoptive therapy after allo‐HSCT.     

Donor natural killer (NK1.1+) cells do not play a role in the suppression of GVHD or in the mediation of GVL reactions after DLI.

Donor regulatory T cells (CD3+ alphabetaT-cell receptor [TCR]+) derived from the repopulating host thymus have been shown to be primarily responsible for suppression of GVHD following DLI therapy in murine BMT models. However, natural killer (NK) T cells also have regulatory properties, and a role for NK T cells in suppression of GVH reactivity has not been completely excluded. NK cells may also contribute to the graft-versus-leukemia (GVL) effect associated with DLI therapy. In this study, we used a murine BMT model (C57BL/6 into AKR) to study whether depletion of donor NK cells had any impact on the suppression of GVH reactivity after DLI or on the DLI-induced GVL effect against acute T-cell leukemia. Depletion of donor NK cells was accomplished in vivo by giving DLI-treated bone marrow chimeras multiple injections of anti-NK1.1 monoclonal antibody (MoAb). The chimeras treated with anti-NK1.1 MoAb had significantly fewer splenic NK1.1 cells than nontreated chimeras, and splenocytes from anti-NK1.1-treated mice were deficient in the ability to generate lymphokine-activated lytic activity. Results presented here showed that NK-cell depletion had no effect on the suppression of GVH reactivity after DLI. When DLI-treated chimeras were challenged with an acute T-cell leukemia, NK-cell depletion had no discernible effect on GVL reactivity. These preclinical data suggest that donor NK cells do not have a significant role in the suppression of GVHD after DLI or in the mediation of GVL reactivity induced by DLI.     

Allogeneic hematopoietic transplantation and natural killer cell recognition of missing self

Summary:  Although the optimal donor for allogeneic hematopoietic stem cell transplantation (HSCT) is a human leukocyte antigen-matched sibling, 75% of patients do not have a match, and alternatives are matched unrelated volunteers, unrelated umbilical cord blood units, and full-haplotype-mismatched family members. To cure leukemia, allogeneic HSCT relies on donor T cells in the allograft, which promote engraftment, eradicate malignant cells, and reconstitute immunity. Here, we focus on the open issues of rejection, graft-versus-host disease (GVHD), and infections and the benefits of natural killer (NK) cell alloreactivity and its underlying mechanisms. Donor-versus-recipient NK cell alloreactivity derives from a mismatch between inhibitory receptors for self-major histocompatibility complex (MHC) class I molecules on donor NK clones and the MHC class I ligands on recipient cells. These NK clones sense the missing expression of the self-MHC class I allele on the allogeneic targets and mediate alloreactions. HSCT from 'NK alloreactive' donors controls acute myeloid relapse without causing GVHD. We review the translation of NK cell recognition of missing self into the clinical practice of allogeneic hematopoietic transplantation and discuss how it has opened innovative perspectives in the cure of leukemia.     

Infusion of select leukemia-reactive TCR Vbeta+ T cells provides graft-versus-leukemia responses with minimization of graft-versus-host disease following murine hematopoietic stem cell transplantation.

T-cell receptor (TCR) Vbeta-expression analysis by complementarity-determining region 3 (CDR3)-size spectratyping can identify the reactive populations in an immunologic response. This analysis was used in this study to characterize the Vbeta responses of C57BL/6 (B6) CD4+ and CD8+ T cells directed to either alloantigen (against [B6xDBA/2]F1; anti-H2d) or the syngeneic myeloid leukemia MMB3.19. Vbeta families exhibiting reactivity to the leukemia cells were then enriched for and administered in both syngeneic and allogeneic hematopoietic stem cell transplantation (HSCT) models to assess in vivo graft-versus-leukemia (GVL) potential. In syngeneic transplants, enrichment for pools of selected Vbeta families (Vbeta7, -11, and -13) of T cells or for a single Vbeta family (Vbeta7) of CD4+ T cells conveyed a beneficial GVL response to the recipients. Furthermore, in the haploidentical allogeneic model, both Vbeta6,7-enriched donor B6 T cells and Vbeta7-enriched CD4+ T cells exhibited significant GVL responses with concomitant minimization of graft-versus-host disease (GVHD) development compared with equal numbers of unfractionated T cells. These results suggest that CDR3-size spectratype analysis of and subsequent selection from donor T-cell repertoires can be an effective approach to separate GVL and GVHD potential following allogeneic HSCT.     

Quantity and Quality Reconstitution of NKG2A+ Natural Killer Cells Are Associated with Graft-versus-Host Disease after Allogeneic Hematopoietic Cell Transplantation

The immune mechanism underlying graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (HSCT) remains unclear. Natural killer (NK) cells play a crucial role in mediating pathogen-specific immunity and are the first donor-derived lymphocytes reconstituted post-HSCT. However, NK cells vary at different stages after HSCT. Here, we found that the absolute NKG2A⁺ subset cell counts and the percentages of NKG2A⁺ among NK cells were significantly reduced in GVHD patients after HSCT compared with those from non-GVHD patients. Moreover, the reduction in NKG2A⁺ NK cells in post-HSCT GVHD patients was ascribed to increased apoptosis and a decreased proliferation capacity while retaining a strong graft-versus-leukemia effect. In vitro assays showed that co-culture of T cells with NKG2A⁺ NK cells significantly reduced IFN-γ secretion by T cells and increased IL-4 secretion. Moreover, the CD25 expression level was decreased, whereas the number of cells with the CD4⁺CD25⁺FOXP3⁺ phenotype was increased. In addition, the NKG2A⁺ NK cells induced T cell apoptosis and decreased T cell proliferation during the co-culture process. Importantly, NKG2A⁺ NK cells mainly regulated activated but not resting T cells. In vivo assays showed that the serologic IL-10 level was evidently lower in GVHD than in non-GVHD patients, whereas the IL-1β, IFN-γ, and tumor necrosis factor-α levels were higher in GVHD patients. Furthermore, the NKG2A⁺ NK cell ratio from GVHD patients was markedly increased by the presence of exogenous IL-10 but not by other cytokines. In contrast, the NKG2A⁺ cell ratio from non-GVHD patients was not increased by IL-10. Therefore, post-HSCT GVHD may be ascribed to the reduced induction of NKG2A⁺ NK cells by IL-10, which further overactivates T cells.     

Levels of granzyme B, perforin and Fas ligand antigens and mRNAs in patients following allogeneic hematopoietic stem cell transplantation

In order to monitor the immunological status of patients after allogeneic hematopoietic stem cell transplantation(HSCT), granzyme B(GrB), perforin(PRF) and Fas ligand(L) antigens and mRNAs were measured by flow cytometry and real time RT-PCR, respectively. Cytoplasmic antigens were detected in whole blood after fixation and pretreatment with saponin. Real time PCR was carried out using extracted RNA from buffy coat. We measured these substances in a cytotoxic T cell clone, a natural killer cell line, and peripheral blood collected from 11 patients after HSCT. Although changes in antigen levels were not detected, increased levels of GrB and Fas L mRNAs were quantitatively measured in CTLs and NK cells stimulated by IL-2 combined with IL-12. Increased levels of GrB and/or PRF antigens were detected in four of five patients with chronic GVHD. Increased mRNA levels were also observed in one or more of GrB, PRF or Fas L in four of five patients with cGVHD, although there was a discrepancy between antigen and mRNA positivity. Four of six patients without cGVHD were positive for apoptosis-inducing factors, either by antigen detection or RT-PCR. One of these four had relapsing leukemia, and another had herpes zoster infection, while the reasons for positive results in the other two patients are not clear. Although changes in antigen levels did not parallel those in mRNA, measurement of these parameters may assist in predicting GVHD, GVL and infections following HSCT.