Graft engineering of G-CSF-mobilized allogeneic leukapheresis products by counterflow centrifugal elutriation after CD34 column adsorption.

A major hindrance to the use of PBSC in allogeneic transplantation is the high rate of contamination with T lymphocytes, resulting in a considerable risk of GvHD. Natural killer (NK) cells are active against tumor cells but do not contribute to the development of GvHD. After adsorption of CD34+ cells of mobilized allogeneic leukapheresis products on a Ceprate column, we studied the separation of CD34 unadsorbed cells by counterflow centrifugal elutriation (CCE). Up to 1.0 x 10(10) cells were clearly separated into lymphocytes (fractions 110 and 140 ml/min), monocytes, and polymorphonuclear cells (fraction rotor off). Characterized by flow cytometry, T cells were distributed nearly equal to fractions 110 and 140. NK cells were concentrated 3.4-fold in fraction 140 as compared with the unseparated cells. The ratio of NK cells/T cells was improved by 33%. These results indicate that CCE is an effective method to enrich NK cells and to reduce T cells in stem cell separation products. Therefore, it is an option for adoptive therapy of cancer patients after transplantations (e.g., CML in relapse).     

Extracorporeal photopheresis reduces the number of mononuclear cells that produce pro-inflammatory cytokines,when tested ex-vivo

Extracorporeal photopheresis (ECP) has been shown to be clinically effective in the treatment of many T cell-mediated conditions. ECP's mechanism of action includes the induction of apoptosis and the release of pro-inflammatory cytokines. Recently, we have observed early lymphoid apoptosis, detectable immediately post ECP. We were interested to determine what influence ECP has on pro-inflammatory cytokine secretion at this early pre-infusion stage. Samples from 6 cutaneous T cell lymphoma (CTCL) and 5 graft versus host disease (GvHD) patients were taken pre ECP and immediately post ECP, prior to re-infusion. Following separation, the PBMCs were added to a cell culture medium and stimulated with PMA, Ionomycin, and Brefeldin A for 6 hours. Using flow cytometry, intracellular cytokine expression of IFNgamma and TNFalpha was determined in the T cell population. The monocytes were evaluated for IL6, IFNgamma, IL12, and TNFalpha. For both patient groups, the number of IFNgamma-expressing T cells fell significantly at re-infusion, whilst both T cell- and monocyte-expressing TNFalpha levels were reduced at re-infusion. All other cytokines tested showed no significant change post ECP. For GvHD, pro-inflammatory cytokines have a pathological role. Their down-regulation may have a direct clinical benefit. However, the reduction in the number of IFNgamma- and TNFalpha-expressing mononuclear cells means, at this early stage, it is unlikely that these cytokines assist in the removal of the malignant Th2 cells present in CTCL.     

Differential effect of cryopreservation on natural killer cell and lymphokine-activated killer cell activities

The use of lymphokine-activated killer (LAK) cell therapy in delayed treatment requires the use of cryopreserved effector cells. The purpose of this study was to determine the optimal cryopreservation protocol for the maintenance of cytotoxic activity in mononuclear cells (MNCs). MNCs were cryopreserved with dimethyl sulfoxide or 1,2-propanediol before and after 3 days of culture with recombinant interleukin 2. The effects of cryopreservation on cell recovery, LAK cell and natural killer (NK) cell cytotoxic activities, and surface antigen markers were studied. Recovery of nonactivated MNCs was higher with 1,2-propanediol than with dimethyl sulfoxide (p < 0.05). Cytotoxic activities, measured with a 51Cr release assay, significantly decreased after thawing, on both activated cells (76.3%; range, 35.8-92.2%) and fresh cells (54.6%; range, 17.5-75.4%). A 6-day kinetic test was used to compare the cytotoxic activity of cryopreserved and fresh cells. The results showed different patterns for NK cells (cryopreserved cells had lower levels of activity than fresh cells) and LAK cells (cryopreserved cells had higher levels of activity than fresh cells). Phenotype changes of effector cells in culture, with and without cryopreservation, were monitored by flow cytometry using monoclonal antibodies. These results were compared with changes in the cytotoxicity of cells with and without cryopreservation. After thawing, there was a decrease in MNCs expressing CD14 and CD56. Recovery of the CD56 marker correlates with increased cytotoxic activity. Despite some loss of NK cell activity, it is concluded that MNCs may be successfully cryopreserved before their use in immunotherapeutic treatment.     

恶性肿瘤患者化疗前后免疫功能的变化

目的探讨恶性肿瘤患者化疗前后免疫功能的变化。方法运用流式细胞仪检测恶性肿瘤患者化疗前后免疫活性细胞,采用全自动免疫比浊术检测其免疫球蛋白。结果化疗前CD3 ,CD4 ,CD8 及NK细胞明显降低,化疗后CD3 ,CD4 及NK细胞显著增高,达到正常对照组水平,而免疫球蛋白在化疗前后无明显变化。结论免疫活性细胞的检测对恶性肿瘤患者的治疗与预后判断具有重要意义。     

自体活化脾细胞输注后T细胞亚群及PBMC细胞活性的临床观察

目的：细胞因子诱导的杀伤细胞具有可以快速增殖、高效和抗瘤谱广的特点。但是有关该细胞在肿瘤患者临床应用的报道甚少。本文旨在探讨自体脾细胞经黄芪皂甙（Astragalus Saponin）和小剂量rIL-2活化后过继免疫治疗对肝癌伴有脾功能亢进的患者免疫功能的影响。方法：16例患者均接受肿瘤切除、脾脏切除、皮下肝动脉泵埋入术和自体活化脾细胞过继免疫治疗。患者于手术前、后及过继免疫治疗后采集外周血Ficoll-Hypaque gradient分离单个核细胞，流式细胞仪检测CD44＋、CD44＋／CD8＋和NK细胞水平；MTT法检测PBMC细胞活性。结果：患者手术前、后CD44＋、CD44＋／CD8＋和NK细胞水平明显低于过继免疫治疗后（P〈0．01），而CD8＋细胞水平明显高于过继免疫治疗后（P〈0.01）；自体活化脾细胞过继免疫治疗后PBMC细胞活性明显高于手术前、后（P〈0．05）。结论：自体活化脾细胞过继免疫治疗可有效地提高患者的细胞免疫功能，且无副作用，对肝癌伴脾亢患者是一安全而有效的治疗方法。     

流式细胞术检测NK细胞活性在获得性噬血细胞性淋巴组织细胞增多症诊断中的应用

本研究目的旨在建立一种准确、稳定的NK细胞杀伤活性检测方法,应用于获得性噬血细胞性淋巴组织细胞增多症诊断。21例疑似患者及20例健康对照者纳入本研究。根据HLH-2004标准将疑似患者分为确诊组和排除组,将pEGFP—N1质粒转染人NK细胞的天然靶细胞K562,经过G418筛选后进一步单克隆,得到稳定、均一表达绿色荧光蛋白的EGFP—K562细胞,将EGFP—K562细胞与人外周血单个核细胞按10：1的比例混合孵育2小时后,用碘化丙啶（PI）标记,用流式细胞仪分析呈红、绿色荧光的细胞数,最后计算NK细胞的杀伤效率;同时采用LDH释放法测定外周血NK细胞对单纯K562细胞的杀伤活性,并进行比较。结果表明：获得了稳定表达绿色荧光蛋白的EGFP—K562细胞;流式细胞仪检测表明健康人NK细胞杀伤率与病人组之间存在明显差异,流式细胞仪技术检测NK细胞杀伤活性与传统LDH释放法检测NK细胞杀伤活性具有显著相关性。结论：EGFP—K562细胞株作为靶细胞用于流式细胞术检测NK细胞杀伤活性,无需预先染色和标记靶细胞,操作简便、省时、稳定、重复性高,可应广泛应用于获得性噬血细胞性淋巴组织细胞增多症诊断。     

Hematopoietic lineage-restricted minor histocompatibility antigen HA-1 in graft-versus-leukemia activity after donor lymphocyte infusion

Immunocompetent alloreactive donor lymphocytes directed against minor histocompatibility antigens are supposed to be responsible for graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activity after allogeneic stem cell transplantation. The authors describe the detection of HA-1-specific T cells by peptide-loaded dimers and flow cytometry in the peripheral blood of a patient in complete remission but without GvHD after donor lymphocyte infusion for chemotherapy-resistant Philadelphia chromosome-positive acute lymphoblastic leukemia. The HA-1-specific T cells were sorted and an alloreactive, polyclonal T-cell line with specific lytic activity against HA-1-positive target cells, including leukemic cells, was established. Although P190 bcr/abl peptide-specific CD8positive T cells were detected in the peripheral blood at the same time, these T cells could not be expanded. Furthermore, no P190 bcr/abl peptide-specific T-cell response could be induced in vitro, even when peptide-loaded dendritic cells were used as stimulator cells. The authors conclude that in the absence of GvHD, HA-1-specific rather than P190 bcr/abl-specific T cells are responsible for ongoing GvL activity.     

Suppression of graft-versus-host disease and amplification of graft-versus-tumor effects by activated natural killer cells after allogeneic bone marrow transplantation.

Bone marrow transplantation (BMT) is currently used for the treatment of a variety of neoplastic diseases. However, significant obstacles limiting the efficacy of allogeneic BMT are the occurrence of graft-versus-host disease (GvHD) and tumor relapse. Natural killer (NK) cells exert a variety of immunologic and homoeostatic functions. We examined whether adoptive transfer of activated NK cells of donor type would prevent GvHD after allogeneic BMT in mice. Lethally irradiated C57BL/6 (H-2(b)) mice, were transplanted with MHC incompatible BALB/c (H-2(d)) bone marrow cells and spleen cells and rapidly succumbed to acute GvHD. In contrast, mice that also received activated NK cells of donor type exhibited significant increases in survival. In determining the mechanism by which the NK cells prevented GvHD, mice were concurrently treated with a neutralizing antibodies to the immunosuppressive cytokine TGFbeta. Anti-TGFbeta completely abrogated the protective effects of the activated donor NK cells indicating that TGFbeta plays an important role in the prevention of GvHD by NK cells. We then examined whether activated NK cells of donor type after allogeneic BMT would induce graft-versus-tumor (GvT) effects without GvHD in mice bearing a murine colon adenocarcinoma (MCA-38). 10 d after receiving the tumor, in which the mice had demonstrable lung metastases, recipients received an allogeneic BMT with or without activated NK cells. Administration of activated NK cells resulted in significant GvT effects after allogeneic BMT as evidenced by increases in median survival and fewer lung metastasis. No evidence of GVHD was detected compared with recipients receiving spleen cells alone which also developed fewer lung metastases but in which all had succumbed to GVHD. Thus, our findings suggest that adoptive immunotherapy using activated donor NK cells combined with allogeneic BMT inhibits GvHD and promotes GvT in advanced tumor-bearing mice. These results also suggest that GvT and GvHD can be dissociable phenomena.     

Effect of ubenimex on the immune system of patients with hematological malignancies

The effect of in vivo administration of ubenimex (Bestatin) on the immune status of patients with hematological malignancies in remission was studied. Natural killer (NK) cell activities, lymphokine activated killer (LAK) cell activities, production of interferon-gamma (gamma-IFN) and surface antigens of peripheral lymphocytes were examined before and after administration of ubenimex. Analysis of the T, B and NK cell compartment ax conducted by assessing expression of the following antigens: CD3+CD19- (T), CD3-CD19+ (B), CD8+CD11b- (Tc), CD8+CD11b+ (Ts), CD4+Leu8-(Th), CD4+Leu8+(Ti), CD16CD57 (NK) using a 2-color flow cytometric analysis. NK and LAK activity was significantly lower in patients with hematological malignancies as compared to normal subjects. The absolute numbers of lymphocytes and NK cells were also lower than those in healthy controls. The reduced NK and LAK activity, however, was elevated after ubenimex administration. The absolute numbers of helper T cells, cytotoxic T cells and NK cells were also increased after administration of the drug. These findings were not observed in patients treated without ubenimex. Serum levels of IFN-gamma were not markedly changed after ubenimex administration. But peripheral blood mononuclear cells cultured with rIL2 showed appreciable levels of IFN-gamma production, and production increased after ubenimex administration. These results shows that ubenimex is a powerful immunomodulator that augments or restores some immune functions in patients with hematological malignancies.     

Mechanisms of action of extracorporeal photochemotherapy.

Extracorporeal photochemotherapy (ECP) has been shown to be effective in variety of pathologic diseases such as Sezary syndrome, autoimmune diseases, organ graft rejection and graft versus host disease. However, its mechanism of action has remained elusive. Understanding of its mechanisms may be useful to identify the best indications, treatment regimes and to optimize the ECP technique. The first step of the ECP procedure is collection of peripheral mononuclear cells. In this step, several cell environment changes occur. These conditions have been suggested to increase monocyte activation and possibly drive dendritic cell differentiation. The second step of ECP is the cell radiation by UVA in presence of 8-MOP which is presumed to induce cell membrane damage, DNA crosslinking and binding to a variety of cytosolic proteins leading to apoptosis, modification of membrane antigenicity and antigen presenting cell activation. The third step of ECP is the reinfusion of the treated cells to the patient. While it is unclear what exactly occurs in vivo, it is thought that DCs play a critical role by inducing an immunological response against pathogenic cells. The immature DC, activated by ECP, phagocytizes and internalizes the apoptotic cells; processes the antigens and increases the synthesis of class I and II Major Histocompatibility Complex (MHC) molecules. The peptides associated with class II MHC are presented to the CD4+ T helper cells. The final maturation of DC is completed in vivo with the help of these activated T helper cells using a variety of mechanisms including CD40 ligation. Finally, the mature DCs fully loaded with pathogenic T cell peptides migrate to secondary lymphoid organs stimulate the naive CD8+ T cells and induce a cytotoxic response (Th1 immune response) directed against pathogenic clones (tumoral cells of Sezary syndrome). Clinical and haematological improvement after ECP in Sezary syndrome is associated with a shift in Th1/Th2 balance and the increase of Th1 cytokines and IL12. ECP can also down regulate the allo or autoimmune response and induces tolerance by regulatory T cells. The clinical response to ECP in patients with chronic GvHD is associated with increase in NK cells and a shift from DC1 to DC2 and a shift from predominantly Th1 to Th2 immune response. Recruitment and involvement of other immune cells in the mechanism of ECP have been suggested and merit more studies. This immunostimulatory capacity of ECP is the most probable hypothesis of its mechanism but further investigations are necessary to determine the precise players important for this activity.     

可溶性IL-2受体及NK细胞活性在噬血细胞性淋巴组织细胞增多症中的意义

本研究通过检测噬血细胞性淋巴组织细胞增多症(HLH)患者外周血中可溶性IL-2受体(sCD25)及NK细胞活性,探讨它们在HLH中的意义。应用酶联免疫吸附法检测20例HLH患者、15例正常对照者、20例急性髓系白血病(AML)患者及20例系统性红斑狼疮(SLE)患者外周血血清sCD25水平;用流式细胞术CD107a标记法及传统的LDH释放法检测HLH组及正常对照组外周血NK细胞活性。结果显示:HLH组血清sCD25水平较正常对照组、AML组及SLE组均明显升高(P<0.001);HLH组NK细胞活性明显低于正常对照组(P<0.05);将流式细胞术CD107a标记法与传统的LDH释放法检测NK细胞活性进行相关分析,该两种检测方法之间具有显著相关性(r=0.73,P<0.05)。结论:血清sCD25及外周血NK细胞活性检测是HLH重要的辅助诊断指标,流式细胞术CD107a标记法检测NK细胞活性简单、稳定、重复性高,有助于HLH的临床诊断。     

CD3^-CD16^＋CD56^＋细胞在脐血与成人外周血中的初步研究

目的探讨脐血、成人外周血中自然杀伤（NK）细胞分化（CD）抗原百分含量的并同，分析在进行脐血（CB）与成人外周血（PB）造血干细胞移植（HSCT）时的利弊。方法采用流式细胞术（FCM），双色荧光素单克隆抗体标记，检测CB与PB中的NK细胞分化抗原（CD3^-CD16^＋CD56^＋）的百分含量。结果成人外周血中自然杀伤细胞分化抗原的百分含量明显高于脐血，经统计学处理两者差别具有显著性意义（P〈0．01）。结论脐血中NK细胞的含量与成人外周血存在不同程度的差异。这可能与两者进行造血干细胞移植后发生移植物抗宿主病（GVHD）、移植物抗白血病（GVL）作用的强弱相关。     

Acute response of peripheral CCr5 chemoreceptor and NK cells in individuals submitted to a single session of low‐intensity strength exercise with blood flow restriction

The purpose of this study was to compare the peripheral expression of natural killers and CCR5 in a session of low‐intensity strength training with vascular occlusion and in high‐intensity training. Young males were randomized into session groups of a high‐intensity strength training (HI) and a session group of low‐intensity strength training with vascular occlusion (LI‐BFR). The exercise session consisted in knee extension and bicep curl in 80% 1RM (HI) and 30% 1RM (LI‐BFR) with equalized volumes. Blood collection was made before, immediately after and 24 h after each training session. Immunophenotyping was carried out through CD195+ (CCR5) e CD3‐CD16+CD56+ (NK) in peripheral blood and analysed by flow cytometry and presented in frequency (%). Peripheral frequency of NK cells showed no significant difference in LI‐BFR group in time effect, while a gradual reduction of NK cells was identified in HI group in before‐24 h postexercise and after‐24 h postexercise comparison. However, significant differences have been found in relative change of NK cells immediately after exercise between sessions. In addition, HI and LI‐BFR groups showed a significant reduction in the cells expressed CCR5 during 24 h postsession compared to the postsession, but CCR5 also differed when comparing before‐24 h after session in the HI group. No differences were observed amongst the groups. LIO induced CCR5 response similar to the HI session, while the NK cells remained in similar frequency during the studied moments in LI‐BFR, but not in HI group, suggesting that local hypoxia created by the blood flow restriction was able to prevent a change in the frequency of peripheral cells and a possible immunosuppression.     

The role of the extracorporeal photopheresis in the management of the graft-versus-host disease

Over the last decades significant advances have been made in the field of donor selection, alternative transplant sources, immunosuppressive treatment and supportive care, as well as in the better understanding of the immunobiology of allogeneic hematopoietic stem cell transplantation (alloTx). Nevertheless, several factors still affect unfavorably the outcome of the procedure. Graft-versus-host disease (GvHD) remains the leading cause of morbidity, non-relapse mortality and treatment failure post alloTx. So far, steroids are the widely used 1st line treatment for GvHD achieving considerable response rate however, patients who fail to respond to the initial therapy have a dismal prognosis and no standard treatment is well established for them to date. In recent years, extracorporeal photopheresis (ECP) has been proposed as an efficacious and safe treatment for steroid refractory GvHD. Overall responses of 75% have been reported in the cutaneous and mucosal involvement and 45–65% in other organ manifestations (lung, liver and intestinal), allowing reduction and even discontinuation of steroids, thus contributing towards a significant reduction of morbidity. Although the mechanism of action of ECP is not fully understood, it seems that it has an immunomodulatory rather than an immunosuppression effect and induces immunotolerance, preserving the beneficial graft-versus-tumor effect. Given these very promising results in steroid-refractory or steroid-depended GvHD, currently, extracorporeal photopheresis is being investigated as both first-line and prevention therapy also.     

慢性自发性荨麻疹患者外周血SP、NK1R和ECP的表达

［摘要］目的： 探究慢性自发性荨麻疹（CSU）患者外周血P物质（SP）、神经激肽/速激肽受体1（NK1R）及嗜酸性粒细胞阳离子蛋白（ECP）的表达情况。方法： 采用流式细胞仪分析15例CSU患者及15例对照者外周血SP、NK1R表达水平；同时用酶联免疫吸附试验检测两组外周血ECP的表达情况。结果： 1.CSU组SP表达水平比对照组略高［（1.907±0.4211）% vs（1.813±0.2687）%］，两组差异无统计学意义（P值＞0.05）。2. 与对照组相比，NK1R在CSU组的表达显著升高［(14.52±0.9316)% vs(5.836±0.8823)%］，两组差异有统计学意义（P值＜0.05）。3.CSU组和对照组血清中ECP的表达平均值分别为14.54±1.255 ng/mL和7.456±0.982 ng/mL，两者差异有统计学意义（P值＜0.05）。4.经Pearson相关性分析，血清ECP水平和NK1R呈正相关（P值＜0.05)，与SP无相关性。结论： CSU患者SP的表达与对照组比较无明显升高；而NK1R与ECP表达均升高，NK1R可能促进嗜酸性粒细胞释放ECP，参与CSU的发生发展。     

恶性肿瘤患者化疗前后细胞免疫功能的监测

应用流式细胞仪对恶性肿瘤患者化疗前后进行Ｔ细胞亚群和自然杀伤（ＮＫ）细胞活性的检测，结果发现恶性肿瘤患者的ＣＤ３＾－、ＣＤ４＾－细胞、ＮＫ细胞活性和ＣＤ４＾－／ＣＤ８＾－细胞比值明显低于对照组，而ＣＤ８＾－和细胞高于对照组。化疗后各检测指标有升有降，其中多数患者ＮＫ细胞活性明显升高ＣＤ３＾－、ＣＤ４＾－细胞同步减少者较负步增多者有较高的感染率。流式细胞术对恶性肿瘤患者免疫功能的监测，具有快速，灵敏     

The effect of extracorporeal photoimmunotherapy (ECP) on serum TNF-a level in chronic graft versus host disease (GvHD).

Graft versus host disease (GvHD) is the most prominent cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (Allo-HCT). Extracorporeal photoimmunotherapy (ECP) is an alternative therapeutic modality in steroid and/or cyclosporin-A refractory GvHD developing after Allo-HCT. The aim of this study was to evaluate whether there was any relation between serum TNF-a levels and the response to ECP in patients with steroid refractory of extensive chronic GvHD. Between March 2001 and August 2003, seven patients (male: 1, female: 6) had ECP for treatment of steroid refractory extensive chronic GvHD. Five age and gender matched healthy volunteers were included in this study as the control group. The age of the patients ranged from 18 to 49 years. All patients were allografted from HLA-identical sibling donors. The median number of ECP sessions was 10 (8-36), consisting of two sequential cycles monthly. For measurement of serum TNF-a levels, blood samples were obtained both prior to ECP (basal) and after the first and second in all patients and in five patients after the 10th session. Serum TNF-a levels (Quantakine HS, R&D system, UK) were measured in peripheral venous blood samples by an ELISA method. ECP was given at a median of 5.8 months (1-14 months) after allo-HCT. No complications were seen during or after the ECP procedures. The median time of an ECP session was 183 minutes. The median volume of Uvadex used per session was 4.40 ml (3.61-5.61). The basal mean level of TNF-a was higher in patients than in the control group (2.47+/-0.83 pg/ml vs. 1.75+/-0.06, p=0.05). The mean TNF-a levels decreased from 2.47+/-0.83 pg/ml to 1.77+/-0.93 pg/ml after the initial session (p=0.045) and from 2.32+/-0.92 pg/ml to 1.69+/-0.93 pg/ml after the second day (p=0.015). After completion of the ECP sessions, extensive chronic GvHD recovered in only three patients. In three clinically responsive patients, the TNF-a levels were significantly reduced after both the second and tenth sessions. In contrast, in two patients not responding to ECP therapy, TNF-a levels were increased. In order to report whether these changes in TNF levels is an early predictor for evaluation of the efficacy of ECP in extensive chronic GvHD, TNF-a levels should be studied in a larger series.     

Induction of lymphokine-activated killer and natural killer cell activities from cryopreserved lymphocytes

Lymphokine-activated killer (LAK) and natural killer (NK) cells were studied for their capacity to retain cytotoxicity after cryopreservation. LAK cells were generated by a 4-day culture of lymphocytes with recombinant interleukin-2 (rIL-2). Cytotoxicity was measured by 51Cr-release assay at effector:target ratios of 10:1 to 80:1. Cryopreserved LAK cells retained 58.8 to 87.4 percent of cytotoxicity, as compared with that in fresh control cells. Cryopreserved NK cell activity against K562 and Molt-4 targets was 45.7 to 67.9 percent of the respective values of the fresh control cells. The responsiveness of NK cells to polyinosinic-polycytidilic acid (poly I:C), interferon-alpha (IFN-alpha), or rIL-2 remained intact. Activated NK cell activity after poly I:C or IFN-alpha stimulation and that after rIL-2 were, respectively, comparable to and higher than the endogenous NK cell activity of the fresh cells. The composition of lymphocyte subsets as determined by flow cytometry using monoclonal antibodies did not change after cryopreservation, indicating that cellular loss of the given subsets did not occur during the procedure. The retention of substantial levels of cytotoxicity in cryopreserved LAK and NK cells may make them promising candidates as cytotoxic effector cells.     

Mobilization of dendritic cells and NK cells in non-Hodgkin's lymphoma patients mobilized with different growth factors

Conflicting results have been reported regarding the effect of various growth factors on the mobilization of natural killer (NK) cells and dendritic cells in patients undergoing stem cell mobilization for autotransplantation. We compared the extent of mobilization of NK cells and dendritic cells in non-Hodgkin's (NHL) patients undergoing mobilization with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, or GM-CSF followed by G-CSF. Overall, 35 patients were studied. NK cells and dendritic were quantitated by flow cytometry. NK cells were defined as the sum of CD56(+) cells and CD56/CD16(+) cells. Dendritic cells were defined as the sum of CD80(+) and CD80(+)/CD14(+) cells. NK activity was determined by by microcytotoxicity assay. NK activity correlated well with the total amount of CD56(+) cells mobilized to the peripheral blood. Patients in the three arms of the study mobilized similar amounts of NK cells and NK activity, and patients who lacked NK activity in the peripheral blood, before mobilization, lacked NK activity in their apheresis collections. In contrast to NK cell mobilization, mobilization of dendritic cells/kg was three- to five-fold higher in patients mobilized with GM-CSF-containing regimens compared to patients mobilized with G-CSF alone. We conclude that GM-CSF-containing mobilization regimens are superior for dendritic cell mobilization but similar in the mobilization of NK cells. Therefore, we recommend using GM-CSF-containing regimens for patients undergoing ex vivo or in vivo manipulation of dendritic cells.