The Role of Amnion Membrane-Derived Mesenchymal Stem Cells on Differentiation and Expansion of Natural Killer Cell Progenitors Originated From Umbilical Cord Blood Mononuclear Cells

Abstract Background: Natural killer (NK) cells are members of the innate immune system. Their unique properties, including recognition of viral infected and tumor cells without major histocompatibility complex (MHC) restriction or prior sensitization, make them a suitable choice for immunotherapy. Low numbers of NK cells in circulating blood is the most important obstacle for this goal. Objectives: The aim of this study was to make an optimum in vitro condition to proliferate and differentiate cord blood (CB)-NK cell progenitors to mature NK cells, which can be used for cell therapy. Materials and Methods: In our study, CB-Mononuclear Cells’ (MNCs) CD3+ lymphocytes were positive depleted using immunomagnetic microbeads. This CD3-depleted (CD3-dep) CB - MNCs compartment was used for in vitro expansion with or without a layer of amnion membrane mesenchymal stem cells (MSCs) in combination with cytokines that are essential for NK cells expansion (IL-2, IL-3, IL-15, and FLT3 ligand). The expansion period lasted for one week. On day seven, immunophenotype and fold expansion of differentiated cells were measured. Results: Combination of cytokines and MSC layer yielded significant fold expansion in comparison with cytokines without feeder conditions (day 7: 5.2 ± 1.12 and 2 ± 0.78, respectively, P < 0.05). CD3-/CD56+ cells percentage increased during the culture period in MSCs/with cytokine and cytokine/without feeder, respectively (day 0: 4.4 ± 0.42% and day 7: 22.9 ± 3.6% and 13.9 ± 1.92 % for MSC/with cytokine and cytokine without feeder, respectively). Conclusions: Our results suggested that CB-NK cells progenitors could proliferate and differentiate on feeder layer of amnion membrane MSCs in combination with specific cytokines to produce NK cells for immunotherapy. Keywords: Umbilical Cord Blood, Natural Killer Cell, Mesenchymal Stem Cel

HLA-E polymorphisms and NK cell reconstitution after allogeneic hematopoietic stem cell transplantation

Background: Natural killer (NK) cells are an important component of the innate immune response. The study presented here suggests that NK cells can significantly influence the outcome after allogeneic hematopoietic stem cell transplantation (HSCT) - such as the incidence and severity of acute and chronic graft-versus-host disease (GvHD) and importantly the incidence of relapse. This study also discusses phenotypic features and functional activity of these cells after HSCT - in particular the importance of human leukocyte antigen (HLA)-E polymorphisms and NK cell reconstitution to clinical outcomes after HSCT. Methods: The study group included 56 patients undergoing allogeneic HSCT with one-year follow-up investigation. All patient-donor pairs were genotyped for HLA-E locus using a sequence-specific primer polymerase chain reaction (SSP-PCR) strategy. Analyses relevant to the association of HLA-E polymorphisms and post transplant outcomes including incidence and severity of acute and chronic GvHD, risk of infection, transplant-related mortality (TRM), relapse and survival rate were performed. Phenotypic and functional experiments were carried out in 27 patients with either acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) in complete remission (CR) pre-HSCT and their relevant donors. The samples were obtained prior to HSCT and at various time points during the first year post-HSCT. The expression of NK cell receptors such as natural cytotoxicity receptors (NCRs), NKG2D and CD16 as well as inhibitory killer Immunoglobulin-like receptors (KIRs) and NKG2A was examined using flow cytometry. The cytolytic activity of NK cells was also evaluated using a standard 51 chromium (Cr) release assay. Results: Phenotypic analyses of pre-HSCT NK cells compared to healthy donors showed similar numbers and frequencies of NK cells as well as the normal expression of activatory and inhibitory receptors. The cytotoxic analyses of NK cells prior to HSCT also exhibited comparable results in patients and donors. Analyses of post-HSCT NK cells compared to relevant donors demonstrated an elevated expression of NCRs and NKG2A in addition to a decreased expression of CD16 and inhibitory KIRs. Furthermore, an impaired cytotoxic activity of NK cells was also detected after HSCT. All these phenotypic and functional abnormalities normalized by the 12th month after HSCT. The results also revealed a delayed recovery of CD3+CD56- T cell frequency and number compared to those of CD3-CD56+ NK cells. The effect of HLA-E polymorphisms on transplant outcomes was also investigated. There was a lower frequency of acute GvHD (grade II or more) (p=0.03) and a lower frequency of extensive chronic GvHD (p=0.01) in the patients who had the HLA-E*0103/0103 genotype. There was also a lower incidence of transplant-related mortality (TRM; p=0.02) and better overall survival (p=0.04) in the patients with this genotype. Further experiments demonstrated, for the first time, a higher expression of activatory NKp30 (p<0.01), NKp46 (p<0.001) receptors and a lower expression of inhibitory KIRs (p<0.001) on NK cells as well as an increased NK cell cytotoxicity (p<0.001) in the patients with the HLA-E*0103/0103 genotype compared to the HLA-E*0101/0101 and HLA-E*0101/0103 genotypes. The impaired cytotoxicity and abnormal phenotypic characteristics of NK cells normalized much earlier in the patients with the HLA-E*0103/0103 genotype compared to those with the other genotypes. Conclusions: The study presented here showed for the first time a direct association between the HLA-E genotypes and phenotypic reconstitution of NK cell receptors as well as their cytolytic activity against MNCs of leukemic patients. These findings also supported other observations made here which exhibited a relation between HLA-E polymorphisms and transplant outcomes suggesting a protective role for the HLA-E*0103/0103 genotype. Further studies are required to investigate the exact molecular mechanisms involved in the association between the HLA-E*0103/0103 genotype and better outcomes of HSCT. These investigations will be relevant to a better understanding of the anti-leukemic effect of NK cells after HSCT that may have considerable clinical importance

HLA-E polymorphisms and NK cell reconstitution after allogeneic hematopoietic stem cell transplantation

Background: Natural killer (NK) cells are an important component of the innate immune response. The study presented here suggests that NK cells can significantly influence the outcome after allogeneic hematopoietic stem cell transplantation (HSCT) - such as the incidence and severity of acute and chronic graft-versus-host disease (GvHD) and importantly the incidence of relapse. This study also discusses phenotypic features and functional activity of these cells after HSCT - in particular the importance of human leukocyte antigen (HLA)-E polymorphisms and NK cell reconstitution to clinical outcomes after HSCT. Methods: The study group included 56 patients undergoing allogeneic HSCT with one-year follow-up investigation. All patient-donor pairs were genotyped for HLA-E locus using a sequence-specific primer polymerase chain reaction (SSP-PCR) strategy. Analyses relevant to the association of HLA-E polymorphisms and post transplant outcomes including incidence and severity of acute and chronic GvHD, risk of infection, transplant-related mortality (TRM), relapse and survival rate were performed. Phenotypic and functional experiments were carried out in 27 patients with either acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) in complete remission (CR) pre-HSCT and their relevant donors. The samples were obtained prior to HSCT and at various time points during the first year post-HSCT. The expression of NK cell receptors such as natural cytotoxicity receptors (NCRs), NKG2D and CD16 as well as inhibitory killer Immunoglobulin-like receptors (KIRs) and NKG2A was examined using flow cytometry. The cytolytic activity of NK cells was also evaluated using a standard 51 chromium (Cr) release assay. Results: Phenotypic analyses of pre-HSCT NK cells compared to healthy donors showed similar numbers and frequencies of NK cells as well as the normal expression of activatory and inhibitory receptors. The cytotoxic analyses of NK cells prior to HSCT also exhibited comparable results in patients and donors. Analyses of post-HSCT NK cells compared to relevant donors demonstrated an elevated expression of NCRs and NKG2A in addition to a decreased expression of CD16 and inhibitory KIRs. Furthermore, an impaired cytotoxic activity of NK cells was also detected after HSCT. All these phenotypic and functional abnormalities normalized by the 12th month after HSCT. The results also revealed a delayed recovery of CD3+CD56- T cell frequency and number compared to those of CD3-CD56+ NK cells. The effect of HLA-E polymorphisms on transplant outcomes was also investigated. There was a lower frequency of acute GvHD (grade II or more) (p=0.03) and a lower frequency of extensive chronic GvHD (p=0.01) in the patients who had the HLA-E*0103/0103 genotype. There was also a lower incidence of transplant-related mortality (TRM; p=0.02) and better overall survival (p=0.04) in the patients with this genotype. Further experiments demonstrated, for the first time, a higher expression of activatory NKp30 (p<0.01), NKp46 (p<0.001) receptors and a lower expression of inhibitory KIRs (p<0.001) on NK cells as well as an increased NK cell cytotoxicity (p<0.001) in the patients with the HLA-E*0103/0103 genotype compared to the HLA-E*0101/0101 and HLA-E*0101/0103 genotypes. The impaired cytotoxicity and abnormal phenotypic characteristics of NK cells normalized much earlier in the patients with the HLA-E*0103/0103 genotype compared to those with the other genotypes. Conclusions: The study presented here showed for the first time a direct association between the HLA-E genotypes and phenotypic reconstitution of NK cell receptors as well as their cytolytic activity against MNCs of leukemic patients. These findings also supported other observations made here which exhibited a relation between HLA-E polymorphisms and transplant outcomes suggesting a protective role for the HLA-E*0103/0103 genotype. Further studies are required to investigate the exact molecular mechanisms involved in the association between the HLA-E*0103/0103 genotype and better outcomes of HSCT. These investigations will be relevant to a better understanding of the anti-leukemic effect of NK cells after HSCT that may have considerable clinical importance

Irradiation of platelets in transfusion medicine: risk and benefit judgments

Irradiation of platelet products is generally used to prevent transfusion-associated graft-versus-host disease (TA-GvHD) as well as transfusion-transmitted infections. As an essential prerequisite, gamma-irradiation of blood products prior to transfusion is required in patients who may develop TA-GVHD. Most studies suggest that gamma irradiation has no significant effect on the quality of platelet products; however, more recent studies have shown that the oxidative effects of gamma irradiation can lead to the induction of platelet storage lesion (PSL) and to some extent reduce the efficiency of transfused platelets. As the second widely used irradiation technique, UV-illumination was primarily introduced to reduce the growth of infectious agents during platelet storage, with the advantage that this method can also prevent TA-GvHD. However, the induction of oxidative conditions and platelet pre-activation that lead to PSL is more pronounced after UV-based methods of pathogen reduction. Since these lesions are large enough to clearly affect the post-transfusion platelet recovery and survival, more studies are needed to improve the safety and effectiveness of pathogen reduction technologies (PRTs). Therefore, pointing to other benefits of PRTs, such as preventing TA-GvHD or prolonging the shelf life of products by eliminating the possibility of pathogen growth during storage, does not yet seem to justify their widespread use due to above-mentioned effects. Even for gamma-irradiated platelets, some researchers have suggested that due to decreased 1-hour post-transfusion increments and increased risk of platelet refractoriness, their use should be limited to the patients who may develop TA-GVHD. It is noteworthy that due to the effect of X-rays in preventing TA-GvHD, some recent studies are underway to examine its effects on the quality and effectiveness of platelet products and determine whether X-rays can be used as a more appropriate and cost-effective alternative to gamma radiation. The review presented here provides a detailed description about irradiation-based technologies for platelet products, including their applications, mechanistic features, advantages, and disadvantages

The Role of Amnion Membrane-Derived Mesenchymal Stem Cells on Differentiation and Expansion of Natural Killer Cell Progenitors Originated From Umbilical Cord Blood Mononuclear Cells

Abstract Background: Natural killer (NK) cells are members of the innate immune system. Their unique properties, including recognition of viral infected and tumor cells without major histocompatibility complex (MHC) restriction or prior sensitization, make them a suitable choice for immunotherapy. Low numbers of NK cells in circulating blood is the most important obstacle for this goal. Objectives: The aim of this study was to make an optimum in vitro condition to proliferate and differentiate cord blood (CB)-NK cell progenitors to mature NK cells, which can be used for cell therapy. Materials and Methods: In our study, CB-Mononuclear Cells’ (MNCs) CD3+ lymphocytes were positive depleted using immunomagnetic microbeads. This CD3-depleted (CD3-dep) CB - MNCs compartment was used for in vitro expansion with or without a layer of amnion membrane mesenchymal stem cells (MSCs) in combination with cytokines that are essential for NK cells expansion (IL-2, IL-3, IL-15, and FLT3 ligand). The expansion period lasted for one week. On day seven, immunophenotype and fold expansion of differentiated cells were measured. Results: Combination of cytokines and MSC layer yielded significant fold expansion in comparison with cytokines without feeder conditions (day 7: 5.2 ± 1.12 and 2 ± 0.78, respectively, P < 0.05). CD3-/CD56+ cells percentage increased during the culture period in MSCs/with cytokine and cytokine/without feeder, respectively (day 0: 4.4 ± 0.42% and day 7: 22.9 ± 3.6% and 13.9 ± 1.92 % for MSC/with cytokine and cytokine without feeder, respectively). Conclusions: Our results suggested that CB-NK cells progenitors could proliferate and differentiate on feeder layer of amnion membrane MSCs in combination with specific cytokines to produce NK cells for immunotherapy. Keywords: Umbilical Cord Blood, Natural Killer Cell, Mesenchymal Stem Cel

Reactive oxygen species generation as marker of platelet activation in PRP derived platelet concentrates during storage: brief report

Background: Platelet storage is complicated by deleterious changes that cause progressive structural and functional damages, so-called platelet storage lesion (PSL). PSL is commonly manifested by augmented platelet activation which is also associated with the increased levels of reactive oxygen species (ROS). Whether ROS generation increases during the storage of platelet concentrates and whether it will be correlated with P-selectin expression as a valid marker of platelet activation was investigated in this study. Methods: In our experimental study, six PRP-platelet concentrates were randomly obtained from Iranian Blood Transfusion Organization (IBTO). All the platelet products met the standard quality assessment based on AABB guidelines. Washed platelets were subjected to flow cytometry analysis for the evaluation of P-selectin expression and intracellular ROS production using DHR 123 in day 0, 1, 3 and 5 after storage. Statistical data were analyzed by Kruskal-Wallis test with Dunn&rsquo;s multiple comparison test. For correlations, linear regression analysis was applied. P values of less than 0.05 were considered to be significant. Results: Platelets ROS generation significantly increased from day 0 to day 5 of storage (P= 0.0002). This observed gradual increase was also directly correlated with the increasing levels of P-selectin expression during platelet storage (r= 0.72, P= 0.0001). Conclusion: Our study showed significant increases in ROS generation during the storage of platelet concentrates correlated with the increments of P-selectin expression as an important marker of platelet activation. This finding suggests that the analysis of ROS generation can also be considered a marker of platelet activation during storage. However, whether ROS generation first induces platelet activation or platelet activation during storage triggers ROS generation is still remain to be determined

The impact of HLA-E polymorphisms in graft-versus-host disease following HLA-E matched allogeneic hematopoietic stem cell transplantation

The non-classical MHC class-I mainly involves in the regulation of innate immune responses where HLA-E plays a significant role in the cell identification by natural killer cells. HLA-E is a main regulatory ligand for natural killer cells and given the importance of these effector cells in hematopoietic stem cell transplantation, we investigated the effect of HLA-E polymorphisms on post-hematopoietic stem cell transplantation outcomes. The study group included 56 donor-patient pairs with underlying malignant hematological disorders undergoing HLA-E matched allogeneic hematopoietic stem cell transplantation. They were genotyped for HLA-E locus using a sequence specific primer-polymerase chain reaction. The median follow-up was 20.6 months (range 0.2-114.8) and the parameters assessed were acute and chronic graft-versus-host disease and overall survival. We showed a lower frequency of acute graft-versus-host disease (grade II or more; p=0.02) and chronic graft-versus-host disease (extensive; p=0.04) in the patients with HLA-E*0103/0103 genotype compared to other genotypes of HLA-E. There was also an association between HLA-E*0103/0103 and improved overall survival (p=0.001)

The expression levels of platelet adhesive receptors in PRP derived platelet concentrates during storage

Background: Major platelet adhesive receptors that contribute significantly to thrombus formation include platelet receptor glycoprotein Ib&alpha; (GPIb&alpha;) of the GPIb-IX-V complex and platelet glycoprotein VI (GPVI). GPIb&alpha; plays a crucial role in platelet tethering to sub-endothelial matrix, which initiates thrombus formation at arterial shear rates, whereas GPVI is critically involved in platelets firm adhesion to the site of injury regardless of shear condition. During storage, platelets experience some changes that deleteriously affect the expression levels of platelet receptors, which in turn can alter platelet functional behaviors. Considering the important roles of GPIb&alpha; and GPVI in platelet adhesion, it seems that any dramatic changes in the expression levels of these receptors can influence adhesive function of transfused platelets. Thereby examining GPIb&alpha; and GPVI expression during the storage of platelet concentrates may provide some useful information about the functional quality of these products after transfusion. Methods: In our experimental study, 5 PRP-platelet concentrates were randomly obtained from Iranian Blood Transfusion Organization (IBTO). All the platelet products met the standard quality assessment based on AABB (American Association of Blood Banks) guidelines. Washed platelets were subjected to flowcytometry analysis for the evaluation of GPIb&alpha; and GPVI receptor expression in day 1, 3 and 5 after storage. Data were presented as mean fluorescence intensity (MFI) and analyzed by Kruskal-Wallis test with Dunn&rsquo;s multiple comparison test. Results: The GPIb&alpha; expression on first day (MFI=86&plusmn;5.9) was reduced three days after storage (MFI= 69&plusmn;6.9). The expression levels continued to reduce until day 5 in which GPIb&alpha; expression was markedly decreased to (MFI= 61&plusmn;7.7) (P= 0.0094). GPVI expression on the days 1, 3 and 5 after storage were 20.6&plusmn;3.3, 24&plusmn;2.5 and 14&plusmn;4.9, respectively. The results showed a significant decrease of expression on day 5, compared to that in day 3 after storage (P= 0.0213). Conclusion: Our study showed significant decreases in the expression of platelet receptors GPIb&alpha; and GPVI after 5 days storage, suggesting a major defect in adhesive function of platelets during this term

GPVI modulation during platelet activation and storage: its expression levels and ectodomain shedding compared to markers of platelet storage lesion

Platelet storage is associated with deleterious changes leading to the loss of platelet reactivity and response. During storage, platelets experience increased expression and shedding of P-selectin and CD40L as specific markers of platelet activation, whereas GPIbα decreases due to ectodomain shedding. As an important adhesive receptor, GPVI contributes significantly to thrombus formation while its expression and shedding levels during storage of platelet products have not been well characterized yet. This study investigated the modulation of GPVI during platelet storage. For this study, samples obtained from 10 PRP-platelet concentrates (PCs) were subjected to flow-cytometry analysis to examine the expression of platelet activation markers and GPVI on days 1, 3, and 5 post-storage. To examine the levels of etcodomain shedding of these molecules, microparticle (MP)-free supernatants were also analyzed by either ELISA or Western blot methods. According to results, the expression levels of P-selectin and CD40L as well as the amounts of their soluble forms significantly increased during storage. The expression of GPIbα and GPVI decreased whereas their shedding significantly increased post-storage. The expression and shedding levels of these two receptors were significantly correlated. Negative correlations between the expressions of GPIbα or GPVI and P-selectin have been observed whereas their shedding levels were significantly relevant together. In a control study, the use of biotinylated platelet resuspended in Tyrode’s buffer in the presence of ionophore with/without EDTA, confirmed the role of calcium in receptors shedding. In citrated PRP-PCs, recalcification of platelets also enhanced shedding levels of both GPIbα and GPVI. Intriguingly, the shedding levels of GPVI in stored PRP-PCs were much higher than those of ionophore-treated controls obtained from washed platelets. The ratios of sGPVI in stored platelet to ionophore-treated controls were also at least six times higher than those of GPIbα during storage. In conclusion, here we showed significant decreases of GPVI expression associated with its increasing levels of shedding during storage, suggesting GPVI as a valid marker of platelet storage lesion. Importantly, we found higher levels of GPVI shedding in stored platelets than those of ionophore-treated non-stored control samples. This suggests whereas platelet receptor shedding is mainly modulated by calcium-dependent signals, either platelet–surface interactions with the container walls during storage or induced shear stress under long-term agitation, might be also involved in the excessive shedding of GPVI during the storage of PCs

Ex vivo expansion of CD3depleted cord blood-MNCs in the presence of bone marrow stromal cells; an appropriate strategy to provide functional NK cells applicable for cellular therapy

Considering umbilical cord blood (UCB) as a rich source of hematopoietic stem cells, we introduced a cost-effective approach to expand CD3depleted UCB-MNCs into functional NK cells. CD3depleted UCB-MNCs were expanded in the presence or absence of a feeder [bone marrow stem cells (BMSCs) or osteoblasts], with or without cytokines and their differentiation into NK cells was determined by flow cytometry. NK cell function was quantified by LAMP-1/CD107a expression, TNF-α/IFN-γ release, and LDH release/PI staining in targets. Higher expansion of NK cells was observed after two weeks in the presence of BMSCs and cytokines (104 ± 15) compared to osteoblasts and cytokines (84 ± 29, p < 0.05). On day 14, CD3depleted UCB-MNCs in the presence of BMSCs and cytokines showed lower expression of CD3, CD19, CD14, CD15 and CD69 as well as higher expression of CD2 and CD7, which were suggestive of cell differentiation into mature NK cell lineage. Strong cytotoxicity of expanded cells was also identified with higher LDH release and PI% in targets. Significant upregulation of LAMP-1 with decreased release of IFN-γ and TNF-α from effectors were observed. We demonstrate an effective expansion of UCB-NK cells that maintained their functional capabilities applicable for cellular therapies